Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics.Trichinellosis is a re-emerging infectious disease, caused by T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate.The full-length cDNA encoding a putative cathepsin F-like protease in 50 value of E64 was 135.50 ± 16.90 nM.Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 μM and Vmax = 6.12 RFU/s μM at pH 5.5, and the ICT. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.TsCF1 was expressed in all stages of  Trichinella spiralis is a zoonotic nematode featured by causing chronic, debilitating infections with considerable morbidity and mortality. The life-cycle stages of T. spiralis constitute by adults worm (Ad), muscle larvae (ML) and newborn larvae (NBL). After ingestion of meat contaminated with T. spiralis ML, the parasite can invade into intestinal epithelium, and develop Ad via four molts within 3 to 4 days. NBL are produced at 4 to 10 days post-infection (dpi) according to the host species. NBL then migrate through the lymphatic and blood vessels and invade into host striated muscle cells and further develop into the muscle larvae, which can be infective to new host. The processes of migration and invasion are involved in a complex host-parasite interaction, and this peculiar stage is maintained until being ingested by a new host, thereafter the next generation begins [Parasite proteases may be involved in a wide variety of adaptive functions and play key roles as virulence factors in many parasite infections. Cysteine proteases have been well characterized in numerous helminths and could be key molecules in host-parasite interactions –6. They T. spiralis, most of them are serine protease [T. spiralis [T. spiralis (TsCF1) was cloned and identified. The expression, immunolocalization of TsCF1 and biochemical properties of recombinant TsCF1 protein were also characterized.Although several proteases have been reported in protease –17 and fspiralis , 19. In Animals were treated in strict accordance with the Animal Ethics Procedures and Guidelines of the People’s Republic of China. All animal procedures were approved by the Animal Ethics Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.T. spiralis (ISS534) ML were isolated from infected mice at 35 dpi by artificial digestion with pepsin-HCl. The protocols of isolation of Ad from infected rats intestines and NBL from female adults were followed as previous study [T. spiralis and excretory/secretory antigen were prepared according to the method reported [us study . Solublereported .Trichinella spiralis AND cathepsin F” were input for Gene database of GenBank search. Three cathepsin F-like genes  were acquired and named TsCF1, TsCF2 and TsCF3 respectively. Total RNA was extracted from the ML at 35 dpi, Ad and NBL of T. spiralis using Trizol (Invitrogen). Primer PR1 (5′-GCGAATTCTGCAGGATCCAACTTTTTTTTTTTTTTTTTT-3′) was used as the reverse transcription primer. Primer 1 (5′-CATCATTATGGTTTCCGTA-3′) and primer 2 (5′-GCGAATTCTGCAGGATCCAAC-3′) were used to amplify TsCF1 gene and study the transcription at different developmental stages. The PCR products were purified, ligated into pMD18-T vector  and sequenced. Analysis of the deduced amino acid sequence was conducted with DNASTAR  and http://www.expasy.org/tools/.Key words “T. brucei rhodesiense) and 3BCN  were selected as templates for homology modeling. Alignment between the target sequence and templates were performed using SALIGN [http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=index.html) (PROCHECK program) and the Structural Analysis and Verification Server (http://services.mbi.ucla.edu/SAVES/) (ERRAT program), and generated a full set of structural analysis for models.Homology modeling of the mature domain of TsCF1 was performed with MODELLER v9.5 . In termg SALIGN  in MODEL5 and 2.5 × 106, respectively. The conformations with lowest binding free energy of all docking results were selected and hypothesized to be a representative binding mode of ligand and receptors. Energy minimizations were performed to produce a series of more meaningful complex conformations for analysis. The complex structures for analysis were minimized with the steepest descent and conjugate gradient by Discovery Studio 2.5.Ligands E64 and K11777 were docked into the activity pocket of the receptor TsCF1 using AUTODOCK 4.0 . LamarckT. spiralis TsCFs and cathepsin Fs of other helminths, a rooted Bayesian (BI) phylogenetic tree was constructed by MrBayes v3.2.2 (https://www.phylo.org/) under the best-fit model WAG + I + G [http://tree.bio.ed.ac.uk/software/figtree/).To explore the phylogenetic relationships involving  + I + G , 25. The + I + G . For eacXho I and Bam HI restriction enzyme sites: PF2 (5′-CGCGGATCCTTGCCAATGAAGCAAAAGAGA-3′) and PR2 (5′- CGCCTCGAGTTAATCAATCACAACTGA-3′) with pMD18T-TsCF1 plasmid as the template. Then the amplified PCR product was ligated into pET-30a vector  which was digested by Xho I and Bam HI and the recombinant plasmid was transformed into E. coli BL21 (DE3) cells (Transgen). The expression of the recombinant TsCF1 protein (rTsCF1) was induced by adding isopropyl-1-thio-β-d-galactopyranoside (IPTG) to a final concentration of 1 mM and analyzed on Coomassie-stained SDS polyacrylamide gel electrophoresis (SDS-PAGE) gels. After SDS-PAGE, protein bands were excised from SDS-PAGE zymography gels and stored in ultrapure water. MALDI -TOF/TOF-MS/MS was performed by 4800 Plus MALDI TOF/TOF TM Analyzer  in Shanghai Applied Protein Technology Co., Ltd (Shanghai). The MS data were used to search against the non-redundant protein database .The TsCF1 prodomain and the entire mature domain were amplified using the specific primers carrying l-arginine, 5 mM reduced glutathione, 1 mM oxidised glutathione and gently stirred at 4 °C for 20 h as described previously [To purify rTsCF1, the cells were harvested at 4 h after incubation with IPTG at 37 °C, and then the pellet of cells from 2,000 ml of culture were suspended in 8 M urea lysis buffer. The rTsCF1 was purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography , and the purity was analyzed by SDS-PAGE. Refolding of the purified rTsCF1 was performed as follows: Ni-NTA affinity purified rTsCF1 (10 mg) was slowly added into 1 L of 100 mM Tris–HCl (pH 8.0) containing 1 mM EDTA, 250 mM eviously .Two New Zealand rabbits were used to produce the polyclonal antibodies against rTsCF1. Rabbits were primarily immunized subcutaneously at two locations with 1 mg rTsCF1 mixed with Complete Freund′ Adjuvant. Booster immunizations were performed at 3 week intervals with 1 mg rTsCF1 emulsion of incomplete Freund′ Adjuvant. The blood samples were collected prior to each immunization, and 7 days after the last immunization, the antibody titers were checked by ELISA.T. spiralis Ad, ML and E/S antigens as well as rTsCF1 were separated on 12.5 % SDS-PAGE before being electrophoretically transferred onto Hybond C extra membranes . The membrane were then blocked and incubated with 1:200 diluted anti-rTsCF1 rabbit sera. After washing, the membranes were incubated with goat anti-rabbit immunoglobin G (IgG) alkaline phosphatase (AP) conjugate . Finally, the bands were detected using NBT/BCIP .The extracts from T. spiralis were recovered from infected mice by the acid-pepsin digestion of striated muscles at 35 dpi. The parasites were fixed in cold acetone (−20 °C) for 10 min and then centrifuged for 5 min at 1000 g, followed by washing three times in PBS. After treated with 0.1 % Triton X-100 for 10 min at room temperature, the parasites were blocked with 5 % normal goat serum in PBS and then incubated in a moist chamber at 37 °C for 1 h with a 1: 10 dilution of immune and normal rabbit sera. After washing three times in PBS, the larvae were incubated with a 1: 20 dilution of FITC-labeled goat anti-rabbit IgG  and 0.01 % Evan’s blue for 1 h, washed five times in PBS, then transformed to slides and added 90 % Buffered Glycerol then observed under a fluorescent microscope .ML of Enzyme activity was assayed fluorometrically as the hydrolysis of Z-Phe-Arg-AMC . Briefly, 0.2 μg of enzyme was added to 190 μl of sodium acetate buffer (pH 5.5) containing 2 μM Z-Phe-Arg-AMC and 10 mM DTT, and the release of fluorescence  over 20 min at room temperature was assessed with a SpectraMax M5 .50) values of E64 of rTsCF1 was measured in a fluorescence endpoint assay using Z-Phe-Arg-AMC as a substrate [For evaluation of the inhibitory kinetic features of rTsCF1, a known inhibitor E64, was selected for assay . The 50 ubstrate . The assT. spiralis  , TsCF1, TsCF2 and TsCF3. The active pocket contains the core residues Gln, Cys and His. Phylogenetic analysis of cathepsin F family proteins from different helminth species revealed that two clans existed, one was nematoda clan and the other was trematoda clan . After induction with IPTG, a fusion protein was expressed as inclusion body form. SDS-PAGE analysis of rTsCF1 revealed a 45 kDa band which was consistent with the predicted size (Fig. T. spiralis adult, muscle larvae and excretory/secretory products of muscle larvae (Fig. T. spiralis.A cDNA fragment of TsCF1 coding the proregion and C1A mature domain was cloned into pET-30a and transformed into ize Fig.  and MALDvae Fig. . And theThe immunolocalization result with the whole parasite showed that the intense green fluorescent staining using anti-rTsCF1 was observed at the cuticle and also slightly at some stichocytes of muscle larvae Fig. .Fig. 8Im50 value of 135.50 ± 16.90 nM.The specific synthetic peptide substrate for cathepsin L like group, Z-Phe-Arg-AMC, was used to investigated the function of the rTsCF1. The peptide was hydrolyzed by rTsCF1 at pH 5.5 with an apparent of Km = 0.5091 μM and Vmax = 6.12 RFU/s μM Fig. . The pepT. spiralis could be recognized by early T. spiralis infection sera and suggested to be a target for vaccine and immunodiagnostic antigen [T. spiralis genome draft was published in 2011 [T. spiralis genome database was mined for searching cathepsin F and three cathepsin F-like proteases were identified. In human, only one cathepsin F was found which is different from other cysteine proteases in that there is a cystatin like domain in the unique very long proregion [T. spiralis infection in human.Cysteine proteases originated from parasite have a critical role in pathogenesis involved in molting, tissue penetration, nutrition metabolism, immune evasion and immune modulation . It is r antigen . T. spir in 2011  and it iroregion . This geroregion . The strSchistosoma japonicum and Schistosoma mansoni only one cathepsin F similar to human cathepsin F was identified. Fasciola hepatica and Fasciola gigantica did not express cathepsin F by database retrieval. However Opisthorchis viverrini parasitized in the bile duct of host, expressed one cathepsin F cysteine protease. Intriguingly, the cathepsin F genes in Clonorchis sinensis and Paragonimus westermani are existed in a pattern of superfamily, which may be correlated with gene expansion events and the function in parasitism. In the nematode, the expression of cathepsin F varies significantly. Three cathepsin F genes were predicted to exist in the genome of Strongyloides stercoralis. As to the other filarial nematode Loa loa, two cathepsin isoforms existed in the genome; and only one cathepsin F gene was existed in Ascaris suum, Brugia malayi, Teladorsagi circumcincta, Dictyocaulus viviparus and Caenorhabditis elegan. When the genome database of Toxocara canis and Trichuris trichura were searched, no cathepsin F was obtained. This may be due to imperfect gene annotation or cathepsin F was authentic in existence. According to the cestode genome data, cathepsin F was not existed within cestode, it may have alternative protease to substitute function of cathepsin F. This phenomenon may be related to the specific anatomical structure. Previously reports demonstrated that cathespin F was associated with nutrition uptake and cathepsin F existed in the gut of parasite. Whereas cestode does not have alimentary canal and oral cavity, which has distinct morphological and histological characterizations thus depending on the body wall for nutrients uptake, and cathepsin B and cathepsin L were abundantly expressed in the tapeworms. In phylogenetic analysis, TsCFs were positioned just at the junction of nematode and trematode. This coincides with the phylogenetic position of T. spiralis since T. spiralis belongs to the clade I of nematode, located just adjacent to platyhelminth trematode species [While the distribution of cathepsin F varies significantly among parasitic helminths, which means that there is a significant diversity among species. As to platyhelminth flukes, the expression pattern of cathepsin F in trematode is variable. In  species .F. hepatica previously, the trematode exerted different functions to survive in the specific tissues of parasitized host. A variety of cathepsin Ls existed in F. hepatica as a result of gene duplication and divergent evolution events [T. spiralis is related to a gene expansion event or not requires futher study.The analogous expansion of cathepsin Ls were identified in n events . WhetherT. circumcincta and CsCP derived from C. sinensis showed immunogenic characteristic [T. spiralis ML and thus might play a pivotal role in interaction between parasite and host.To date, cathepsin Fs have been functionally characterized in parasitic trematode and nematode –13. Cytoteristic . TsCF1 wT. spiralis developmental stages including ML, Ad and NBL at expression level. Western blots analysis showed that anti-rTsCF1 serum recognized the native protein in crude antigens of Ad, ML and ES antigens of ML, indicating that TsCF1 is one component of both the crude and ES proteins from T. spiralis. These coincide with the data from the global transcriptome analysis of T. spiralis [E. coli system as inclusion body form and showed substantial enzyme activity after renaturation. This activity could be inhibited by inhibitor E-64.RT-PCR results indicated TsCF1 was consecutively transcribed during all spiralis . The rTsDifferentiation in the binding modes of E64 to respective enzymes may be due to different pocket structures induced by different residues of each pocket. Because of the similar structure of the Cα backbone chains of pocket, in all complexes, there is a set of similar interactions between ligands and TsCF1 active pocket. According to the previous research, K11777 was a potent specific inhibitor of cathepsin B and preliminary analysis predicted that K11777 could not inhibit TsCF1 activity.T. spiralis. TsCF1 was expressed in all stages of T. spiralis, and localized in the cuticle and stichosome. The recombinant TsCF1 protein demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. These results indicated that TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection. The biological function of the enzyme involved in host-parasite interface in vivo needs to be further studied.This is the first work to characterize the cathepsin F-like protease TsCF1 from


Noise-induced hearing loss (NIHL) is one of the most reported occupational diseases internationally. The occurrence of audiometric notches is emphasized in both American and European guidelines for the diagnosis of NIHL. The aim of this study was to describe the prevalence of notched audiograms among railway personnel with and without noise exposure to better assess the usefulness of such notches in the diagnosis of NIHL.The most recent audiogram from 1994 to 2011 of a total of 12,055 railway workers, age 20 to 65 years, was obtained from the medical records of the occupational health service of the Norwegian State Railways (NSB). The prevalences of three types of notched audiograms, Coles notch, notch index, and 4 kHz notch, were computed, in relation to age, sex, and occupational noise exposure.p < 0.001). The corresponding figures for the 4 kHz notch were 31% versus 21% (p < 0.001). For the notch index, 61% of the exposed and 51% of the controls had a notched audiogram (p < 0.001). For female workers, the prevalence of audiometric notches was lower, and the differences between noise exposed and nonexposed was smaller than those in men. Increasing age led to an increased prevalence of notches.Coles notch in either ear was found in 63% of the male railway maintenance workers, exposed to noise levels of 75 to 90 dB(A), compared with 53% of the non-noise exposed (<70 dB(A)) traffic controllers (Audiometric notches commonly occur among both noise-exposed and those not exposed to noise in railway personnel. The usefulness of audiometric notches in the diagnosis of NIHL is therefore limited. Noise induced hearing loss (NIHL) is one of the most reported occupational diseases internationally. The occurrence of audiometric notches is emphasized in both American and European guidelines for the diagnosis of NIHL. The aim of this study was to describe the prevalence of notched audiograms among railway personnel with and without noise exposure in order to better assess the usefulness of such notches in the diagnosis of NIHL. Our results indicate that audiometric notches commonly occur among both noise-exposed and those not exposed to noise in railway personnel. Thus, we conclude that the usefulness of audiometric notches in the diagnosis of NIHL is limited. Noise is one of the most common causes of hearing loss . Noise-iIn developed countries, hearing loss due to occupational noise exposure is decreasing . This isIt is difficult to differentiate between age-induced hearing loss and NIHL . The occDespite the fact that audiometric notches commonly occur among non-noise-exposed individuals, there are still some who consider a notch as proof of NIHL . This caEach country has its own guidelines. Many have pointed out the lack of international guidelines for the diagnosis of NIHL as a problem which also makes it difficult to compare research . The aimThree groups of railway employees were chosen: Train drivers and conductors, train and track maintenance workers, and a reference group doing traffic controlling and other types of office work. All these groups have to perform a periodic audiometric test as a part of the mandatory health examination at 1- to 5-year intervals depending on age. All the tests are conducted by the occupational health service (OHS) of the Norwegian State Railways (NSB).An extensive noise exposure assessment program done by the OHS department has revealed a mean (8-hour Leq) noise exposure of 70 to 85 dBA for the train drivers and conductors, 75 to 90 dBA for the maintenance workers, and <70 dBA for the reference group of traffic controllers. Most of the train drivers, conductors, and traffic controllers are recruited at the age of 20+ years, and because railway employees have very little turnover, age minus 20 years is a good proxy for the exposure time. Because the maintenance workers use hearing protection during the most exposed types of work, the exposure level may be somewhat lower than that indicated by the noise measurements.The hearing sensitivity of the train drivers and conductors has been found to be approximately the same as for the reference group and not different from that of a national reference population of non-noise-exposed persons , which rThe results for the maintenance workers disclosed a small hearing loss in the 3 to 6 kHz area in the order of 3 to 5 dB in another study not yet published.Madsen Xeta Otometrics pure-tone audiometric testing using TDH-39P earphone headsets in a soundproof booth at frequencies 0.25, 0.5, 1, 2, 3, 4, 6, and 8 kHz was performed by trained nurses. The testing was done in-line with standard procedures according to the We used three types of notch definitions. The Coles notch was defined as hearing thresholds at 3 or 4 or 6 kHz of 10 dB or more compared with that at 1 or 2 kHz and 6 or 8 kHz. The criteria established by Coles et al. have been proven to correlate well with clinical assessments .The Notch index > 0 was defined as a mean hearing threshold of 2, 3, and 4 kHz minus the mean of 1 and 8 kHz > 0. It was used in a study of Rabinowitz to follow the progression of notched audiograms and may be more robust against the diminishing of notches caused by the hearing loss of 8 kHz in presbycusis . It is qThe 4 kHz notch was defined as a hearing threshold of 4 kHz at least 10 dB greater than that at 2 and 8 kHz and has been regarded as the signature notch in audiograms of NIHL .According to Norwegian regulations, an application to the regional ethics committee is not necessary because the audiograms have been obtained as a part of regular OHS work where risk assessment of NIHL is an important task.2 tests for categorical variables and analysis of variance for continuous variable. For the sex and age adjustments of audiometric findings, the UNIANOVA procedure in SPSS was used, and for the multivariate analysis of categorical variables binary logistic regression was used.The data analysis was performed by using SPSS (IBM Statistics version 20). Descriptive statistics were reported. Groups were compared using χIn the study, we used the most recent audiogram, obtained in the period 1994–2011, for 12,055 railway workers, 9881 men and 2174 women . The 4 kHz notch was found in 31% of male maintenance workers compared with 21% in the reference group (p < 0.002), and notch index > 0 was detected in 61% of the male maintenance workers compared with 51% in the reference group (p < 0.001). The prevalence of notched audiograms among male train drivers and conductors is in between the prevalences among the maintenance workers and in the reference group. This shows a dose–response relationship between noise exposure and the occurrence of notches, but the results also show that notches are indeed common among employees not exposed to occupational noise.Table For the female maintenance workers and train drivers and conductors, the prevalences of notches and the differences compared with the reference group were smaller and significant for the Coles notch only.Unilateral notches are more frequent than bilateral ones Table . The preBecause age, sex, and exposure to noise appear to be associated with notched audiograms, we analyzed the data in a logistic regression model Table . The anaLast, we examined the hearing threshold between the three occupational groups adjusted for age and sex in relation to the occurrence of notched audiograms or not. Table This study shows that notched audiograms are commonly occurring both in workers exposed and not exposed to noise. The prevalence varies with the type of notch. In male workers, Coles notch and notch index > 0 occur in a similar manner, about 60% in noise-exposed and 50% in nonexposed. The prevalence of the 4 kHz notch is lower, 31% in noise-exposed and 21% in nonexposed male workers. Unilateral notches are more common than bilateral. In men, the prevalence of notches increases with increasing noise exposure and age. The prevalence for Coles notch and 4 kHz notch is highest in the age group 45 to 54 years, and then declines. The prevalence of the notch index > 0 continues to increase with age.In women, it is different. The prevalences of notches is about half of that in men. The relationship with noise exposure is weaker than for men.The findings of increased prevalences of notches among train drivers and conductors compared with the reference group was surprising because they have a hearing acuity highly similar to the reference group and a noise exposure level that is unlikely to have any significant effect on hearing. We found that train drivers and conductors had slightly worse hearing at 6 kHz (2.1 dB) and a slightly better hearing at 8 kHz (1.4 dB) compared with the controls. Because 8 kHz is central to all definitions of audiometric notches, this small difference probably explains the occurrence of more notches in the train driver and conductor group than in the reference group, despite the small difference in hearing between the groups and the reference group.This study has some strengths. The number of participants is large. Because a hearing test is compulsory for individuals in all participating groups defined as safety personnel, we believe that the participation rate is close to 100%. To our knowledge, no other studies of this size have looked at the prevalences of notches in relation to age, sex, and noise exposure. The noise exposure data make it possible to provide good estimates of the prevalences of notches at different exposure levels. In addition, we have a large group of nonexposed workers with a hearing acuity comparable with national figures  and inteThis study is a cross-sectional study; longitudinal data would be desirable. We cannot rule out selection mechanisms such as those related to health requirements for jobs, including hearing acuity. The health requirements are, however, the same for the three occupational groups, and it is very unusual that someone will leave jobs because of hearing impairment because the health requirements are not very strict. In the jobs under study, one usually starts working at the age of 20 to 30 years and there is little turnover. We therefore believe that the lack of confounder control has not affected the results substantially.Our results are in accordance with those of Wilson examined the prevalence of 4 kHz notches in a group of military veterans and found a similar prevalence of notches as in the present group. The prevalence among the veterans for either ear was in excess of 30% in men 40 to 59 years of age compared with 30 to32% in our total material of men 45 to 64 years . Wilson Wilson, however, found a slightly lower prevalence of a modified version of Coles notch compared with our study. The 4 kHz notch definition was the same as in our study, but slightly different for the Coles notch that probably explains the higher prevalence in our study . The stuVarious types of audiometric notches are widely prevalent in workers not occupationally exposed to noise and almost as common as in exposed subjects. This makes the diagnosis of NIHL difficult. The prevalence of notches is emphasized in some studies, and some use occurrence of a notch as proof that there is an NIHL . RecentlThe prevalence of Coles notch and notch index in the present study is approximately 60% in the exposed and 50% in the nonexposed group. This implies that the specificity of one notch as criterion for NIHL diagnosis is very low, as 5 of 6 notches may be unrelated to occupational noise exposure. The general rule that a hearing loss with a notch indicates an NIHL and the lack of a notch speaks against must be used with great caution. The attributable fraction of hearing loss caused by noise at work has decreased in recent years  in the iIn conclusion, audiometric notches commonly occur among noise-exposed and non-noise-exposed workers in a Norwegian train company. Age and sex also play a certain role for the prevalence of audiometric notches. The usefulness of audiometric notches as a criterion of the diagnosis of NIHL is therefore of limited value.


Research on the effects of perinatal human immunodeficiency virus (HIV) exposure on hearing sensitivity is increasing. This is important because hearing loss can delay language and communication development in young children. The purpose of this project was to evaluate hearing sensitivity characteristics in HIV+ and HIV- children.Children were recruited from outpatient clinics within Tygerberg Children’s Hospital in Cape Town, South Africa. Caregiver report of middle ear infections was obtained through a questionnaire. Bilateral otoscopy, tympanometry, then pure-tone testing was completed. Hearing loss was defined as a pure-tone average (PTA) of 500, 1000, 2000, and 4000 Hz of >15 decibels of hearing level (dB HL) in the poorer ear. Unilateral hearing loss was defined as one normal ear and one ear with hearing loss. Conductive hearing loss was defined as an air-bone gap of ≥15 dB at a minimum of two frequencies in either ear. The percentage of children with hearing loss and mean PTA were compared between HIV+ and HIV- children. The effect of WHO status on hearing loss was examined for HIV+ children.Sixty-one children: 37 HIV+, 24 unexposed uninfected (HIV-), mean age 7.1 years (SD=1.6) were assessed. HIV+ children had a higher rate of reported middle ear infections and more conductive hearing loss. The risk for hearing loss was higher for HIV+ children (p=0.18), who also had a significantly higher mean PTA in the poorer ear than HIV- children . Six HIV+ children had unilateral hearing loss whereas one HIV- child had a unilateral hearing loss. Risk for hearing loss was higher for HIV+ children with WHO stage IV disease compared to other stages, but not significant.There were more middle ear infections, a higher risk of hearing loss, and more unilateral hearing loss in HIV+ than HIV- children; HIV+ children had a significantly higher PTA (worse hearing) in their poorer ear. The risk of hearing loss was higher in those with WHO stage IV disease than those with stage II or III.


Individuals with a unilateral severe-to-profound hearing loss, or single-sided deafness, report difficulty with listening in many everyday situations despite having access to well-preserved acoustic hearing in one ear. The standard of care for single-sided deafness available on the UK National Health Service is a contra-lateral routing of signals hearing aid which transfers sounds from the impaired ear to the non-impaired ear. This hearing aid has been found to improve speech understanding in noise when the signal-to-noise ratio is more favourable at the impaired ear than the non-impaired ear. However, the indiscriminate routing of signals to a single ear can have detrimental effects when interfering sounds are located on the side of the impaired ear. Recent published evidence has suggested that cochlear implantation in individuals with a single-sided deafness can restore access to the binaural cues which underpin the ability to localise sounds and segregate speech from other interfering sounds.The current trial was designed to assess the efficacy of cochlear implantation compared to a contra-lateral routing of signals hearing aid in restoring binaural hearing in adults with acquired single-sided deafness. Patients are assessed at baseline and after receiving a contra-lateral routing of signals hearing aid. A cochlear implant is then provided to those patients who do not receive sufficient benefit from the hearing aid. This within-subject longitudinal design reflects the expected care pathway should cochlear implantation be provided for single-sided deafness on the UK National Health Service. The primary endpoints are measures of binaural hearing at baseline, after provision of a contra-lateral routing of signals hearing aid, and after cochlear implantation. Binaural hearing is assessed in terms of the accuracy with which sounds are localised and speech is perceived in background noise. The trial is also designed to measure the impact of the interventions on hearing- and health-related quality of life.This multi-centre trial was designed to provide evidence for the efficacy of cochlear implantation compared to the contra-lateral routing of signals. A purpose-built sound presentation system and established measurement techniques will provide reliable and precise measures of binaural hearing.http://www.controlled-trials.com/ISRCTN33301739 (05/JUL/2013)Current Controlled Trials  Permanent acquired unilateral severe-to-profound hearing loss, or single-sided deafness (SSD), has been estimated to affect between 12–27 individuals in every 100,000 of the general population with the majority of losses being sudden and idiopathic; that is, a cause has not or cannot be determined . In partIn the UK National Health Service (NHS), the standard of care for SSD is a Contra-lateral Routing of Signals (CROS) hearing aid system. A CROS system picks up sounds arriving at the impaired ear using a remote microphone and presents those sounds to the non-impaired ear through a wired or wireless link . The priAlternatives to a CROS include a Bone-Anchored Hearing Device (BAHD) which also transmits sounds arriving on the impaired side to the non-impaired ear but achieves this by conduction through the cranial bones. There is some emerging evidence that BAHD may provide benefits to speech perception in noise over a CROS system although more high quality trials are needed ,15. BAHDAn alternative treatment for SSD which does have the potential to restore access to binaural cues is cochlear implantation. In cases of SSD where the auditory nerve is intact, impairments of the middle and inner ear can be bypassed and the auditory nerve stimulated electrically. Like CROS and BAHD devices, cochlear implantation in SSD has been found to improve access to sounds on the impaired side and has been associated with a reduction in self-reported difficulties with listening to speech in noise ,18,19. Ualternative to CROS in the NHS. Rather, it may be considered as an intervention for SSD in cases where a CROS system has been found to offer insufficient benefit as in these cases no other treatment options are currently available on the NHS. The current trial was therefore designed to evaluate whether cochlear implantation provides benefit to those patients who report receiving insufficient benefit from a CROS system.In the NHS, cochlear implantation is currently commissioned for adults and children with bilateral severe-to-profound deafness  and is nThis within-subjects longitudinal trial will determine whether cochlear implantation provides significant benefit in patients who have failed to receive sufficient benefit from a Contra-lateral Routing of Signals (CROS) system.• Does a cochlear implant significantly improve sound localisation compared to a CROS hearing aid in patients who report insufficient benefit from a CROS?• Does a cochlear implant significantly improve speech perception in noise compared to a CROS hearing aid in patients who report insufficient benefit from a CROS?• Does a cochlear implant significantly improve quality of life compared to a CROS hearing aid in patients who report insufficient benefit from a CROS?The trial is a within-subjects longitudinal study in which participants will be assessed before and after receiving two interventions delivered in a fixed order. Participants will be recruited from five NHS Trusts in the UK: 1) Nottingham University Hospitals NHS Trust; (2) University Hospitals Birmingham NHS Foundation Trust; (3) Central Manchester University Hospitals NHS Foundation Trust; (4) University College London Hospitals NHS Foundation Trust; and (5) Guy’s and St. Thomas’ NHS Foundation Trust. The trial is being coordinated by the National Institute for Health Research (NIHR) Nottingham Hearing Biomedical Research Unit, Nottingham. After screening against the inclusion/exclusion criteria, participants will progress through three phases: baseline, CROS, and cochlear implantation  was chosen in consultation with clinical colleagues from several UK audiology services to ensure that (a) it was of sufficient duration to allow the benefits from CROS to emerge; (b) any benefits from CROS would have stabilised; and (c) self-reported benefits would be based on patients’ experience of CROS use rather than any pre-conceived expectations (whether positive or negative). The duration of the cochlear implantation follow-up period (9 months) was chosen based on data from previous studies which have observed that (a) a large proportion of the benefit from cochlear implantation is achieved within the first 9 months and additional benefits emerge at a slower rate over the course of several years ,23; and The primary endpoints are the assessments of sound localisation and speech understanding in noise at baseline, after CROS aiding, and after cochlear implantation. Secondary endpoints are questionnaire measures of hearing- and health-related quality of life and the impact of tinnitus at baseline, after CROS aiding, and after cochlear implantation.The trial protocol and the study activities across the five NHS trusts were given a favourable opinion by the National Research Ethics Committee, East Midlands Nottingham - 2, Nottingham, UK (12/EM/0378). The sponsor of the trial is Nottingham University Hospitals NHS Trust, Research and Innovation Department, Nottingham, UK.Participants will be recruited from a notes review at each of the participating hospitals. The primary outcome measure is sound localisation accuracy; specifically, the mean absolute angular error in localising speech sounds. The trial is powered to detect a within-subject improvement in mean localisation accuracy resulting from the use of a cochlear implant compared to, and following the use of, a Contra-lateral Routing of Signals (CROS) hearing aid. A previous study examined the within-subject improvement in localisation accuracy after cochlear implantation compared to brief periods of CROS and simulated BAHD use prior to implantation . The autα=0.05 and with 95% power would require a sample size of 7. A final sample size of 10 was chosen to achieve the desired statistical power and to allow for some attrition due to the length of the follow-up period after cochlear implantation (9 months). The desired power reflects the fact that a study of this nature is unlikely to be repeated due to the substantial cost of providing the participants with a cochlear implant.Arndt et al.  reported• Eighteen years of age or older at the time of entry to the trial• Able and willing to undertake all assessments required by the trial• Good understanding of written and spoken English• Poorer ear (ear to be implanted): – Acquired  severe-to-profound sensorineural hearing loss of <10 years duration– Severe-to-profound deafness defined as having hearing threshold >90 dB HL at 1 & 4 kHz, and >65 dB at 0.5 kHz– Minimal benefit from a hearing aid (see below)• Better ear : Normal or near-normal hearing. For the purposes of this trial this is defined as hearing thresholds with a pure-tone average (PTA) of ≤30 dB HL at 0.5, 1, & 2 kHz.The process that will be used to determine minimal benefit from a hearing aid is illustrated in Figure For those patients who do not use a hearing aid at the time of the screening interview, an assessment will be made as to whether it would be clinically appropriate to provide amplification in the impaired ear. This assessment process will reflect current standard clinical practice. If the provision of a hearing aid would be clinically appropriate, the patient will be referred for hearing aid fitting to confirm whether the level of gain being called for can be obtained using any of the hearing aid models available on the NHS, and whether the prescription can be achieved. Verification of the prescription will be determined using real-ear measurements . If the Participants may have previous experience with using a CROS system. However, participants must have ceased use of any CROS system prior to entry into the study so that they may be evaluated in their unaided state during the baseline phase.• Evidence of middle-ear pathology based on otologic examination and tympanometry• Medical or psychological conditions that contraindicate undergoing surgery• Tinnitus as primary motivation for treatment• Ossification or any other cochlear anomaly that might prevent complete insertion of the electrode array• Hearing loss of neural or central origin, including auditory neuropathy and neurofibromatosis type II• Additional handicaps that would prevent or restrict participation in the audiological evaluations• Unrealistic expectations on the part of the participant regarding the possible benefits, risks, and limitations which are inherent to the surgical procedure and prosthetic device• Unwillingness of the participant to comply with all of the trial requirements• Any known factor which would limit the benefit obtainable from a cochlear implantA participant will be withdrawn from the trial if there is a failure of the cochlear implant device or they experience a serious or intolerable device-related adverse event leading to the explant or discontinued use of the cochlear implant device. Participants may withdraw themselves from the trial at any time and without giving a reason. Participants who withdraw voluntarily during the CROS or cochlear implantation phases may continue to use their current device (CROS or cochlear implant) indefinitely.The CROS hearing aid comprises a conventional acoustic hearing aid and a remote microphone. The remote microphone is worn on the impaired ear and the hearing aid is worn on the non-impaired ear. Sounds arriving at the impaired ear are picked up by the remote microphone and sent via a wireless link to the hearing aid which delivers the sounds via air conduction to the non-impaired ear. The acoustic coupling for the hearing aid is selected to have the smallest possible impact on the sound arriving at the non-impaired ear.The trial will use the Phonak CROS system . The system will be fitted by an experienced audiologist using the standard fitting software provided by Phonak (Target™3.0). The fitting procedure ensures that the acoustic information at the non-impaired ear is similar regardless of whether a sound source is incident to the impaired or non-impaired ear ,27. The A cochlear implant comprises an externally-worn sound processor and a wholly-implantable receiver-stimulator. The receiver-stimulator is connected to a micro-electrode array that is surgically-inserted into the inner ear (cochlea). Sounds are picked up by one or more microphones in the sound processor and are separated into frequency bands containing both slow changes in amplitude over time (envelope) and rapid changes in frequency around the centre frequency of each band (fine structure). The sound processor extracts the envelope in each frequency band and uses the envelopes to define the pattern of pulses to be delivered by each electrode. The pulse patterns are delivered through the scalp to the implanted receiver-stimulator via a radio-frequency coil.TM NucleusⓇ Cochlear Implant System . The NucleusⓇ CI442 implant will be implanted by experienced ENT surgeons. The implant system will be activated once the wound has healed, typically 3–6 weeks after surgery, and the sound processor will be fitted by an experienced cochlear implant audiologist. The programme of post-activation fitting and rehabilitation appointments will reflect the current standard care delivered to traditional cochlear implant candidates in the UK. Participants will be encouraged to use their cochlear implant for as long as possible each day and to record their actual usage in their user diary. Participants will also be encouraged to follow a rehabilitation programme which will include listening to speech materials fed directly into the sound processor of the implant via an accessory cable.The trial will use the CochlearThe CROS fitting procedure aims to preserve the resonance characteristics of the non-impaired ear and to overcome the head shadow which would otherwise attenuate sounds arriving at the impaired ear. The procedure requires the use of Real Ear Measurements (REMs). REMs will be carried out according to recommendations from the British Society of Audiology  and manuThe probe tube will be calibrated prior to commencing the fitting procedure. The procedure involves making three probe measurements : 1. A real-ear unaided response (REUR) while the loudspeaker is positioned 45 degrees towards the non-impaired ear. The probe tube and reference microphone are both situated at the non-impaired ear. The CROS system is not worn during this measurement.2. A real-ear aided response (REAR 1) measured using the same setup as for the REUR. The CROS system is worn and turned on during this measurement.3. A second real-ear aided response (REAR 2) measured while the loudspeaker is positioned 45 degrees towards the impaired ear. The probe tube is still situated at the non-impaired ear but the reference microphone is situated at the impaired ear.The measurement REAR 1 will be compared to the REUR to confirm that the coupling of the hearing aid to the non-impaired ear is largely acoustically transparent. Any gross dissimilarity will be addressed by checking and reconsidering the method of acoustic coupling. The REAR 1 and REAR 2 measurements will be compared and checked to be within ±3 dB across the available frequency range. Any deviations between the two measurements will be addressed by adjusting the gain of the CROS system and repeating the measurement of REAR 2.The available fitting hardware and software at each of the five trial sites may not permit the separation of probe tube and reference microphone on opposite sides of the head as required for the measurement of REAR 2. An alternative procedure has been described based on a stored equalisation method  and willThe cochlear implant fitting procedure will follow the manufacturer-recommended procedure for fitting using the Custom SoundⓇ software. The default programming parameters specified by the manufacturer will be used unless the individual needs of a patient require a deviation from those parameters to maximise benefit. In general terms, the fitting procedure will involve checking electrode impedances, disabling electrodes based on impedance measurements and/or intra-operative findings, creating a mapping between input frequency bands and electrodes (MAP), and establishing the minimum detectable (threshold) stimulation level and the maximum comfortable stimulation level for each electrode.Cochlear implant fitting is an iterative process to maximise hearing function, reflecting the emergence of hearing function over time due to a gradual acclimatisation and adaptation to electrically-delivered information. Participants will therefore attend several programming sessions in the initial months after activation of their implant through which their MAP, threshold levels, and comfort levels will be fine tuned to maximise speech understanding and acceptability to the patient.Assessments of the ability to localise sounds and understand speech in noise will be administered using a purpose-built test setup -30 in anSound localisation will be measured in anechoic conditions and a range of reverberant conditions following established methods using a visual-pointing technique . On eachThe original speech waveforms will be presented after being convolved with the room impulse of a simulated room, the methods and parameters of which have been described previously . The ratSpeech understanding in noise will be measured in terms of a speech-reception threshold (SRT) which represents the signal-to-noise ratio (SNR) at which 50% of sentences are reported correctly. Sentences will be drawn from a recording of the IEEE sentence corpus  spoken bSpeech section asks about difficulties with understanding speech in a range of everyday situations. The Spatial section asks about difficulties with localising sound sources and tracking moving sounds. The Qualities section asks about difficulties with the perceived naturalness of sounds, separating sounds, and the amount of effort required to listen. The SSQ has been found to be capable of distinguishing between groups of unilateral and bilateral hearing aid [The Speech, Spatial and Qualities of Hearing Scale (SSQ)  containsring aid  and cochring aid  users, cring aid .Glasgow Hearing Aid Benefit Profile (GHABP) has been developed as a patient-centred outcome measure designed to measure the disability caused by a patient’s hearing loss before and after hearing aid fitting and the impact that the disability has on a patient’s everyday life ,44. The The Glasgow Benefit Inventory (GBI) is an instrument for measuring changes in quality of life as a result of otorhinolaryngological interventions . The GBITinnitus Functional Index (TFI) has been designed to measure treatment-related changes in tinnitus intrusiveness and severity. The development of the TFI has included the definition of the minimum clinically-important change in TFI score (13 points) and identified a test-retest reliability of 0.78 .A user diary will also be provided in booklet form for the 3-month trial of the CROS system and for the 9-month follow-up period after cochlear implantation. Participants will be instructed to record the duration of their device use on a daily basis and will also be encouraged to record any comments about situations in which either intervention was particularly helpful or unhelpful.The Health Utilities Index Mark 3 (HUI3) is a generic preference-based method for describing health status and measuring health-related quality of life . The HUIThe EQ-5D is a generic method for describing and valuing health states ,55. The Analysis of the trial data will include all patients who pass the inclusion and exclusion criteria and will be conducted on an intention-to-treat basis. Participants who are withdrawn at any stage will be replaced. Participants who choose to leave during the baseline or CROS phases of the trial will be replaced. Participants who choose to leave after receiving a cochlear implant will not be replaced. Data from participants who choose to leave will be retained and included in the analyses of individual trial phases; i.e. baseline, CROS, and cochlear implantation. Due to the small sample size, missing data will not be imputed when conducting within-subject comparisons of the incremental change from baseline to CROS and from CROS to cochlear implantation. Drop-out rates at each phase of the trial will be reported as a marker of potential bias in the between-phase comparisons.Baseline characteristics of the participants will be reported including (but not limited to) age, gender, audiometric thresholds, aetiology and history of hearing loss, duration of severe-to-profound deafness, and history of hearing aid use.t-tests, Wilcoxon signed-rank tests, and repeated-measures analysis of variance, as appropriate. For each outcome measure, summary measures of the size and variance in the incremental benefit from cochlear implantation (difference between cochlear implantation and CROS) and CROS (difference between CROS and baseline) will be reported.The primary endpoint and analysis of the trial data will be at the end of the 9-month follow-up period after cochlear implantation. The analysis will compare outcomes in the cochlear implant phase to outcomes in the CROS phase. Outcomes in the CROS and baseline phases will also be compared. Comparisons will be performed for both the primary and secondary outcome measures. Comparisons both within and between trial phases will be performed using paired Student’s The reporting of adverse and serious adverse events will follow the standard operating procedure (SOP) for trials of medical devices specified by the sponsor organisation .Self-report and behavioural data confirm that individuals with SSD experience difficulties with listening to sounds on the side of their impaired ear and in determining the location of sounds in space -4. CurreThe current trial has been designed to provide evidence for the efficacy of cochlear implantation as an intervention in SSD compared to the current standard of care, a CROS system. A cochlear implant will be offered to those who receive insufficient benefit from a CROS system and for whom few other treatment options currently exist on the UK NHS. Participants will be evaluated prior to any intervention (baseline), after 1 and 3 months of CROS use, and at 3 and 9 months after cochlear implantation. The use of established measurement techniques  and a soThe trial is currently in recruitment phase.BAHD: Bone-Anchored Hearing Device; CROS: Contra-lateral Routing Of Signals; HUI3: Health Utilities Index Mark 3; GBI: Glasgow Benefits Inventory; GHABP: Glasgow Hearing Aid Benefit Profile; NHS: National Health Service (UK); NIHR: National Institute for Health Research; NRES: National Research Ethics Service; PTA: Pure-Tone Average; REUR: Real-Ear Unaided Response; REAR: Real-Ear Aided Response; REM: Real Ear Measurement; SD: Standard Deviation; SOP: Standard Operating Procedure; SNR: Signal-to-Noise Ratio; SRT: Speech-Reception Threshold; SSQ: Speech, Spatial and Qualities of Hearing Scale; TFI: Tinnitus Functional Index; VAS: Visual-Analogue Scale.This study is supported by infrastructure funding from the National Institute for Health Research (NIHR). PTK, GMOD and BUS were awarded an industry grant from Cochlear Europe Ltd  to cover the surgical, device, and rehabilitation costs of cochlear implantation for participants in the trial. The CROS hearing aids are being provided by Phonak Group Limited .All authors  developed the protocol. PTK, GMOD, MEJ and BUS drafted the manuscript. All authors read and approved the final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6815/14/7/prepub


TOTAL) at each test frequency was due to a combination of cochlear gain loss, or OHC dysfunction (HLOHC), and inefficient IHC processes (HLIHC), all of them in decibels. HLOHC and HLIHC were estimated from cochlear I/O curves inferred psychoacoustically using the temporal masking curve (TMC) method. 325 I/O curves were measured and 59% of them showed a compression threshold (CT). The analysis of these I/O curves suggests that (1) HLOHC and HLIHC account on average for 60–70 and 30–40% of HLTOTAL, respectively; (2) these percentages are roughly constant across frequencies; (3) across-listener variability is large; (4) residual cochlear gain is negatively correlated with hearing loss while residual compression is not correlated with hearing loss. Altogether, the present results support the conclusions from earlier studies and extend them to a wider range of test frequencies and hearing-loss ranges. Twenty-four percent of I/O curves were linear and suggested total cochlear gain loss. The number of linear I/O curves increased gradually with increasing frequency. The remaining 17% I/O curves suggested audiometric losses due mostly to IHC dysfunction and were more frequent at low (≤1 kHz) than at high frequencies. It is argued that in a majority of listeners, hearing loss is due to a common mechanism that concomitantly alters IHC and OHC function and that IHC processes may be more labile in the apex than in the base.Identifying the multiple contributors to the audiometric loss of a hearing impaired (HI) listener at a particular frequency is becoming gradually more useful as new treatments are developed. Here, we infer the contribution of inner (IHC) and outer hair cell (OHC) dysfunction to the total audiometric loss in a sample of 68 hearing aid candidates with mild-to-severe sensorineural hearing loss, and for test frequencies of 0.5, 1, 2, 4, and 6 kHz. It was assumed that the audiometric loss (HL TOTAL) for a given test frequency may be conveniently expressed as the sum of two contributions: one associated with cochlear mechanical gain loss, or OHC dysfunction (HLOHC), and one associated with inefficient IHC transduction, or IHC dysfunction (HLIHC), where HLTOTAL, HLIHC and HLOHC are all in decibels (dB)  and partly due to other factors (akin to HLIHC). Individual across-frequency estimates of HLOHC and HLIHC would be highly useful to optimize individualized treatment with hearing aids  listeners. Their results were broadly consistent with the common view that HLOHC is greater and more frequent than HLIHC, but they also reported some cases with substantial HLIHC at low frequencies. Jürgens et al. (OHC) was proportional to HLTOTAL but 10–15 dB lower (p. 189). More recently, we have proposed a more refined method for estimating HLOHC and HLIHC from the analysis of TMC-based input/output (I/O) curves. We concluded that HLOHC and HLIHC account on average for 60 and 40% of HLTOTAL with large variability across cases; indeed, percentages were sometimes reversed  and for test frequencies of 0.5, 1, 2, 4, and 6 kHz. A second objective was to investigate to what extent HLIHC and HLOHC vary across test frequencies to examine potential structure-function correlations; that is, to examine the potential correspondence between HLOHC and HLIHC with existing evidence regarding physical loss or injury and/or dysfunction of OHCs and IHCs and their distribution across frequency. A third objective was to investigate the degree of variability of HLOHC and HLIHC across listeners. We used virtually the same approach as in our recent study , whereas the present analysis is extended to all I/O curves; second, the present study is for a much larger subject sample and for a wider range of frequencies; third, the present study included participants with hearing losses from mild to severe, hence more representative of the hearing-aid candidate population.The main aim of the present study was to assess HLOur approach was virtually identical to that of Lopez-Poveda and Johannesen . The detTOTAL, HLOHC, and HLIHC are all in dB. In what follows, HLOHC and HLIHC will be referred to as “OHC loss” and “IHC loss,” respectively, and should be interpreted as contribution to audiometric loss (in dB) rather than as anatomical lesions or reduced cell counts.where HLOHC can be found using the OHC dysfunction model of Plack et al. (OHC can be found by comparing the CT of a given hearing-impaired (HI) listener with a reference CT for normal hearing (NH) listeners  as the difference between HLOHC and HLTOTAL. For a sufficiently large OHC dysfunction, all the cochlear gain is lost, the I/O curve becomes linear (absent CT) and HLOHC is assumed to be equal to the NH gain.We further assumed that HLk et al. . In thisk et al. . An assuIHC. For these cases, it is assumed that Equation (1) does not hold and that HLTOTAL ~ HLIHC. In other words, it is assumed that even though HLOHC may occur, it does not contribute to the audiometric hearing loss  with symmetrical sensorineural hearing loss participated in the study. Their ages ranged from 25 to 82 years (median = 61 years). Air conduction absolute thresholds were measured using a clinical audiometer (Interacoustics AD229e) at the typical audiometric frequencies  ANSI, . Bone coThe data from our previous related study was also included in the present analysis  of 0.5, 1, 2, 4, and 6 kHz. Maskers and probes were sinusoids. The duration of the maskers was 210 ms including 5-ms cosine-squared onset and offset ramps. Probes had durations of 10 ms, including 5-ms cosine-squared onset and offset ramps with no steady state portion, except for the 500-Hz probe, whose duration was 30 ms with 15-ms ramps and no steady state portion. The level of the probes was fixed at 10 dB above the individual absolute threshold for the probe. Masker-probe time gaps, defined as the period from masker offset to probe onset, ranged from 5 to 100 ms in 10-ms steps with an additional gap of 2 ms. Masker levels sometimes reached the maximum permitted sound level output (105 dB SPL) after a few time gaps. If the number of measured data points was insufficient for curve fitting (see below), masker levels were measured for additional intermediate gaps . In a few cases, masker levels were atypically low for a time gap of 100 ms. In these cases, masker levels were measured for additional gaps in the range 110–140 ms.Stimuli and procedure were similar to those of Lopez-Poveda and Johannesen . On-freqpf or 0.5pf. The selection of linear reference condition depended on the listener's hearing loss at the linear-reference probe frequency and on the maximum permitted sound level output (105 dB SPL). Following the indications of earlier studies,  and delivered to the listeners through Sennheiser HD-580 headphones. Sound pressure levels (SPL) were calibrated by placing the headphones on a KEMAR equipped with a Zwislocki DB-100 artificial ear connected to a sound level meter. Calibration was performed at 1 kHz only and the obtained sensitivity was used at all other frequencies.Masker levels at threshold were measured using a two-interval, two-alternative, forced-choice adaptive procedure with feedback. The inter-stimulus interval was 500 ms. The initial masker level was set sufficiently low that the listener always could hear both the masker and the probe. Masker level was then changed according to a two-up, one-down adaptive procedure to estimate the 71% point on the psychometric function Levitt, . An initAbsolute thresholds for the probes and maskers were measured using a similar procedure except that the adaptive procedure was one-up, two-down.TMC and absolute threshold measurements took between 12 and 15 h per participant in total and were distributed in several (1- or 2-h) sessions on several days.Linear-reference and on-frequency TMCs were fitted before they were used to infer I/O curves. Linear reference TMCs were fitted with a double exponential function with four parameters  was used to infer I/O curves. This average linear reference TMC was also used for reanalysis of four cases from our previous study  than for the NH reference group or the HI listeners used in our previous study . The difference for conditions ① and ⑤ was close to being significant (p = 0.055) but did not reach significance. Therefore, we concluded that all linear-reference TMCs had statistically comparable slopes and that it was reasonable to use them to infer I/O curves.Lopez-Poveda and Alves-Pinto  argued tad Table , and comFigure OHC and HLIHC may be reliably obtained from an I/O curve only if the I/O curve in question shows a CT. They nonetheless hinted that the shape of the I/O curves may be indicative of the type and extent of HLOHC or HLIHC . Here, each I/O curve was analyzed in search for HLOHC and HLIHC using their reasoning and following the logic outlined in Figure Lopez-Poveda and Johannesen  argued tand (2) that the residual cochlear gain of the mean I/O curve (estimated as described below) was greater than zero. The median CT of the Monte-Carlo simulated I/O curves was taken as the final CT.A CT was first sought for each I/O curve. Lopez-Poveda and Johannesen  arbitrarA large proportion of I/O curves showed a CT Table . Many otGain was defined here as the difference in sensitivity for low and high input levels, as illustrated in the right panels of Figure AL, BL, CL, and DL were defined as in Figure where If gain was not significantly different from zeroTable OHC and HLIHC. Type 1 I/O curves were analyzed as suggested by Lopez-Poveda and Johannesen  . This procedure required having mean reference CT and c values for NH listeners at each of the test frequencies . Lopez-Poveda and Johannesen , HLhannesen  providedOHC (top) and HLIHC (bottom) as a function of HLTOTAL. Note that HLTOTAL is defined here as the difference between a participant's absolute threshold for the masker and the mean absolute masker threshold of the reference NH group . Interestingly, these percentages were approximately constant across test frequencies, as shown by the slopes of the regression lines. The individual variability of the contributions HLOHC and HLIHC can be assessed from the confidence limits for new single observations. The confidence intervals for HLOHC and HLIHC were around ±9 dB at 0.5 kHz and around ±6 dB over the range 1–6 kHz. In all cases, the confidence intervals were almost independent of the HLTOTAL. Recall that these results were only for Type 1 I/O curves.The linear regression functions in Figure OHC) separately for each frequency. The vertical dotted red line (at HLOHC = 0 dB) indicates the hypothetical location of cases whose hearing loss was exclusively due to IHC dysfunction (pure IHC loss). The blue diagonal line depicts the hypothetical location of cases whose hearing loss was exclusively due to cochlear gain loss (pure OHC loss). The blue-dotted diagonal lines show 5–95% confidence intervals for gain loss as calculated from the reference NH listeners  plus an additional component (mixed OHC/IHC loss). The results from I/O curves with a CT are depicted as blue circles in the top panels of the figure. For completeness, also shown are the results for listeners with NH (black circles) and mild-to-moderate hearing loss (red circles) from our earlier study , and was equal to 35.2, 43.5, 42.7, 42.7, 42.7 dB at 0.5, 1, 2, 4, and 6 kHz. HLIHC was then obtained using Equation (1).Linear I/O curves were assumed to be indicative of total gain loss. Hence, HLIHC > 0 dB.Results for these cases are shown as blue crosses in the top panels of Figure TOTAL was equal to HLIHC.As explained above, I/O curves that were either compressive straight lines  was estimated by subtracting the obtained gain estimate from the reference gain for NH listeners (see the previous section). The bottom panels of Figure OHC was probably smaller than estimated, hence that symbols should be to the left of their position in the figure, and closer to the red-dotted line indicative of pure IHC loss. The figure reveals two important results: first, that most of these cases are indicative of mixed IHC and OHC dysfunction . As shown in the bottom panels of Figure The previous analyses have focused mostly on the relative contribution of HL2 tests were used to test if the above described frequency trends were statistically significant. The null hypothesis was that for each I/O curve type, the frequency distribution followed the distribution of the total number of cases .The top part of Table The present analysis was based on the hearing loss model of Plack et al.  whereby The aim of the current study was threefold: (1) to assess to what extent the audiometric loss is due to a reduction in cochlear gain (or OHC dysfunction), and/or to an additional component, referred here to as IHC dysfunction; (2) to investigate the frequency distribution of the two potential contributions; and (3) to investigate the degree of variability of the two contributions across listeners. Our approach was based on the analysis of behaviorally inferred cochlear I/O curves, as we proposed elsewhere  suggest that on average IHC and OHC dysfunction contribute 30–40 and 60–70% to the audiometric loss, respectively, and that these percentages hold approximately constant across the frequency range from 500 Hz to 6 kHz Figure . Cases fIHC and smaller HLOHC. In the present context, this means that if the assumptions were not valid, the contribution of HLIHC to the total hearing loss might be higher than reported in Figure In inferring I/O curves from TMCs, the assumption has been made that the post-mechanical rate of recovery from forward masking is independent of masker frequency and level  because their hearing losses at the linear reference probe frequencies Table  were so OHC was set equal to the mean cochlear gain of the reference, NH group. If the latter were inaccurate, this could have affected the present estimates of HLOHC  seem reasonable at high frequencies but are less certain at low frequencies.In summary, the NH gain values used here to quantify HLt-test, p = 0.014) and then gain remained constant at higher frequencies (42.7 dB at 4 kHz). This pattern differed slightly from that reported by . Small conductive losses might have increased probe absolute threshold and hence TMC masker levels by an amount equal to the conductive loss at the corresponding probe frequencies. The influence on the inferred I/O curve would be an upward vertical shift of the I/O curve equal to the conductive loss at the frequency of the linear reference probe and a rightward horizontal shift equal to the conductive loss at the frequency of the on-frequency masker. The CT would be affected only by the horizontal shift. Therefore, conductive loss at the particular frequency might lead to an overestimate of HLA “dead region” is “a region in the cochlea where the IHCs and/or neurons are functioning so poorly that a tone which produces peak BM vibration in that region is detected via an adjacent region where the IHCs and/or neurons are functioning more efficiently”  or the percentage of cases for which HLOHC ~ HLTOTAL (the percentage of pure OHC dysfunction) are small, they are both larger for frequencies ≤1 kHz and decrease with increasing frequency  does not hold for cases where IHC dysfunction is so significant that it makes it impossible to measure a CT. In these cases, it was assumed that HLTOTAL may be explained fully in terms of HLIHC even though concomitant cochlear gain loss did probably occurred  and hair cell pathophysiology in humans. Discussing potential relationships might be nonetheless useful.TOTAL is due to HLOHC and 30–40% is due to HLIHC, and that these percentages are roughly constant across frequencies , 60–70% of HLs Figure . Second,s Figure . This his Figure , and a rs Figure . The preWe have also shown, however, that the percentage of Type 2, linear I/O curves increases with increasing test frequency Figure . If metaLastly, we have also shown that the percentage of Type 3 I/O curves is greatest for test frequencies ≤1 kHz and decreases with increasing frequency. This trend of more frequent IHC dysfunction at apical sites remains intriguing. A few studies have reported similar trends. For example, apical IHCs were found to be more labile than basal IHCs in guinea pigs treated with polypeptide antibiotics Kohonen, . SimilarUnfortunately, confirmation of these conjectures was not possible due to the lack of accurate information regarding the etiology of hearing loss for the present participants.1. For cases where a CT is present, IHC and OHC dysfunction contribute on average to 30–40 and 60–70% to the total audiometric loss, and these contributions are approximately constant across the frequency range from 0.5 to 6 kHz.2. The individual variability of the relative contributions of IHC and OHC dysfunction to the audiometric loss is, however, large particularly at low frequencies or mild-to-moderate hearing losses.With regard to the contribution of IHC and OHC dysfunction to the audiometric loss, the main conclusions are:3. The large majority of cases suffer from mixed IHC and OHC dysfunction, even though in some cases with presumably substantial IHC dysfunction, any concomitant OHC dysfunction does not contribute to the audiometric loss.4. The percentage of cases for which the audiometric loss can be explained exclusively in terms of cochlear gain loss or of inefficient IHC processes  is higher at frequencies ≤1 kHz and decreases gradually with increasing frequency.5. The percentage of cases suffering from total cochlear gain loss  increases gradually with increasing frequency.With regard to the incidence of dysfunction types, the main conclusions are:Overall, the present results undermine the common view that high-frequency loss is typically due to greater physical damage of basal OHCs, and suggest that in a large percentage of cases, it is due to a common mechanism that concomitantly affects IHCs and OHCs, possibly reduced endocochlear potential. They further suggest that IHC processes may be more labile in the apex than in the base and/or that IHC dysfunction may have a greater impact on auditory threshold than cochlear gain loss at low frequencies.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycoplasma hyopneumoniae commercial vaccines and their relationship with field strains, the authors attempted to identify genetic subtypes of some M hyopneumoniae bacterins, and to compare them with M. hyopneumoniae field strains. Six commercial M hyopneumoniae bacterins and 28 bronchoalveolar lavages from pigs at slaughter from three herds were analysed by Multiple-Locus Variable number tandem repeat Analysis (MLVA) on p146R1, p146R3, H4, H5 and p95 loci. The results obtained showed the presence of more than one M hyopneumoniae genotype in some pigs and also in one of the bacterins analysed. It is also worth noting that MLVA typing allowed the distinction among circulating field strains and also when comparing them with vaccine strains, which, knowing the relatedness among them, could be useful in the research of the efficacy of the vaccines.Because of the lack of information about both the genetic characteristics of  Mycoplasma hyopneumoniae is the primary agent involved in porcine enzootic pneumonia. Infections with M hyopneumoniae are highly prevalent in almost all swine-producing areas, causing significant economic losses to the pig industry worldwide , according to the international guidelines of the Council for International Organizations of Medical Sciences (CIOMS).M hyopneumoniae bacterins that are commercialised in Argentina  were analysed. In order to know the M hyopneumoniae strains used for the bacterins, information was retrieved from vaccine manufacturer companies. Bacterins A and D corresponded to strain J, but for bacterins B, C, E and F, no information was available. DNA from the M hyopneumoniae bacterins was extracted with a commercial kit  to avoid the interference of possible PCR inhibitors.Six M hyopneumoniae bacterins with M hyopneumoniae field strains, DNA from 28 bronchoalveolar lavages (BAL) positive for the pathogen were included in the analysis. BAL corresponded to 22-week-old pigs at slaughter from three herds from centre-south of Córdoba province, and DNA was extracted using a commercial kit  according to the manufacturer’s instructions. Before MLVA, DNA from M hyopneumoniae bacterins and field strains were tested by a species-specific nested PCR  with the primers used in the second round  and four BAL samples .In a few loci were observed more than one allele in the same sample, in the case of a bacterin  without killing animals or performing invasive sampling (Although pathogen . Sensitipathogen  and/or cpathogen . This wosampling .M hyopneumoniae vaccine strains using PCR-restriction fragment length polymorphism (RFLP) due to the absence of amplification of the p146 gene. In the present paper, the use of the nPCR format for the p146R3 locus was shown to be advantageous and allowed the typing from all samples.M hyopneumoniae genotypes in the same sample. An unspecific annealing of the primers is considered highly unlikely. The presence of different genotypes of M hyopneumoniae has been reported previously. The authors found two probable alleles present in one pig BAL sample when analysing the locus H4 from M hyopneumoniae in a previous study in the authors’ country , but bacterin B seems to be made from more than one strain, since three possible alleles were found in each of the two loci (p146R1 and H4). Bacterins B and C could be distinguished with regard to the alleles present at locus p146R1.Regarding bacterins B, C and F, they all showed a 21-serine repeat motif in the rgentina , 2013, brgentina  that werM hyopneumoniae virulence  could be associated with irulence , at presM hyopneumoniae typing in clinical samples and bacterins was demonstrated, particularly when a high-sensitivity method such as nPCR is used. Furthermore, the results obtained showed the presence of more than one M hyopneumoniae genotype in some pigs and also in one of the bacterins analysed. It is also worth noting that MLVA typing allowed the distinction among circulating field strains and also when comparing them with vaccine strains, which, knowing the relatedness among them, could be useful in the research of the efficacy of the vaccines.In the present paper, the utility of MLVA for


In this study, we investigated the effects of varying intensity levels of repeated moderate noise exposures on hearing. The aim was to define an appropriate intensity level that could be repeated several times without giving rise to a permanent hearing loss, and thus establish a model for early adulthood moderate noise exposure in rats. Female Sprague-Dawley rats were exposed to broadband noise for 90 min, with a 50 % duty cycle at levels of 101, 104, 107, or 110 dB sound pressure level (SPL), and compared to a control group of non-exposed animals. Exposure was repeated every 6 weeks for a maximum of six repetitions or until a permanent hearing loss was observed. Hearing was assessed by the auditory brainstem response (ABR). Rats exposed to the higher intensities of 107 and 110 dB SPL showed permanent threshold shifts following the first exposure, while rats exposed to 101 and 104 dB SPL could be exposed at least six times without a sustained change in hearing thresholds. ABR amplitudes decreased over time for all groups, including the non-exposed control group, while the latencies were unaffected. A possible change in noise susceptibility following the repeated moderate noise exposures was tested by subjecting the animals to high-intensity noise exposure of 110 dB for 4 h. Rats previously exposed repeatedly to 104 dB SPL were slightly more resistant to high-intensity noise exposure than non-exposed rats or rats exposed to 101 dB SPL. Repeated moderate exposure to 104 dB SPL broadband noise is a viable model for early adulthood noise exposure in rats and may be useful for the study of noise exposure on age-related hearing loss. Hearing impairment is one of the most common disabilities in modern society, according to National Institute on Deafness and Other Communication Disorders NIDCD, . Of the stria vascularis and fibrocytes in the spiral ligament are affected  starting from the age of 9 weeks were used in this study. Animals were kept three to four in wire-meshed cages with free access to food and water in the same holding unit on a 12/12-h day and night cycle. All animal procedures followed the local ethical regulations at Karolinska Institutet and were in consistence with national regulations for care and use of animals, approval numbers N33/07, N12/10, and N300/11.n = 8), 104 (n = 8), 107 (n = 7), and 110 (n = 7) dB SPL. Age-matched animals that were not exposed served as a control group (n = 10). Exposures were repeated every 6 weeks for up to six times or until the animals received a permanent hearing loss . The noise, generated using Brüel & Kjær 3560-C PULSE hardware and a LAB 300 amplifier with PULSE LabShop Version 13.1.0.246 software , was presented through a transducer  with a Beyma TD-360 horn  centrally positioned inside the box, 85 cm above the animal cages. The noise was calibrated using a microphone  placed between the cages and adjusted to the desired level before each exposure. Prior to the experiments, the noise level was recorded at each of the four corners of the cages and shown to vary by ±2 dB SPL.For repeated moderate noise exposures, the rats were exposed to broadband free-field noise, 2–20 kHz, at a 50 % duty cycle (500 ms of exposure each second) for 90 min at intensity levels of 101, 104, 107, or 110 dB SPL. Groups that showed no permanent threshold shift after six repetitions  and the non-exposed control group were then exposed to a high-intensity noise of 110 dB SPL  for 4 h.The animals were anesthetized using an intraperitoneal injection of a 2.7 ml/kg mixture of 1 ml Hypnorm®, 1 ml Dormicum (5 mg/ml Midazolam®), and 2 ml sterile water. ABR recordings were randomly made in either left or right ear, and the same ear was subsequently used throughout the study. Before the first ABR recording was performed, the tragus cartilage was removed in order to place the microphone close to the eardrum. The eardrums were inspected using an otoscope, and animals with middle ear infections were excluded. After placing the animal on a non-electric heating pad inside a soundproof box, subcutaneous needle electrodes were positioned at the vertex (active), behind the recorded ear (reference), and in the hind leg (ground). Acoustic stimuli were generated by a Tucker-Davis Technologies system , presented through an electrostatic speaker , which was connected to the ear canal of the rat. Frequency-specific tone bursts of 3.5, 7, 14, and 28 kHz were presented at a duration of 2, 1, 0.5, and 0.25 ms for the respective frequencies and with a 1, 0.5, 0.25, and 0.125 ms raise and fall time.(p-p) amplitude difference from the peak of wave I (P1) to its trough (T1)  Fig. . The latP < 0.05. Data analysis was performed using SigmaPlot for Windows Version 11.0. All data are presented as mean ± standard deviation (SD).Differences between groups were compared statistically by one-way ANOVA followed by a Holm-Sidak comparison, and comparisons within the groups over time were analyzed using one-way repeated measures ANOVA followed by Holm-Sidak comparison on repeated measurements. When normality test failed, Kruskal-Wallis one-way ANOVA on ranks using Dunn’s method as an all pair-wise multiple comparison procedure was used. The significance level was set to H(3) = 18.6, P < 0.05 for both 110 dB compared to 101 and 104 dB SPL as well as 107 dB SPL compared to 101 dB SPL). After 2 weeks, all threshold shifts decreased in all subjects, but the animals exposed to 107 and 110 dB SPL continued to display an increased threshold shift of about 15 dB at all tested frequencies  = 16.2, P < 0.001) both for 107 and 110 dB SPL compared to 101 and 104 dB SPL. At 7 kHz; F(3) = 12.6, P < 0.001 for 107 dB SPL compared to 101 and 104 dB SPL, P = 0.001 for 110 dB SPL compared to 101 dB SPL and P = 0.004 for 110 dB SPL compared to 104 dB SPL. At 14 kHz one-way ANOVA on ranks, H(3) = 9.77, P < 0.05 for 107 dB SPL compared to 104 dB SPL. At 28 kHz one-way ANOVA on ranks, H(3) = 8.71, P < 0.05 for 107 dB SPL compared to 104 dB SPL) than those obtained in animals exposed to the lower intensities (101 and 104 dB SPL), which had a mean threshold shift of about 2 dB. The animals exposed to 104 or 101 dB SPL differed neither from each other nor from the non-exposed control group regarding threshold shifts.After the first exposure to moderate noise, the rats’ auditory function was affected. For animals exposed to 107 or 110 dB SPL, ABR threshold shifts were around 20 dB  at 24 h after the first exposure Fig. . At 7 kHies Fig. , and theThe animals exposed to intensity levels of 107 and 110 dB SPL displayed permanent threshold shifts after the first exposure, and these groups were thus not further exposed. The remaining groups, exposed to intensity levels of 101 and 104 dB SPL, could be exposed for at least six repetitions without achieving permanent threshold shifts  = 7.34, Holm-Sidak test for comparison over time, representative measurements from 7 kHz shown in Fig. F(6) = 2.78, Holm-Sidak test for comparison over time, representative measurements from 7 kHz shown in Fig. F(6) = 4.909, Holm-Sidak test for comparison over time, representative measurements from 7 kHz shown in Fig. For the groups exposed to the lower intensity levels (101 and 104 dB SPL), the effect of the repeated moderate noise exposures were explored in detail 5 weeks after each exposure  and compared to the non-exposed control group . Also here, no statistical differences were found between the groups.F(2) = 3.95, compared to the previously non-exposed group; Holm-Sidak test, P = 0.013). Neither the wave I p-p amplitudes nor latencies of the ABR wave differed at any time point between any of the groups (data not shown).Even though no permanent hearing thresholds were observed in the groups repeatedly exposed to 101 and 104 dB SPL, a possible change in noise susceptibility was investigated. The animals, including the previously non-exposed group, were thus exposed to 110 dB SPL narrowband noise for 4 h, an exposure known to induce permanent threshold shifts in this rat strain. The resulting threshold shifts peaked after the more intense exposure to around 40–50 dB at 24 h following the exposure Fig. , decreasIn this study, we investigated the functional effects of moderate noise exposures at different intensity levels in early adulthood of the female Sprague-Dawley rat in order to determine the levels that could be repeated several times without causing permanent threshold shifts. In the present model, 104 dB SPL was the highest exposure intensity that could be repeated up to six times without causing a permanent change in hearing thresholds. In addition, exposures at this level provided a certain protective effect from subsequent acoustic overstimulation of higher-intensity noise exposure. We suggest that repeated exposures of 104 dB SPL can be used as a model for studying the influence of early adulthood noise exposures on age-related hearing loss.A broadband noise exposure with intensity levels of both 101 and 104 dB SPL, for 90 min at a 50 % duty cycle, could be repeated up to six times with no permanent effect on hearing thresholds. The same type of exposures with intensity levels of 107 or 110 dB SPL produced a permanent threshold shift following the first exposure. The exposures started when the female rats were young adults (10 weeks old) and continued until the rats were middle-aged (about 10 months of age). At this age, the non-exposed rats displayed normal hearing thresholds without any signs of an incipient age-related hearing loss. It is possible that the first exposure could fall within an early vulnerable period for the rat. However, previous studies indicate that the most sensitive period for the rat are around 20–25 days  when their amplitudes were significantly reduced; if compared to humans, it would correspond to early mid-life. In human studies, the peak to peak amplitude of the ABR waves also becomes reduced at early ages, when hearing thresholds are still normal . The highest threshold shift was found at frequencies close to the frequency span of the exposure-noise. The group previously exposed repeatedly to 104 dB SPL had slightly lower hearing thresholds than the other groups, which were significant at 28 kHz compared to the non-exposed group, and moreover, at this frequency the thresholds had returned to normal. This group of animals had previously also displayed a decrease of ABR amplitudes later than the other groups. These results indicate that this intensity level of repeated moderate noise exposures made the animals somewhat more resistant to intense noise exposure later in life. Two types of “training” phenomena are described in the literature, both of them reducing inner ear susceptibility to noise. In one type, a low level of continuous non-damaging noise is used prior to a more intense noise exposure and is referred to in the literature as “sound conditioning” (Canlon et al. The long-term auditory effects of non-damaging sound exposures throughout the life span are relatively unknown. The present animal model using repeated moderate noise exposures to mimic human early adulthood exposures may be used to explore these effects and their influence on age-related hearing loss. In daily life, individuals are regularly exposed to moderate sound levels during both working hours and leisure activities. The exposures may lead to a slight, often not detected, temporary hearing loss, which declines after a few days. Our observations indicate that these short-term effects do not impair hearing sensitivity. However, it should be noted that little is known of the long-term effects on onset and severity of age-related hearing loss. Other investigators have studied the effects of noise exposure at young age on age-related hearing loss (Kujawa and Liberman