The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8 Successful immunotherapy against cancer requires the induction of a robust cytotoxic T lymphocyte (CTL) response in combination with the appropriate immune environment and T cell help . The iniin vivo (+ T lymphocytes both in vitro and in vivo have since been published (The antigen-presenting cell types most able to provide co-stimulation are dendritic cells (DCs) and macrophages (MΦs). Initially, DCs were thought to be the only cell type competent to prime naive T cells in vivo . Howeverublished –13. Moreublished  and partublished , 14, 15.Stichodactyla heliantus, to induce a strong antigen-specific CTL response in mice  stycholysin II (StII) from the sea anemone  in mice . This li in mice . These p+ T lymphocytes in vivo, as well as the cross presentation pathway enrolled are not known. The aim of this study was to elucidate the pathway by which OVA, co-encapsulated with StII in DPCC:Chol liposomes, induces OVA cross-presentation and which populations of murine APCs are responsible for CD8+ T cell activation in vitro and cross-priming of CTL in vivo. The results provide evidence that bone marrow-derived macrophages (BM-MΦs), but not bone marrow-derived dendritic cells (BM-DCs), are able to cross-present OVA co-encapsulated with StII into liposomes. The antigen cross-presentation in BM-MΦs seems to occur through the vacuolar pathway. In addition, MΦs are responsible for the priming of CTL responses induced by Lp/OVA/StII in vivo.The particulate antigens have to be taken up and cross-presented by APCs to initially activate effector CTL, an event known as cross-priming –5, 17. IStichodactyla helianthus and characterized as previously described by Lanio et al. (257−264 peptide (SIINFEKL peptide) used in CTL experiments in vivo was synthesized by the Center for Genetic Engineering and Biotechnology, Havana, Cuba and stored in phosphate-buffered saline  at −20°C until use. The SIINFEKL peptide used in cross-presentation experiments in vitro was purchased from Invitrogen .OVA grade V used in immunization protocols in soluble form or encapsulated into liposomes and OVA grade II used to coat ELISA plates were purchased from Sigma-Aldrich . DPPC and Chol were purchased from Sigma-Aldrich and egg L α- phosphatidylcholine (ePC) from TRANSFERRA Nanosciences Inc. . StII (Swiss Protein Data Bank P07845) was purified from the sea anemone o et al. . StII pro et al. . The proBrefeldin A (BFA) and cytochalasin D were obtained from Sigma-Aldrich. Clodronate for liposome production, epoxomicin, and inhibitors of cathepsin B (CA-O74Me) and cathepsin S (Z-FL-COCHO) were purchased from Calbiochem . Cathepsin general inhibitor (Z-FA-fmk) and leupeptin were purchased from ENZO life Science, Inc.  and Invitrogen, respectively.g for 15 min .Liposomes encapsulating OVA with or without StII were obtained using a vesicle dehydration and rehydration procedure developed by Kirby and Gregoriadis . BrieflyLiposomes encapsulating clodronate (Lp/Clodronate) were obtained by a simple dispersion method. The appropriate amounts of lipids ePC and Chol (12 μmol each lipid/per dose) dissolved in chloroform were mixed and evaporated thoroughly at 50°C. Multilamellar vesicles were prepared by hydration of the dried lipid mixture with 120 μg of clodronate dissolved in MilliQ water. Six cycles of freezing and thawing were carried out to improve the clodronate encapsulation and homogenize the vesicle size. The removal of untrapped clodronate was performed by centrifugation at 10,000 g for 15 min . The vesicles corresponding to each mouse administration dose were resuspended in 200 μL of PBS.b) mice, 6 to 8 weeks of age, were purchased from the Center for Laboratory Animal Production  and further kept in the animal facilities of the Center of Molecular Immunology  under standard animal housing conditions. Immunizations were performed subcutaneously (s.c.) in the inferior right limb by delivering 200 μl of liposomal preparation, containing 50 μg of OVA, 6.25 μg of StII and 19 μmol of total lipids (DPPC and Chol) per dose. Two injections of Lp/OVA/StII or PBS with 12 day interval were given.Female C57BL/6  were prepared as previously described by Celada et al. with some modifications . BrieflyBone marrow derived-DCs (BM-DCs) were prepared as described in Lutz et al. . Briefly257−−264 peptide was detected using the B3Z CD8+ T cell hybridoma cell line that expresses β-galactosidase under the control of the IL-2 promoter (5 BM-DCs or 5 × 104 BM-MΦs per well in 96-well flat bottom plates were cultured in RPMI containing antibiotics and 10% (vol/vol) FBS at 37°C in 5% CO2. After 24 h, the medium was removed and cells were incubated with or without chemical inhibitors dissolved in RPMI medium without FBS for 30 min before the addition of the antigen. BM-DCs and BM-MΦs were pulsed, in the presence or absence of inhibitors, with OVA in soluble form (OVA) or encapsulated with or without StII into liposomes containing DPPC and Chol in a 1:1 or 2:1 ratio  for 90 min at 37°C in 5% CO2. BM-DCs and BM-MΦs incubated with the SIINFEKL peptide alone were used as control. After stimulation, cells were washed and co-cultured with 1 × 105 B3Z CD8+ T cells at 37°C, 5% CO2. After 24 h of incubation the supernatant was removed and 100 μl of LacZ buffer containing 0.15 mM chlorophenol red-β-D-galactopyranoside (Sigma-Aldrich) in PBS plus 0.13% Nonidet P-40, 9 mM MgCl2, and 0.1 mM β-mercaptoethanol, were added to the wells. The plates were incubated for 5 h at 37°C. Absorbance was measured at 570/620 nm using a SpectraMax Plate Reader . IL-2 secretion was measured in cell culture supernatants by ELISA.Antigen cross-presentation of the OVApromoter . Briefly+ T cells, 96-well polystyrene flat-bottom high binding microtiter plates  were coated with 10 μg/ml of monoclonal antibodies specific to IL-2 (BD Biosciences Pharmingen), diluted in 0.05 M sodium carbonate buffer pH 9.6, and incubated overnight at 4°C. After washing four times with PBS with 0.05% Tween 20 (PBS/T), the plates were blocked with 10% (v/v) of FBS in PBS (200 μl/well) for 2 h at 37°C. One hundred microliters of culture supernatants diluted 1:2 with 2% FBS in PBS (buffer samples) were added and plates were incubated overnight at 4°C. Serial dilutions (1:2) of recombinant IL-2 were used as standard curve. After four washes with PBS/T, 100 μl of 5 μg/ml of an IL-2-specific biotin-conjugated antibody  was added. Following 2 h of incubation at 37°C, plates were washed six times. Bound antibodies were detected with streptavidin -HRP (BD Biosciences Pharmingen) diluted 1:1,000 in buffer samples. Plates were washed six times and 100 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution (Invitrogen) were added. The reaction was stopped with 50 μl of 1 M H2SO4 after 5 min of incubation and absorbance was measured at 450 nm using a SpectraMax Plate Reader.To quantify the IL-2 secreted by B3Z CD82. Peptide-pulsed target cells were extensively washed to remove free peptide. Subsequently, pulsed (target) and non-pulsed  cells were simultaneously labeled with the fluorescent dye CFSE at 5 μM (CFSEbright) and 0.33 μM (CFSEdull), respectively, during 5 min at 37°C. Labeled cells were washed, resuspended in PBS, and mixed at 1:1 ratio. A total of 6 × 107 labeled splenocytes in 200 μl of PBS were injected i.v. into immunized mice [Lp/OVA/StII (1:1)] or control mice (PBS) 7 days after the last immunization. Sixteen h later, mice were sacrificed and inguinal lymph nodes closest to the site of immunization were removed, and the total events corresponding to both fluorescent intensities (CFSEdull and CFSEbright) were determined by flow cytometry. The percentage of lysis for each mouse was calculated as: % Lysis = 100–[ immunized mice × 100 ×  PBS].Total splenocytes from naive C57BL/6 mice were pulsed or not with 1 μM of SIINFEKL peptide for 60 min at 37°C and 5% COAll procedures were performed in compliance with the protocols approved by the Institutional Committee for the Care and Use of Laboratory Animals of the CIM . Animals were sacrificed by cervical dislocation, minimizing their suffering.post-hoc test to assess statistical significance between the means of more than two groups. Data not normally distributed or without equality of variance, even after scale transformation, were analyzed using the Kruskal-Wallis non-parametric test with Dunn as post-hoc test. For comparing the means of two independent groups with or without normal distribution, the unpaired test and the Mann-Whitney U test were used, respectively.Statistical analysis was performed using the SPSS software version 16.0 (SPSS). The Kolmogorov-Smirnov test was used to verify normal distribution of data and the Levene test to determine the homogeneity of variance. Data with normal distribution and equality of variance were analyzed with one-way variance analysis (ANOVA) simple classification, with Dunnett as + T cell proliferation and CTL response in vivo    Figure . HoweverI Figure . The CD8A Figure . In addin Figure . BM-MΦs s Figure . Notably+ T lymphocyte in vivo than liposomes containing half quantity of Chol (Lp/OVA/StII (2:1) (manuscript in preparation). To explore the influence of reducing the Chol amount in the DPPC liposomes on the ability of StII to induce OVA cross-presentation in BM-MΦs, we compared the CD8+ T cell activation by BM-MΦs stimulated with these two liposome formulations. Irrespectively of the level of Chol in the formulations, the presence of StII enhanced the CD8+ T cell activation  was blocked by both cathepsin S and B inhibitors. However, the cross-presentation induced by Lp/OVA/StII (2:1) was only affected by the cathepsin B inhibitor Figure . These rProcessing and presentation of both endogenous and exogenous antigens often requires nascent MHC molecules , but can+ T lymphocyte proliferation and CTL differentiation in vivo . In contrast, mice depleted of macrophages and immunized with Lp/OVA/StII (1:1) exhibited similar quantities of both cell populations  liposomes leads to CD8 in vivo . After ss Figure . A quant+ T cell response and potent anti-tumor immunity resulting in a promising vaccine platform for cancer immunotherapy , the hemolytic protein of Listeria monocytogenes, carried with the OVA into liposomes the antigen cross-presentation is mediated by the cytosolic pathway  Figure . These r pathway –51. Our  pathway , who sho pathway . Further pathway .kiss and run” events, acquiring superior degradative properties. Formation and subsequent maturation of phagosomes in APCs are related to the characteristics of the ingested particulate antigens. Degradation of the phagocytosed particulate vaccines generates peptide antigens for loading on MHC molecules and subsequent antigen presentation at the cell surface .While the antigen cross-presentation induced by both liposomal/StII formulations was sensitive to lysosomal protease inhibitors Figure , these e surface . Since b surface , 52. The surface , 54. The surface . InteresThe results obtained here show the partial inhibition of the cross-presentation of Lp/OVA/StII by BFA Figure , suggestin vitro was also assessed in vivo through MΦs depletion with liposomes encapsulating clodronate. The lower percentage of CTL induced by Lp/OVA/StII in mice depleted of MΦs (Figure in vitro suggests a decrease relevance of these cells in the in vivo process. Nevertheless, future experiments with specific exhaustion of DCs should be carried out in order to determine their contribution at the CTL in vivo response induced by Lp/OVA/StII.The MΦ role in antigen cross-presentation induced by Lp/OVA/StII s Figure  suggestes Figure . We canns Figure . Howeverin vivo, macrophages played a key role. These events described in this work should be determining the efficacy of the liposomal formulation encapsulating StII as a vaccine platform for enhancing CTL response. Nevertheless, further studies are required to completely elucidate the mechanism underlying the enhancement of antigen cross-presentation induced by StII carried by liposomes.In conclusion, our study provides evidence that OVA co-encapsulated with StII into liposomes is internalized by phagocytosis and cross-presented through the vacuolar pathway by BM-MΦs but not by BM-DCs. Moreover, in the CTL priming induced by the liposomal formulation containing StII YC-L Conception and design of the work, acquisition, analysis and interpretation of the data and writing of the manuscript. DG Acquisition, analysis and interpretation of the data and critical revision of the manuscript. CN Acquisition, analysis and interpretation of the data and critical revision of the manuscript. IL-G Acquisition, analysis, interpretation of data and revision of the manuscript. AdV Acquisition, analysis and interpretation of data. FE Acquisition, analysis and interpretation of data. RL Analysis, interpretation of data and revision the manuscript. CA Analysis and interpretation of the data and revision of the manuscript. LF Critical scientific support, interpretation of the data, revision of the manuscript. MS Critical scientific support, interpretation of the data. DH Critical scientific support, interpretation of the data, revision of the manuscript and final approval of the manuscript. ML Conception and design of the work, interpretation of the data, writing, critical revision and final approval of the manuscript.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.


Traditional hearing conservation programs are based on the premise that noise dose, as measured by the time-weighted average noise level, is the primary risk factor associated with occupational hearing loss and that permanent threshold shifts are the most relevant outcome measures for determining when a noise-related hearing loss has occurred. However, recent studies in animal models have suggested that significant neurological damage to the hearing system can occur from noise exposures even when they are not severe enough to result in permanent threshold shifts. This has led to a number of studies attempting to relate subjective measures of noise exposure to subjective measures of hearing difficulty and suprathreshold measures of hearing performance . In this study, 3,330 U.S. service members volunteered to complete a survey on noise exposure, subjective hearing complaints, and tinnitus in conjunction with their annual hearing tests. Two questions were also included about the frequency and duration of temporary hearing losses that may have been experienced by the service member. The results show that subjective reports of temporary threshold shifts were substantially more predictive of tinnitus and other hearing complaints than more traditional questions based on the frequency of noise exposure. Noise exposure is an occupational risk in many professions, but some of the most hazardous noise exposures occur in military environments. In addition to continuous noise exposures from vehicles and machinery, service members (SMs) may also experience repeated doses of impulsive noise from small and large caliber firearms and occasional doses of very high-level noise or blast overpressure from heavy weapons fire, grenades, demolition explosions, improvised explosive devices, or incoming enemy fire.Traditional hearing conservation programs are based on the assumption that the primary risk associated with noise exposure is sensorineural hearing loss, which manifests as a shift in the intensity level of the quietest pure tone a listener can detect.The Occupational Safety and Health Administration (OSHA) and the National Institute for Occupational Safety and Health (NIOSH) rules require noise-exposed individuals to receive periodic hearing tests to ensure that no shifts in pure-tone threshold have occurred, and these rules presume that no noise-related hearing loss has occurred until an individual experiences a standard threshold shift (STS) . An STS permanent threshold shifts (PTSs). Most individuals who are exposed to the maximum allowable dose of noise under OSHA guidelines  Diagnosis: Participants were asked “Have you ever been diagnosed with a TBI or concussion?” and were given the options “No,” “Only mild TBIs or concussions,” or “One or more moderate or severe TBIs.” A positive mTBI Diagnosis was counted if the participant selected “Only mild TBIs or concussions.”>mTBI Diagnosis: Same as the mTBI Diagnosis question. A positive > mTBI Diagnosis was counted if the participant selected “One or more moderate or severe TBIs.”Continuous Noise: Participants were asked “How often are you exposed to continuous noise that is so loud you need to raise your voice to be heard by a person standing three feet away?” and were given the options “Never,” “Less than once per year,” “Once per year,” “Several times a year,” “Several times a month,” “Several times a week,” and “Daily.”Small Arms Noise: Participants were asked “How often are you exposed to noise from small-caliber firearms (up to 12.7 mm)?” and were given the options “Never,” “Less than once per year,” “Once per year,” “Several times a year,” “Several times a month,” “Several times a week,” and “Daily.”Heavy Weapons Noise: Participants were asked “How often are you exposed to heavy weapon fire  or explosions?” and were given the options “Never,” “Less than once per year,” “Once per year,” “Several times a year,” “Several times a month,” “Several times a week,” and “Daily.”Hearing Protection: Participants were asked “How often did you wear hearing protection?” and were given the options “Never,” “Some of the time,” “Most of the time,” or “Always.” This question was asked separately for Continuous Noise, Small Arms Noise, and Heavy Weapon Noise. Because the hearing protection question was meaningless for individuals who indicated they were never exposed to a certain type of noise, a decision was made to analyze only one hearing protection variable which was defined as the maximum hearing protection response across all three noise categories. A very small number (<5%) of participants reported they were never exposed to any type of noise. These subjects were assigned an “N/A” response for the hearing protection question that assigned a numerical value equal to the mean response of all subjects who did have a response, which was numerically roughly halfway between the “Some of the Time” and “Most of the Time” responses.Far Blast Exposure: Participants were asked two questions about blast exposure: “Have you ever been exposed to an explosive blast in combat?” and “Have you ever been exposed to an explosive blast in training?” The participants were given the options “No,” “Yes, but not close enough to feel the heat or pressure wave,” or “Yes, close enough to feel the heat or pressure wave.” A positive Far Blast Exposure was counted if the participant selected “Yes, but not close enough to feel the heat or pressure wave” for either the combat or training question.Close Blast Exposure: Same as the Far Blast Exposure questions. A positive Close Blast Exposure was counted if the participant selected “Yes, close enough to feel the heat or pressure wave” for either the combat or training question.TTS Frequency: Participants were asked “Have you ever experienced a temporary change in your hearing (dullness or a muffled sound) after exposure to a loud noise?” and were given the options “Never,” “Once in my lifetime,” “A few times in my lifetime,” “Several times a year,” “Several times a month,” “Several times a week,” and “Every day.” TTS Max Duration: Participants who responded that they had experienced at least one TTS were asked “What is the longest time it has taken for your hearing to recover back to normal?” and were given the options “A few seconds,” “A few minutes,” “A few hours,” “A few days,” and “It never fully recovered.” These responses were assigned the values 1 to 5 on this scale. Participants who indicated they had never experienced a TTS were automatically assigned a 0 value on this question.Perceptual Tinnitus: Participants were asked “Have you ever experienced tinnitus  that has lasted more than 2 to 3 minutes?” and were given the options “Never,” “Once in my lifetime,” “A few times in my lifetime,” “Several times a year,” “Several times a month,” “Several times a week,” and “Every Day.” Note that this question was derived from the tinnitus and hearing survey . the THS . The parSSQ Survey: This questionnaire consisted of six questions derived from the Spatial and Sound Qualities (SSQ) questionnaire . For analysis purposes, the SSQ values were subtracted from 10 to make them consistent with the other measures in the study .ionnaire . The parAuditory Localization: Participants were asked “How well are you able to determine the locations of sounds that you hear?” and were asked to select the number that represented where they fell on a range of 0 to 10 .Noise Sensitivity: Participants were asked “Over the last week, sounds were too loud or uncomfortable for me when they seemed normal to others around me” and were asked to circle the number that represented where they fell on a range of 0 to 10 . Note this question was derived from the THS , as measured from the Defense Occupational and Environmental Health Readiness System-Hearing Conservation  hearing test using a CCA-200 audiometer .WE PTA Low: The average of the worse ear thresholds at 500 Hz, 1 kHz, and 2 kHz (in dB HL).BE PTA High: The average of the better ear thresholds at 3 kHz, 4 kHz, and 6 kHz (in dB HL).WE PTA High: The average of the worse ear thresholds at 3 kHz, 4 kHz, and 6 kHz (in dB HL).TTS Freq × Duration: This was a derived variable obtained by multiplying the TTS Frequency score by the TTS Max Duration score. Both variables were on a 0 to 6 range, so the TTS Freq × Duration variable ranged from 0 to 36.y axis is shown on a normal probability scale, which means that variables that fall on a straight line in the plot are exhibiting an approximately normal distribution.Individual responses are shown by “+” symbols. Thus, the difference between cumulative probability for the highest “+” value in each category and the cumulative probability for the lowest “+” value in each category is an indicator of how many participants made each possible response. These distributions provide some useful insights into the noise exposure experience of the military population tested in this experiment. For example, in terms of perceptual tinnitus , and 10% reported no exposure to small arms fire. As expected, exposure to heavy weapon fire (third panel) was much less frequent, with only 18% reporting exposure at least once per year and 45% reporting no exposure.Self-reported TTS was also relatively infrequent as shown in x greater than 12 with the value log(x − 11) + 12.In almost all cases, the multiple-choice questions selected in this experiment produced response distributions that fell on or near a straight line in the normal distribution plot, which suggests that the responses can be viewed as discretely sampled values of variables with underlying Gaussian distributions. There are two notable exceptions. The first was the TTS Frequency × Duration value .The second variable that was not intrinsically approximated by a normal distribution was the Hearing Protection question (x with x0.65 + (9 − 90.65). The resulting distributions are shown in The PTAs of the participants were obtained from their annual hearing screenings and used to calculate Low-Frequency and High-Frequency PTAs for the left and right ears. In raw form, these PTAs were not normally distributed, so a transformation was applied to normalize them prior to their use in the regression analysis. This transformation replaced each PTA value greater than 9 dB HL p value less than .05. Finally, the relative contribution of each predictor value in the final model was determined by repeating the stepwise regression for all the variables in the model excluding that variable and determining how much the variance of the residual changed σout2 when that variable was excluded. This difference was expressed as a ratio σin2 in percent, with larger ratios indicating that the unique contribution that particular variable made to the overall model was greater than the unique contributions of the other variables.The data from the study were analyzed using the statistics toolkit in MATLAB. Each analysis consisted of a set of predictor values and a single output variable selected from the data variables described in the previous section. First, a simple linear correlation was conducted between each predictor variable and the output variable. Next, a stepwise multivariate regression was performed. In this analysis, the predictor variables were added to the linear regression model of the output one variable at a time until no variables remained where the correlation with the residual variable had a The first question of interest was to identify the demographic and environmental exposure variable that best predicted the frequency and duration of self-reported TTS.r” values in the second column of the table show the univariate correlations between each predictor value and the TTS Frequency. This analysis shows that all the predictor variables were significantly correlated with TTS Frequency.p value column shows the p value of the component in the final stepwise regression model. Only those variables with p values less than .05 were retained in the final model, and these are shown in bold type. The last column shows the ratio of variance in the residual of the prediction when that variable was excluded or included in the model. This can be viewed as an estimate of the relative contribution each variable made to the prediction. Note that the predictor variables that were included in the final model are ranked in order of this ratio, and that variables that were not included in the model are ranked in order of increasing p value.The right half of the table shows the results of the stepwise regression analysis. The “Order” column shows the order in which the variables were added to the regression. The “Coeff” column shows the coefficient of the variable, which by itself is not meaningful because the different predictor values represent different units. The r) of the final prediction, which in this case was .408.The bottom row of the table shows the correlation coefficient , indicate that Age and TTS Freq × Dur, respectively, were the strongest predictors of the Low-Frequency PTAs, with variance ratios 2.5 to 5 times as high as those measured for Continuous Noise. The use of Hearing Protection was also a significant factor, indicating that those who reported that they frequently used hearing protection were less likely to have elevated low-frequency thresholds than those who did not.There was also a contribution of TTS Max Duration. Note, however, that the coefficient for this variable was opposite in sign from the TTS Freq × Dur Variable, indicating that it was correcting for an overestimation of the influence of duration in the combined TTS Freq × Dur. If a single variable of TTS Freq × Dur − TTS Dur was included in the regression, it was the only significant TTS variable and its variance ratio increased roughly 50%. This suggests that TTS is the most dominant predictor of low-frequency hearing loss in this population of SMs.Gender made a small contribution to both ears, with slightly higher thresholds for males than for females.better low-frequency thresholds than those who were not. The reasons for this are unclear.OIF/OEF deployment also made a slight contribution to both ears, but, somewhat counterintuitively, the sign of the coefficient was negative, indicating that individuals deployed in OIF/OEF had The analysis of tinnitus is generally separated into perceptual tinnitus, where the listener perceives a phantom tone, buzz, or other sound, and bothersome tinnitus, when the phantom sound becomes distracting enough to interfere with daily activities. In this study, we only looked at perceptual tinnitus, which was defined by how frequently the participant reported hearing buzzing or ringing in the ear that lasted for more than 3 minutes.We contend that the goal of a hearing conservation program is to avoid hearing damage that results in a degradation in overall quality of life, regardless of whether that degradation is accompanied by an associated increase in pure-tone threshold. Under this model, some of the most meaningful outcome variables are the subjective hearing complaints experienced by SMs who are noise exposed. We used four different instruments to assess hearing complaints in this study. better ear and the Low-Frequency PTAs in the worse ear. However, with the SSQ, there was a significant contribution of TBI diagnosis, with those diagnosed with more than a mild TBI scoring, on average, almost one full point (0.908) worse on the SSQ. There was also a significant effect of hearing protection, with improved SSQ scores for those who reported using hearing protection more often.Although the data clearly show that there is a strong relationship between self-reported incidents of TTS and subjective hearing complaints, there is certainly some room for concern that this relationship might be explained by psychological factors related to the way the questions were asked. For example, one possibility is that individuals with hearing complaints might be more likely to remember temporary hearing changes from the past than those who do not have any such complaints. Or, alternatively, people who are more sensitive to or annoyed by hearing problems might be more likely to notice temporary hearing changes when they occur.Although these psychological factors almost certainly played some role in the data from this experiment, there is also some objective evidence from the shape of the audiograms that suggests that there were systematic physiological differences in the hearing profiles of individuals who reported TTS and those that did not.The individual data points plotted in The dark black lines in the figure divide the audiograms in the normal hearing range into those that fall above the 25th percentile in slope for a given PTA value (above the line) and those than fall below the 25th percentile in slope for a given PTA value (below the line). These lines, which are the same in each panel, were calculated by dividing the normal hearing audiograms into five 10-dB wide bins centered at PTA values of 0, 5, 10, 15, and 20 dB HL. The 25th percentile value for each bin was calculated, and a line was fit to these 25th percentile values and extended over the region from −10 dB and up to +80 dB.In the normal hearing range (PTA < 25 dB), all three TTS regions had roughly the same percentage of data points (approximately 25%) under the 25th percentile line. However, in cases where there was some hearing loss , there were substantial differences in the pattern of slopes. In the case where there was no reported TTS (first panel), only 9% of the data points for hearing impaired listeners were under the line. In the moderate TTS case (second panel), 24% were under the line. In the severe TTS case (third panel), 44% were under the line. In the data for individuals who had at least one ear's TM punctured by explosive blast (third panel), 77% of the data points were under the line. Thus, it appears that hearing impaired listeners who report a history of TTS tend to have substantially flatter audiograms (lower slopes at a given PTA) than those who do not report a history of TTS, and that this shift appears to be on a trajectory in the direction of ears that suffered extreme trauma from exposure to explosive blast.A further analysis indicated that the change in audiogram shape seen for hearing impaired SMs with a history of TTS was more strongly related to the frequency of the TTS than its reported duration. Only 19% of hearing impaired SMs who reported experiencing TTS no more than a few times in their lifetime had audiograms that fell under the line in Although these data are not completely conclusive, they do provide some validation that the subjective reports of TTS history obtained in this survey were correlated with objective changes in auditory function that were consistent with what we have seen in military populations with confirmed blast exposures that resulted in perforated TMs .The purpose of this study was to use a brief survey administered to SMs to assess the relationship between the different types of occupational noise exposures, hearing thresholds, and subjective hearing complaints. The results of the study show that self-reports of temporary hearing loss are substantially stronger predictors of perceptual tinnitus and subjective hearing difficulty than self-reported exposure to continuous, small arms, or heavy weapon noise.Self-reported TTS was also as good as or better than self-reported noise exposure for predicting hearing thresholds. This is illustrated in The effect of the maximum reported TTS duration (second panel) was more modest, except for the case where the participants reported a single noise exposure that resulted in a permanent change in hearing. It is notable both that there was a substantial increase in THS score even for those reporting TTSs that resolved within minutes, and that there was not much difference in THS score for those with TTS durations that lasted for hours versus those that lasted for days.The results for Continuous Noise exposure (third panel) are notable primarily in terms of their comparison to the TTS exposures. Individuals who reported experiencing TTS more than once per year had substantially worse THS values than those who reported daily exposure to continuous noise. Individuals who had at least one TTS lasting hours had worse THS scores than those exposed to noise every day. For those exposed to continuous noise, there was not much difference between those exposed monthly and those exposed weekly or daily.Certainly, there are some limitations and caveats associated with these results. Because of our time limitations, our noise exposure questions were somewhat rudimentary, and it is possible that a more comprehensive or better constructed set of noise questions might have increased the apparent contribution of noise exposure to apparent handicap. Also, as was mentioned earlier, it is possible that the sensitivity of THS to reported subjective TTS could represent some underlying confounds. For example, it is possible that people who are more aware of or pay more attention to their hearing would be more likely to notice a TTS after loud noise exposure, and that these same people might also be more likely to report a hearing handicap on a survey like the THS.The results also appear to contradict the results of Nevertheless, it is hard to dismiss the completely dominant role that questions related to TTS appeared to play in predicting virtually all of the subjective measures of hearing complaint, or the fact that the TTS questions were stronger predictors of hearing thresholds than the other questions related to noise exposure. It is also difficult to believe that the systematic relationship between audiogram shape and subjective TTS does not reflect some underlying link between self-perceived TTS and hearing damage.consequences of noise exposure, rather than the assumption that TTS is somehow a more reliable way of assessing quantitative noise dose than a task-based exposure study like the NEQ or NESI. Although we are aware of very few noise surveys that have included questions on TTS, there were three questions very similar to our TTS questions included as potential screening questions in the development of the NEQ. These questions asked about the frequency of exposure to sounds that made your hearing “ring or buzz,” “sound muffled,” or “feel full or hurt.” Ultimately, these three questions were rejected from use in the final 1-minute NES screener because they were not correlated with the Annual Noise Exposure predicted by the full NEQ  what the sources of the TTSs were, and whether that has an effect on outcome and (b) whether there are other objective measures, besides PTA and audiogram shape, that correlate with perceived TTS. This might include objective performance measures, such as speech in noise performance, or objective physiological measures, such as DPOAEs and auditory brainstem response (ABR) Wave I amplitudes. In fact, at least one study has already reported some evidence for slightly better DPOAEs and WAVE I ABRs in patients who reported no history of noise-induced changes in hearing, although this was not accompanied with any differences in perceived handicap or functional performance . A bette


In vitro, a contribution to neuroinflammation and neurotoxicity has been shown for the lysosomal protease cathepsin X; however, its expression and its role in PD remain unknown. Therefore, the current study was designed to address the regional, cellular, and subcellular localization and activity of cathepsin X in hemi-parkinsonian rats with 6-hydroxydopamine (6-OHDA)-induced excitotoxicity in the unilateral medial forebrain bundle (MFB) lesion. We report for the first time that cathepsin X expression and activity are rapidly increased in the ipsilateral SNc after injection of 6-OHDA into the MFB reaching a maximum after 12 h but seem to stay strongly upregulated after 4 weeks after injection. At early time points of 6-OHDA injection into the MFB, the increased cathepsin X is localized in the lysosomes in the neuronal, predominantly tyrosine hydroxylase-positive dopaminergic cells. After 12 h of 6-OHDA induced lesion, only a few activated microglial cells are positive for cathepsin X whereas, in 4 weeks post-lesion accompanied with complete loss of dopaminergic neurons, there is persistent cathepsin X upregulation restricted to activated glia cells. Taken together, our results demonstrate that cathepsin X upregulation in the lesioned dopaminergic system may play a role as a pathogenic factor in PD. Moreover, inhibition of cathepsin X expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in the PD.Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc).  Parkinson’s disease is an age-related neurodegenerative disease with a primary pathology characterized by a progressive degeneration of the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNc) . AlthougCysteine cathepsins, lysosomal peptidases, constitute the largest cathepsin family, comprising 11 members , and possess a conserved active site involving cysteine, histidine, and asparagine residues in a catalytic triad . They arIn the present study, we have investigated the expression pattern and cellular localization of lysosomal cysteine cathepsin X in the rat brain following the unilateral, 6-OHDA–induced lesion in the MFB. Cathepsin X was found to be markedly upregulated in the ipsilateral SNc with a concomitant reduction of dopaminergic neurons. Increased expression of cathepsin X in the ipsilateral SNc was predominantly localized to lysosomes in dopaminergic neurons. However, after an extended 6-OHDA-induced lesion, upregulated cathepsin X was localized only in activated microglia cells. Our results suggest that elevated cathepsin X expression and its activity is implicated in neurodegeneration in the nigrostriatal DA system involved in PD.Male Wistar rats, from the Medical Experimental Center, Medical Faculty, University of Ljubljana, Slovenia, weighing 280–350 g (7–9 weeks old) at the beginning of the experiments were housed at 22 ± 2°C and 60 ± 10% relative humidity under a 12 h light/dark cycle, with free access to food and water. All animal-related procedures were conducted in accordance with the European Communities Council Directive of 2010 (2010/63/UE) for animal experiments and the National Veterinary Institute Guide for the Care and Use of Laboratory Animals. Care was taken to minimize the number of experimental animals and their suffering. The experiments were approved by the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector, and Plant Protection.®; Bayer, Leverkusen, Germany) and 10% ketamine  and were positioned in a stereotaxic frame  with the incisor bar at the level of the ear. 6-OHDA hydrobromide  was infused at a rate of 1 μl/min over 4 min into the right MFB at the following coordinates: anterior 4.4 mm from lambda, lateral 1.0 mm from the midline and ventral 7.8 mm from the surface of dura [stereotaxic coordinates , 24 h (n = 4), 48 h (n = 4), or 4 weeks (n = 4) following the injections of 6-OHDA. The brains were rapidly removed and quickly frozen on dry ice and stored at -80°C in a freezer until cryostat sections could be cut. Coronal sections (10–20 μm), identified using a rat brain atlas . The slides were then vacuum-packed and stored in a freezer at –20°C until being further processed. For the in situ hybridization histochemistry, immunohistochemistry, and double immunofluorescence staining, four 10 μm sections of the striatum and four 10 μm sections of the SNc from each animal were analyzed. For the protein expression and activity analysis, eight 20 μm sections of the SNc were used.The rats were divided into four groups, sacrificed at different times, i.e., 12 h . The coronal brain slices at the level of SNc were analyzed. Only animals with evident downregulation of TH mRNA and TH protein level in the ipsilateral SNc were used in subsequent analyses. Animals sacrificed 4 weeks after 6-OHDA injection were additionally tested for the development of nigrostriatal degeneration by apomorphine test. The 6-OHDA-lesioned animals were treated with directly acting mixed agonist of DA receptors apomorphine hydrochloride  in the fourth post operative week. The rats were placed in plastic cylindrical chambers (40 cm diameter), and the number of contralateral turning was counted manually. The animals showing >200 stereotyped contralateral turns per hour after a single dose of apomorphine were used in the subsequent experiments.The extent of the nigrostriatal dopaminergic cell loss after 6-OHDA lesion was assessed by the tyrosine-hydroxylase (TH) mRNA in situ hybridization histochemistry. The 10 μm sections were incubated with 35S-labeled oligodeoxyribonucleotide “antisense” probes (45 bases long) complementary to the rat TH mRNA (sequence 5′-AAC CAA ACC AGG GCA CAC AGG GAG AAC CAT GCT CTT AAG-3′) and rat cathepsin X mRNA (sequence 5′-AGG TCT CAT CGG GGA TGC CAT GCT TGT GGG CAT ACT CCC ACA CCG-3′). The GenBank accession numbers used to design the probes were TH M23598 and CTSX NM183330. Hybridized sections were exposed to X-ray film  for 2–3 weeks and developed using standard darkroom techniques.The standard procedure described by in situ hybridization were immersed in Ilford K2 emulsion in gel form  and exposed for 9 weeks at 4°C in the dark before being developed with Kodak GBX developer (Sigma-Aldrich)  for 5 min, then fixed in Kodak GBX fixer  for 10 min. After washing with water, the sections were counterstained with 0.2% methylene blue, dehydrated, coverslipped with DPX (Sigma-Aldrich), and photographed under a light microscope  with a digital camera (Olympus DP71).The slides from in situ hybridization signal was semi-quantified using an image analyser software . An intensity threshold was set based on the mean gray values measured within the cerebral peduncle. Mean gray value of the area covered by the above-threshold pixels in the manually outlined region was determined separately for the contralateral and ipsilateral side. In the graph, data are presented as means ± standard deviation (SD) determined from four animals. Differences between ipsilateral and contralateral sides within each experimental group were analyzed using the two-tailed paired Student’s t-test.The 2O2. Brain slices were incubated in blocking buffer containing 4% normal serum, 1% BSA, and 0.1% Triton X-100 in potassium phosphate buffer (KPBS) for 1 h at room temperature. They were then incubated overnight at 4°C with mouse monoclonal antibody against TH , goat polyclonal primary antibody against cathepsin X  or mouse monoclonal antibody against OX-6 , diluted in blocking solution and then with biotinylated anti-mouse or anti-goat secondary antibodies  diluted in KPBS containing 1% BSA and 0.02% Triton X-100 for 1.5 h at room temperature. Avidin–biotin–peroxidase complex  was added for 30 min. Staining was visualized with 3,3′-diamino-benzidine . All sections were immunolabeled simultaneously to ensure the same conditions, such as using identical DAB staining incubation times. Sections were then mounted, dehydrated, and coverslipped with DPX mounting medium . Brain sections were examined and imaged with an Olympus microscope (Olympus IX81) with an attached digital camera (Olympus DP71) using the same system settings for all samples.Coronal brain sections (10 μm) were fixed in cold 100% methanol for 5 min followed by 15 min in cold methanol with 1% Hg at 4°C for 15 min to collect the supernatant. Total protein concentration was determined by DCTM Protein Assay . All the samples were kept at -70°C until used for analysis.For analysis of the protein level of cathepsin X and its activity, the SNc was dissected out from eight 20 μm frozen brain slices of 6-OHDA rat brains, separately from the contralateral (Control) and ipsilateral side (Lesion), using a cryostat at -20°C. Tissue was homogenized in ice-cold lysis buffer  supplemented with a cocktail of phosphatase inhibitors , then sonicated and centrifuged at 15.000 2O2 was added. After 15 min, the reaction was stopped by adding 50 μl of 2 μM H2SO4. The amount of protein was determined by measuring the absorbance at 450 nm using a microplate reader , and the concentration of cathepsin X was calculated from the calibration curve.The protein level of cathepsin X was determined by ELISA, as reported . Briefly2). Results are presented as a change in fluorescence as a function of time (ΔF/Δt), and cathepsin X activity was expressed relative to Control.Cathepsin X activity was measured in tissue lysates with the cathepsin X-specific, intramolecularly quenched fluorogenic substrate Abz-Phe-Glu-Lys(Dnp)-OH synthesized by Jiangsu Vcare Pharmatech Co. (China). An aliquot of 50 μg of the lysate proteins was incubated at 37°C, followed by measurement of cathepsin X activity using 10 μM Abz-Phe-Glu-Lys(Dnp)-OH. The fluorometric reaction was quantified at 37°C at an excitation wavelength of 320 nm and emission wavelength of 420 nm on a microplate reader (Tecan Safiredata not shown). Fluorescence microscopy was performed using a Carl Zeiss LSM 710 confocal microscope . Images were analyzed using Carl Zeiss ZEN 2011 image software.The striatal and nigral brain sections (10 μm) of 6-OHDA treated rats were double-immunostained for cathepsin X localization analysis using the following primary antibodies: goat anti-cathepsin X , the lysosomal marker, rabbit anti-LAMP1 , the neuronal marker, mouse anti-NeuN , the microglia marker, mouse anti-OX-6 , the astrocyte marker, mouse anti-GFAP , and the marker for dopaminergic neurons, mouse anti-TH . The immunofluorescence procedure was performed as reported . Brieflypost hoc analysis for multiple comparisons using Dunnett’s multiple comparisons t-test using GraphPad Prism, version 6 was used for statistical evaluation when two sets of values were compared; P < 0.05 was considered to be statistically significant.Results of cathepsin X protein level and activity are representative of two independent experiments, each performed in duplicate, and are presented as means ± SD. ANOVA test and In vitro study revealed that 6-OHDA induces the protein level and activity of cathepsin X in dopaminergic-like cells , and the upregulation of cathepsin X mRNA was observed also at the 24 h time point after the injection , whereas at 24 h the increase was not significant as was at 12 h after 6-OHDA-induced lesion , microglial cells (OX-6) and astrocytes (GFAP), to elucidate the cell type expressing increased cathepsin X in the degenerated SNc at the 12 h-induced lesion. The majority of cathepsin X-positive cells in the ipsilateral SNc expressed the neuronal marker NeuN Figure , while tTo confirm the upregulation of cathepsin X in dopaminergic neurons in the SNc at the short term-induced lesion, we performed additional double immunostaining with the dopaminergic neuronal marker TH, using brain slices at 12 h of 6-OHDA-induced lesion. As shown in Figure Notably, some single, cathepsin X-positive microglial cells were observed in the ipsilateral SNc at 12 h after the 6-OHDA-induced lesion Figure . The numin vitro models mimicking neurodegenerative processes has been demonstrated; however, its expression pattern and role in the brain with PD remained unknown. Therefore, in the present study, we characterized the regional and cellular expression, as well as the activity of cathepsin X in striatum and SNc of rat 6-OHDA model of PD, in a time course manner. Our results show for the first time that 6-OHDA, injected into the MFB, leads to an increased level of cathepsin X mRNA, as well as protein expression and its activity in the ipsilateral SNc. Upregulated cathepsin X has been at early time points localized in the lysosomes in neuronal cells, with preferential localization in dopaminergic neurons. Nevertheless, on the short-term, specifically at 12 h after 6-OHDA injection, the association of single-microglial cells with cathepsin X has been observed, whereas after a longer period of 4 weeks, cathepsin X was present in glial cells, predominantly in activated microglia concentrated in SNc.The contribution of cathepsin X to neuroinflammation and neurotoxicity in in vitro studies of novel role of cathepsin X in neuroinflammation (in vitro study revealed that cathepsin X promotes 6-OHDA-induced apoptosis and the consequent neuronal toxicity, and cathepsin X inhibition exerts neuroprotection of dopaminergic-like neuronal cells (Recent studies have demonstrated that lysosomal cysteine cathepsin X might play an important role in neurodegenerative processes, through carboxypeptidase activity within the cells or by release from cells as a proenzyme acting extracellularly, independent of its enzymatic activity . In mousammation . Neverthammation . Furtherammation . 6-OHDA,ammation . Neverthal cells . Cathepsin situ hybridization, we obtained pronounced upregulation of cathepsin X mRNA levels in the ipsilateral SNc and VTA region, 12 and 24 h after 6-OHDA-induced lesion, whereas, after 48 h, a decreasing trend in cathepsin X mRNA level in ipsilateral SNc was observed. Using immunohistochemistry, we were able to confirm cathepsin X upregulation in the ipsilateral SNc compared to regions in the contralateral sides, as the protein expression pattern was similar to the mRNA pattern at all three time points used for lesion. Additionally, cathepsin X protein level and activity in contralateral and ipsilateral SNc were determined and the values obtained for protein expression correlated well with microscope observation. Interestingly, the cathepsin X activity was significantly altered, but only slightly increased in ipsilateral SNc at 12 and 24 h after injection into MFB.Injection of 6-OHDA into the MFB results in rapid uptake of the toxin by DA neurons, and a near complete loss of the DA neurons in the SNc. After time, there is extensive DA depletion of SNc target areas on the site of injection, including the striatum . In our There are no reports in the literature regarding the expression of cathepsins in the striatum after 6-OHDA injection into MFB. In the hemiparkinsonian rat model used in our study, the destruction of nigrostriatal neurons has been observed . We founMislocalization and altered secretion of the lysosomal cathepsins have been extensively reported for several pathological conditions [reviewed in Although we were able to show that at early time points the upregulated cathepsin X was seen predominantly in the dopaminergic neurons in the ipsilateral SNc, a few microglial cells also showed a positive signal for cathepsin X expression, whereas astrocytes were devoid of cathepsin X. This profile was observed 12 h after injection of 6-OHDA into the MFB; however, cathepsin X-positive microglial cells increased with the prolonged time-period of 6-OHDA-induced lesion. Following CNS injury, activated microglia appear in the SNc as early as day’s 1–3 post-6-OHDA lesioning, and the rapid microglia response is followed by an astrocytic reaction . Thereafin vitro study, where increased cathepsin X expression and activity in microglial cells resulted in microglia activation-mediated neurodegeneration (Nevertheless, after longer period of time from 6-OHDA injection into the MFB, no neuronal cells were observed at the ipsilateral SNc after 4 weeks of injection, presumably due to complete loss of dopaminergic neurons. On the contrary, microglial reactivity increased and was followed by an astrocyte reaction. Therefore, no neuronal cells positive for upregulated cathepsin X were present in the area of damaged SNc, but dense accumulation of cathepsin X-immunoreactive signals were localized in microglial cells as well in some astrocytes concentrated in the ipsilateral SNc, which correlated well with strongly increased glial reactivity. These results suggest the important role of cathepsin X in processes at an early stage of neurodegeneration, as well in processes in progressive inflammation, which both lead to chronic neurodegenerative disorder resulting from progressive loss of dopaminergic neurons in the SNc. These results are also in line with those of our recent neration .in vitro studies and on the current results, we can propose that cathepsin X is an important causative factor in neurodegenerative processes, resulting in progressive loss of dopaminergic neurons, and is therefore designated as a potential target for therapeutic interventions in PD.We present, for the first time, the distribution and cellular localization of cathepsin X in the striatum and SNc of 6-OHDA-lesioned rats. 6-OHDA injection into the MFB increased cathepsin X expression and its activity in the SNc at the ipsilateral side, with the simultaneous degeneration of dopaminergic nigrostriatal neurons. The prominent cathepsin X upregulation was restricted to dopaminergic neuronal cells at an early time points after the injection, whereas a late time point of 6-OHDA-induced lesion caused the upregulation of cathepsin X restricted to the glial cells concentrated in the ipsilateral SNc. Based on our previous in situ hybridization histochemistry, participated in carrying out the immunohistochemistry and double immunofluorescence analysis, and reviewed the manuscript. GG participated in carrying out the immunohistochemistry and reviewed the manuscript. MŽ performed unilateral 6-OHDA lesions. MŽ and JK coordinated the research and reviewed the manuscript. All authors read and approved the final manuscript.AP and MŽ designed the study. AP prepared coronal sections of early time point of injection, performed immunohistochemistry, ELISA assay, cathepsin X activity assay and double immunofluorescence labeling, generated the data for Figures, and prepared the draft manuscript. LT prepared coronal sections of late time point of injection, performed 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.


Lawsonia intracellularis is an obligate intracellular bacterium which cannot be cultured by conventional bacteriological methods. Furthermore, L. intracellularis needs enriched medium and a unique atmosphere for isolation, cultivation and propagation. Because of this,there are only a few isolates of L. intracellularis available and few studies in vitro demonstrating the susceptibility of this bacterium to antimicrobial agents. The objectives of this study were to isolate South American and Southeast Asia strains of L.intracellularis and to determine the in vitro antimicrobial activity against these isolates. Tested antimicrobials included: chlortetracycline, lincomycin, tiamulin, tylosin and valnemulin(against both Brazilian and Thailand strains) and additionally, amoxicillin, zinc-bacitracin, carbadox, enrofloxacin, gentamicin, sulfamethazine, trimethoprim, spectinomycin and a combination (1:1) of spectinomycin and lincomycin were also tested against the Thai isolates. The minimum inhibitory concentration (MIC) was determined by the antimicrobial activity that inhibited 99% of L. intracellularis growth in a cell culture as compared to the control .L. intracellularis strains tested. Tylosin showed intermediate activity, chlortetracycline had variable results between low and intermediate activity, as well as spectinomycin, spectinomycin and lincomycin, amoxicillin, sulfamethazine and enrofloxacin. L. intracellularis was resistant to lincomycin, gentamicin, trimethoprim, colistin and bacitracin in in vitro conditions.Two strains from Brazil and three strains from Thailand were successfully isolated and established in cell culture. Each antimicrobial was evaluated for intracellular and extracellular activity. Pleuromutilin group  and carbadox were the most active against L. intracellularis strains from South America and Southeast Asia and characterization of the antimicrobial susceptibility patterns of these new strains.This is the first report of isolation of  Lawsonia intracellularis [Proliferative enteropathy (PE), or ileitis, is one of the most important enteric bacterial infectious diseases in grower and finisher pigs. PE was first recognized in the North American swine industry in the early 1930s [llularis . There aL. intracellularis for antimicrobial selection is available [L. intracellularis from infected intestine or fecal samples, requiring experienced personnel and several months for the establishment of a pure L. intracellularis culture. Consequently, the in vitro sensitivities of L. intracellularis for antimicrobials are difficult to obtain in a timely fashion to treat a PE outbreak. Furthermore, the obligate intracellular nature of L. intracellularis prohibits the use of standard antimicrobial susceptibility testing methods. Instead, a complicated tissue culture system has been used to evaluate antimicrobial activity against some isolates of L. intracellularis originated from the United States and two other countries [When a PE outbreak occurs in a herd, antimicrobial therapy is often used to control the disease . Antimicvailable –6. The mountries –7.L. intracellularis can have different antimicrobial sensitivities [L. intracellularis isolated from Latin America and Southeast Asia, where swine production is an important industry and there is documented high prevalence of proliferative enteropathy in these areas [L. intracellularis, additional primary isolates of L. intracellularis from Brazil and Thailand must be obtained, propagated in vitro and then evaluated.A previous study found that isolates of tivities . Therefose areas , 8–11. IL. intracellularis from Latin America and Southeast Asia and to determine the in vitro minimum inhibitory concentration (MIC) of antimicrobials against these new isolates for use as a guideline for antimicrobial selection in the treatment and control of PE.The overall aims of this investigation were to obtain new isolates of L. intracellularis strains BRPHE01_E5, BRPHE02_E8,CUPHE01_SW13, CUPIA01_SW13, and CUPIA02_SW13 were successfully isolated as pure cultures from swine intestines affected by PE. The number of cells heavily infected by each isolate, which is an indicator of viable bacteria, dramatically increased to approximately 100% around passage 5. Moreover, all isolates were continuously maintained and propagated in vitro.L.intracellularis isolates were tested for antimicrobial MICs at passages up to 15. The final concentration of inoculum was approximately between 106 and 107L. intracellularis/ml for all isolates.The L. intracellularis isolates are shown in Table L. intracellularis proliferation. An example of heavily infected cells (HIC) of L. intracellularisgrowth at different concentrations of antimicrobials in the McCoy cells is shown in Fig. The value of extracellular and intracellular MICs of all tested antimicrobials against the Brazilian L. intracellularis isolates, with MICs rangingfrom≤0.125 to 2 μg/ml. Tylosin showed moderate activity against the L. intracellularis isolates with MICs rangingfrom 2 to 8 μg/ml. Chlortetracycline had lower activity with MICs rangingfrom 8 to 64 μg/ml. Lincomycin showed the lowest activity against the L. intracellularis isolates with MICsgreater than 128 μg/ml.For intracellular MIC testing, tiamulin and valnemulin had the highest activity against the Brazilian L. intracellularis isolates with MICs from ≤0.125 to 2 μg/ml. Chlortetracycline had moderate antimicrobial activities against L. intracellularis with MICs ranging from of 32 to64 μg/ml. Tylosin had different results between the two strains, with moderate activity against BRPHE02_E8 (16–32 μg/ml) and low activity against BRPHE01_E5 (> 128 μg/ml). Lincomycin had lowest antimicrobial activities with the MIC of > 128 μg/ml for both isolates.For extracellular activity, the results showed that tiamulin and valnemulin had highest activity against the L. intracellularis isolates is shown in Table L. intracellularis isolates with MICs of ≤0.125 to 1 μg/ml. Amoxicillin, enrofloxacin and tylosin showed moderate activity against all three L. intracellularis isolates with MICs rangingfrom 2 to 32 μg/ml. Zinc-bacitracin, chlortetracycline, colistin, gentamicin, lincomycin, spectinomycin, lincomycin:spectinomycin (1:1), sulfamethazine and trimethoprim showed the lowest activity against all three L. intracellularis isolates with MIC ranging from 64 to > 128 μg/ml.The median value of extracellular and intracellular MICs for all tested antimicrobials against three Thailand L. intracellularis isolates with MICs from 0.25 to 8 μg/ml. Spectinomycin, lincomycin:spectinomycin (1:1), and tylosin had moderate antimicrobial activities against L. intracellularis with MICs of from 2 to 32 μg/ml. Amoxicillin, bacitracin, chlortetracycline, colistin, enrofloxacin, gentamicin, sulfamethazine and trimethoprim had lowest antimicrobial activities, with the MICs ranging from 64 to 128 μg/ml.For extracellular activity, the results showed that carbadox, tiamulin, and valnemulin had highest activity against the three L. intracellularis isolates have been successfully cultured and maintained in vitro worldwide. Of these, only 15 L. intracellularis isolates have been tested for their antimicrobial susceptibilities:three United Kingdom isolates [L. intracellularis from PE field cases and to evaluate its antimicrobial susceptibility in Brazil and Thailand. Five L. intracellularis isolates were successfully established in pure culture, two from Brazil and three from Thailand. Like other continents, a high level of L. intracellularis infection had been found in swine herds in South American and Southeast Asian countries. It was estimated that the herd prevalence of PE in pigs was 77% in Vietnam, 85% in China, 86% in the Philippines, 94% in Japan, and 100% in Korea, Malaysian and Thailand [L. intracellularis infectionindicating direct contact with the pathogen [Although outbreaks of PE occur globally, it has been estimated that less than 25 isolates , 7; one isolates . Our stupathogen .L. intracellularis susceptibility to antimicrobials are limited. Published data have shown MIC endpoints for diverse isolates expressed as both extracellular and intracellular MICs using a tissue culture system [L. intracellularis infection in vivo. The bacteria would be exposed to antimicrobials before and after invasion into intestinal cells . Similar to previous studies [Studies describing e system –7. Both  studies –7, our ML. intracellularis isolates with an MIC of > 128 μg/ml. This might be due to the fact that this antimicrobial has been used more intensively with high dosages for control of other endemic pathogens in swine farms or by the difficulty of extrapolating the in vitro results to the in vivo scenario.Our extracellular and intracellular MIC results showed that carbadox, tiamulin and valnemulin were the most active compounds against the Brazilian and Thai isolates, inhibiting extracellular and intracellular activities with concentrations of ≤0.125–8 μg/ml. Since the use of carbadox is not permitted in Brazil, this component was not tested for the Brazilian isolates. Tylosin had intermediate activity against all the isolates with MICs ranging from 2 to 64 μg/ml for intracellular and extracellular activities, except for one Brazilian isolate, which had an MIC of > 128 μg/ml for extracellular activity. Lincomycin was the least active compound against the Brazilian and Thai L. intracellularis isolates tended to have higher MIC endpoints than the North American, European and Korean isolates [L. intracellularis isolates, similar to the results previously published for North American, European, and Korean isolates [When compared to other data, the MIC values for the Brazilian and Thai isolates , 6. For isolates , 6.Chlortetracycline showed intermediate activity against Brazilian isolates 8–64 μg/ml), whereas Thai isolates less sensitive (64 - > 128 μg/ml). This was the only antibiotic with substantial differences between the Thai and Brazilian isolates, with the exception of the Thai strain CUPIA02_SW13, which had similar results to the Brazilian isolates and to previous studies  μg/ml, w to 64 μgL. intracellularis was exposed to the antimicrobial agent in each of the preparations, the MIC assays were performed as described by Wattanaphansak et al. [L.intracellularis for three consecutive days. Moreover, it is likely that the effect of antimicrobials accumulated inside the cells overtime. This accumulation of intracellular antimicrobial concentration suggests that a one-time antimicrobial treatment may be insufficient to inhibit the growth of L. intracellularisin vitro.Previous reports have shown that extracellular MICs for all tested antimicrobials were higher than the intracellular MICs , 6, and k et al. . ExtraceL. intracellularis generated for isolates from pig farms worldwide. Based on our in vitro results, we confirm that Brazilian and Thai L. intracellularis isolates have a unique in vitro antimicrobial sensitivity pattern, in relation to other regions. Since it is impractical to culture L.intracellularis and perform an antimicrobial sensitivity test during a PE outbreak, our data serve as a guideline for the range of antimicrobial activities against L. intracellularis.In conclusion, our in vitro data expand the antimicrobial susceptibility information for L. intracellularis isolates were obtained from pigs affected with the acute form of PE. For the first isolate,BRPHE01_E5, ileum was obtained from adiarrheic finishing pig from a multi-site commercial farm in the metropolitan region of Belo Horizonte, Minas Gerais state, Brazil, in 2011. The second isolate, BRPHE02_E8, was obtained from a diarrheic finishing pig from a herd located in São Paulo state, Brazil, also in 2011. Affected intestines were submitted to the Veterinary Pathology Laboratory at the Universidade Federal de Minas Gerais, for routine bacteriology examination and immunohistochemistry confirmation of L. intracellularis, which was the only etiologic agent found.L. intracellularis isolates. One intestine had the acute form of the disease, characterized by blood clots in the lumen associated with thickening of the small intestine mucosa, proliferative hemorrhagic enteropathy (PHE), and two hadthe chronic form of the disease, characterized by the thickening of the small intestine mucosa, porcine intestinal adenomatosis (PIA). The intestines were collected from three distinct herds in the Western region of Thailand. The PHE strain, CUPHE01_SW13, was obtained from a gilt that died suddenly with acute bloody diarrhea in a breeding herd in Kanchanaburi province in 2013. Both of the PIA intestines, CUPIA01_SW13 and CUPIA02_SW13, were collected from finishing pigs at the slaughter house in Nakornpathom province in 2012. All three affected intestines were submitted to the Veterinary Diagnostic Laboratory at the Chulalongkorn University, Nakhonpathom, for PCR confirmation of L. intracellularis infection. All three infected intestine samples were PCR positive for L. intracellularis which was the only etiologic agent found.Three PE affected swine intestines were used to obtain L. intracellularis from the infected intestines was modified from a previous study [2:H2:N2 gas mixture, respectively [L. intracellularis was maintained in the McCoy culture until the number of HICwas 90 to 100%.Infected segments of the jejunum or ileum were cut into several pieces of approximately 5 cm and kept at -80 °C until the initiation of the bacterial isolation process. The primary isolation of us study . Brieflyectively . The culectively . L. intrL. intracellularis isolate was used to quantify the inoculum for the antimicrobial MIC assay using a previously described staining protocol [L. intracellularis dilutions, from 100 to 10− 5, was dilutedin PBS. Then, 10 μl of each dilution was applied into 15-well glass slides as duplicates and the slides were allowed to dry at 37 °C. After fixing with acetone at 4 °C, the slides were stained with the modified immunoperoxidase monolayer assay (IPMA) protocol as described by Guedes et al. [L. intracellularis quantity between 50 and 500 bacteria/well was counted using a light microscope with 40X objective lens, and the initial concentration was calculated.After the establishment of a pure culture, eachprotocol , 13. Bris et al.  using raL. intracellularis was tested twice from two independent bacterial passage.For the Brazilian isolates the following antibiotics were used: Chlortetracycline hydrochloride, lincomycin hydrochloride, and tylosin tartrate obtained as pure chemicals from Sigma Aldrich . Tiamulin hydrogen fumarate and valnemulin hydrochloride were supplied as pure chemicals from Novartis Animal Health . For the Thailand isolates, amoxicillin, Zinc-bacitracin, carbadox, enrofloxacin, gentamicin sulfate, polymyxin B (colistin), spectinomycin dihydrochloride, sulfamethazine and trimetroprim were also used and obtained as pure chemicals from Sigma Aldrich . The combination of lincomycin-spectinomycin was prepared as a 1:1 ratio for determination of the combined activity. The working solutions of tested antimicrobials were prepared as previously described . BrieflyL. intracellularis. The intracellular MIC was defined as the effect of antimicrobials on L. intracellularis when intracellular organisms were inside of the enterocytes [The MIC assays were performed as described by Wattanaphansak et al. . The anterocytes . One hunerocytes , the bacL. intracellularis when the bacteria were freely in the gut lumen. For this, a series of two-fold dilutions of stock antimicrobial solutions were diluted 1:10 with culture medium which contained L. intracellularis. The suspension was incubated at 37 °C in a bag for two hours, allowing the bacteria to be exposed directly to the antimicrobials. After incubation, 100 μl of the bacterial suspension was transferred to one-day-old McCoy cells. The medium was removed 24 h after incubation (under microaerophilic conditions) and replaced with 100 μl of fresh culture medium, without any antimicrobials, for three consecutive days. Each test plate contained control culture, containing no antimicrobials.The extracellular MIC testing was performed as described by Wattanaphansak et al. (5) in order to measure the effect of antimicrobial on L. intracellularis proliferation, the infected plates were stained using a modified immunoperoxidase monolayer assay staining method as described previously [After 5 days of incubation for both assays (intracellular and extracellular), supernatant from the infected plates was removed and the cell culture monolayer was fixed with 50 μl of cold 50% acetone and 50% methanol for 1 min. To assess the inhibitory effect of each antimicrobial on L. intracellularis inside the host cells had proliferated to greater than 30 bacteria per cell [L. intracellularis proliferation in the McCoy cells.The infected cells were considered to be HIC if the number of per cell . A compa


Saccharomyces cerevisiae, the meiosis-specific axis proteins Hop1 and Red1 are present nonuniformly across the genome. In a previous study, the meiosis-specific VMA1-derived endonuclease (VDE) was used to examine Spo11-independent recombination in a recombination reporter inserted in a Hop1/Red1-enriched region (HIS4) and in a Hop1/Red1-poor region (URA3). VDE-initiated crossovers at HIS4 were mostly dependent on Mlh3, a component of the MutLγ meiotic recombination intermediate resolvase, while VDE-initiated crossovers at URA3 were mostly Mlh3-independent. These differences were abolished in the absence of the chromosome axis remodeler Pch2, and crossovers at both loci became partly Mlh3-dependent. To test the generality of these observations, we examined inserts at six additional loci that differed in terms of Hop1/Red1 enrichment, chromosome size, and distance from centromeres and telomeres. All six loci behaved similarly to URA3: the vast majority of VDE-initiated crossovers were Mlh3-independent. This indicates that, counter to previous suggestions, levels of meiotic chromosome axis protein enrichment alone do not determine which recombination pathway gives rise to crossovers during VDE-initiated meiotic recombination. In pch2∆ mutants, the fraction of VDE-induced crossovers that were Mlh3-dependent increased to levels previously observed for Spo11-initiated crossovers in pch2∆, indicating that Pch2-dependent processes play an important role in controlling the balance between MutLγ-dependent and MutLγ-independent crossovers.In  Spo11 protein , to enable their proper segregation during the meiotic divisions (reviewed in  protein . In buddMlh1-Mlh3 and Exo1) meiosis-specific resolvase (Yen1 and Slx1-4) to form both NCOs and COs  at high efficiency regardless of chromosomal context  or by ends-in transformation  at six different locations, using primers listed in Table S2. Ends-in transformation was used for inserts at divergently transcribed loci to minimize effects on expression caused by disruption of 5′ untranslated regions. Transformation was performed with overlapping DNA fragments as illustrated in Figure S1. The VRS-arg4 and VRS-103-arg4 constructs are 5.5kb and 8.6kb long, respectively, with ∼3kb sequence homology around the VRS site. This size difference, along with HindIII site differences, enables the detection of the parental and recombinant chromosomes on Southern blots (see All strains (Table S1) used in this study are of SK1 background  and werelots see , below.4 in sporulation medium to induce VDE expression . Other supplementary files, available at the same URL, include:Figure S1: Construction of insertsVDE DSBs, combined CO and NCO data, and NCO/CO ratiosFigure S2: Additional data and analyses. Includes cells completing meiosis I, timing of Table S1: Strain genotypesTable S2: Primers for all reporter inserts. Supplemental material available at Figshare: https://doi.org/10.25387/g3.7800728.All strains are available upon request. The authors affirm that all data necessary for confirming the conclusions of this article are represented fully within the article, tables, figures, and supplementary figures, tables and files. Data underlying graphs in all figures and chromosome coordinates in Hop1-enrichment determines the MutLγ-dependence of meiotic CO formation, six new sites were selected for VRS reporter insertion, one (HSP30) with regional Hop1 levels (average Hop1 occupancy over 10-20kb around the insert location) similar to those at URA3, four  with Hop1 levels similar to those at HIS4, and one (FIR1) with intermediate Hop1 levels . In addition, VDE-initiated DSBs appeared and disappeared with levels and timing similar to those previously seen at HIS4 and URA3 , COs in the same sequences inserted at all other loci were only modestly affected, with COs in mlh3∆ ranging from ∼80% to ∼115% of wild type . Frequencies of NCOs at all eight VRS insert loci in the pch2∆ were similar to those seen in wild type (pch2∆/PCH2 = 111 ± 10%), as were COs (pch2∆/PCH2 = 113 ± 16%). Loss of Mlh3 did not substantially affect NCOs (pch2∆ mlh3∆/pch2∆ MLH3 = 114 ± 14%). However, in pch2∆ mlh3∆ double mutants, COs were reduced 20–35% relative to pch2∆ MLH3 (pch2∆ mlh3∆/pch2∆ = 74 ± 7%), as was previously observed for inserts at URA3 and HIS4  originally examined by HIS4, VDE-initiated COs at all insert loci were largely Mlh3-independent, regardless of whether inserts were at loci in Hop1-enriched or Hop1-depleted regions of the genome. Therefore, our previous hypothesis, that local Hop1 occupancy determines mechanisms of CO formation, is inaccurate, at least for VDE-initiated recombination, in that it was based on analysis of inserts at a locus (HIS4) that appears to be exceptional. We currently do not understand why the genetic dependence of VDE-induced CO formation at HIS4 differs from that seen at the other loci examined, but cannot rule out other effects or interactions on the basis of current data.In this study, we examined VDE-initiated COs at most insert loci are Mlh3-independent, in turn, raises the question of whether or not VDE-initiated recombination events that occur in cells undergoing meiosis can be properly described as being “meiotic”. VDE-initiated NCOs are recovered in excess of COs , which is reminiscent of, although less than, the 5 to 20-fold excess of NCOs over COs seen in budding yeast mitotic recombination (VDE-initiated DSB processing also resembles DSB processing in the mitotic cell cycle, in that break ends are continuously resected over time (Spo11 DSBs (Spo11, VDE frequently cuts both sister chromatids in a single meiosis (HO endonuclease-initiated meiotic recombinants (Mlh3-independence of VDE-initiated COs at seven of the eight insert locations examined, and why the majority of VDE-initiated COs at HIS4 are Mlh3-dependent.The observation that pch2∆ strains, VDE-initiated COs show the same Mlh3-dependence as Spo11-initiated COs, regardless of wild-type Hop1 occupancy levels around insert loci. It therefore seems unlikely that Hop1 redistribution in pch2∆ mutants is the only factor responsible for the increased Mlh3-dependence of COs at most insert loci and the decreased Mlh3-dependence of COs at HIS4. Homolog synapsis, recombinant formation and meiotic divisions are all delayed in pch2∆ mutants; pch2∆ mutants also display a more even distribution of the Zip1 central element protein along chromosomes and reduced CO interference (pch2∆ mutant defects might delay either recruitment of factors promoting MutLγ action at Spo11-initiated events or implementation of CO interference, thus increasing the window of opportunity for these factors to act at VDE-initiated events. It also has been suggested that Pch2-dependent remodeling affects the stability of recombination intermediates (Spo11. Exogenous DNA damage is unable to fully rescue spo11 mutant phenotypes in several organisms (Spo11 and to DSBs formed by exogenous DNA damage (Pch2 might implement mechanisms that distinguish Spo11-initiated recombination events from those initiated by other forms of DNA damage.In contrast, in rference . These oVDE-initiated recombination events are treated differently than are those initiated by Spo11 during wild-type meiosis. VDE-initiated events produce an excess of NCOs over COs and, at seven of eight loci examined, form COs by MutLγ-independent mechanisms, and thus their outcome more closely resembles those of DSB repair events that occur during the mitotic cell cycle. We conclude that the full spectrum of meiotic recombination processes that occur at Spo11-initiated DSBs do not occur at VDE-initiated DSBs, and, by inference, DSBs formed during meiosis by other nucleases. Thus, our findings call for caution in the use of DSBs formed by this nuclease, or by other exogenous means, for inferring factors that control normal meiotic recombination.In summary, the data presented here indicate that


To characterize the audiometric evaluation and acoustic immittance measures in different types of mucopolysaccharidosis.Fifty-three mucopolysaccharidosis patients were evaluated. The classification consisted of type I , type II (Hunter syndrome), type III (Sanfilippo syndrome), type IV (Morquio syndrome), and type VI (Maroteaux-Lamy syndrome). Immittance audiometry and play or conventional threshold tone audiometry were used to obtain auditory thresholds and were chosen according to the patient's chronological age and ability to understand/respond to the procedure. The findings were analyzed using descriptive statistics and considering the recommendations for research involving human beings contained in Resolution CNE N° 466/2012.Fifty-one subjects (96.2%) had hearing loss, and the conductive type was the most frequent. Only two (3.8%) patients presented bilateral thresholds within normal limits, one with type IV mucopolysaccharidosis and the other with type VI. There were 11 individuals (20.8%) with mucopolysaccharidosis type I with mixed hearing loss, 9 (16.9%) individuals with type I with conductive hearing loss and 9 (16.9%) with type VI with conductive hearing loss. Mild hearing loss was most common (37.3%), followed by moderately severe hearing loss (36.3%). The type B tympanometric curve (80.4%) was the most frequent.Most of the individuals with mucopolysaccharidosis types I, II, III, IV and VI presented mixed or conductive hearing losses of mild to moderately severe degree, type B tympanograms and an absence of contralateral acoustic reflexes. Mucopolysaccharidosis (MPS) comprises a group of diseases caused by a deficiency in the lysosomal enzymes involved in mucopolysaccharide metabolism MPS are hereditary and therefore permanent metabolic diseases caused by inborn errors of metabolism (IEM) that lead to altered functioning of certain enzymes Clinical manifestations of MPS vary according to the enzyme that is missing in the affected individual. The most common clinical symptoms are facial dysmorphism, communicating hydrocephalus, hearing impairment, neuropsychomotor developmental delay, obstructive airway disease, skeletal abnormalities, heart abnormalities and hepatomegaly The hearing loss due to MPS may be conductive, sensorineural or mixed. A study Authors One study It is important to note that the appropriate treatment of MPS described in the literature is enzyme replacement therapy. One author It is therefore important to diagnose auditory alterations in MPS patients in all age groups, especially during early childhood, because otological infections can cause irreversible damage to hearing, delaying the child's linguistic development.Although different studies have noted the auditory manifestations of MPS, few have evaluated patients with all types of the disease. Studies most frequently involve patients with a single type of the disease or are case reports.Thus, the present study aimed to characterize the audiometric profiles and acoustic immittance measurements of patients with different types of MPS.This is a descriptive, cross-sectional study conducted at the audiology medical clinic of the hearing disorders program of Universidade Federal de São Paulo- UNIFESP, Departamento de Fonoaudiologia.Sixty individuals were selected, but seven were excluded from the sample because they could not complete the audiometric test battery. Therefore, the final sample consisted of 53 individuals aged 2 to 23 years  who met the following inclusion criteria: diagnosed with mucopolysaccharidosis of any type in the Reference Center in Inborn Errors of Metabolism (CREIM) of the UNIFESP; completion of audiometric tests.To examine the association between audiological results and MPS type, the sample was distributed into five groups according to MPS classification Data collection started only after the project received approval from the Research Ethics Committee (REC) of the Universidade Federal de São Paulo (UNIFESP) under protocol n° 02111/08. The adults included in the sample and the legal guardians of the children signed the informed consent form, authorizing participation in the study.® brand otoscope; pediatric audiometer, PA 2 model, and acoustic immittance meter, AZ7 model, both by Interacoustics; an audiometer, model MA-41, MAICO brand, calibrated according to ANSI 1996 standards; TDH-39 headphones; and MX-41 cushions.Data collection procedures included anamnesis, otoscopy, conditioned or conventional tonal threshold audiometry, and immittance audiometry. The equipment used to perform these procedures were a MissuriThe procedure used to obtain auditory thresholds was chosen based on the chronological age of the child and his/her ability to understand/respond to the procedures. When it was not possible to perform the appropriate procedure for the patient's age, we performed the procedure corresponding to the next youngest age and progressed until the results could be obtained safely.The following procedures were used:warble) from 500 to 4000 Hz at intensities of 80, 60, 40 and 20 dB NA, both in these orders.- Visual reinforcement audiometry (VRA): VRA was performed by means of stimulation-response-visual reinforcement for children aged 6 months to 2 years, as proposed by the authors of previous studies - Conditional tonal play audiometry: auditory thresholds for frequencies from 500 to 4000 Hz by air conduction for children aged 2 years to 6 years and 11 months.- Conventional tonal audiometry: measurement of auditory thresholds for the frequencies from 250 to 8000 Hz by air conduction for individuals aged 7 years and older, and auditory thresholds for the frequencies from 500 Hz to 4000 Hz by bone conduction when the air conduction thresholds were altered at intensities above 15 dB NA in children up to 11 years and 11 months and above 25 dB NA in individuals over 12 years of age.- The results were classified as normal or altered according to standards mentioned in the literature - Acoustic immittance measurements: compliance, volume of the external acoustic meatus and the threshold of the acoustic reflex with contralateral stimulation were verified. Tympanometric results were analyzed according to the classification proposed by Jerger Absolute distributions and univariate and bivariate percentages were obtained for descriptive analysis, and the Kruskal-Wallis test was used for inferential statistics. The margin of error adopted in statistical test decisions was 5%.Fifty-three MPS patients diagnosed at the UNIFESP reference center were evaluated. Twenty-one patients were diagnosed with MPS type I, 12 with type II, one with type III, 4 with type IV and 15 with type VI. The ages of the individuals ranged from 2 to 23 years, with a mean of 12.5 years. Males comprised slightly more than half of the subjects (60.4%).The procedure chosen for the audiological evaluation varied according to the age of the individual. Of the 53 individuals evaluated, 51 (96.2%) had hearing loss, and conductive hearing loss was the most frequent type. Twenty-five individuals (47.2%) had bilateral hearing loss, and 5 (9.4%) had unilateral conductive hearing loss; 19 individuals had bilateral mixed hearing loss (35.8%), and 5 (9.4%) had unilateral mixed hearing loss; one individual (1.9%) had bilateral sensorineural hearing loss, and 2 (3.8%) unilateral sensorineural hearing loss, one of whom presented with conductive hearing loss in the other ear and the other of whom presented with mixed hearing loss in the other ear. Only two (3.8%) participants presented thresholds within normal limits: one had MPS type IV, and the other had type VI.Regarding the most common hearing loss profiles associated with disease type, 11 individuals (20.8%) had MPS type I with mixed hearing loss, followed by 9 (16.9%) who had MPS type I with conductive hearing loss and 9 (16.9%) who had MPS type VI with conductive loss .Concerning tonal audiometry alterations (51 children - 102 ears), there was a high prevalence of mild hearing loss (37.3%), followed by moderate-severe hearing loss (36.3%). Regarding the type of hearing loss, there was a predominance of the conductive type (53.9%), followed by mixed hearing loss (42.2%).Regarding the individuals with hearing loss, acoustic immittance measures were analyzed for only 90 ears because in 12 out of the 102 altered ears (11.7%), it was not possible to perform the examination . A Type B tympanometric curve was most frequent (76.5%). A type A curve was observed in four ears with sensorineural hearing loss and in four ears with thresholds within the normal range.Regarding the relationship between the audiometric tests and the type of MPS, a significant association was found in the variable "audiometric tests results". A total of 63.3% of the ears of patients with MPS type VI had conductive hearing loss. However, the one participant with MPS type III had sensorineural hearing loss in both ears (100%) .Individuals with MPS type I had better thresholds at 3000 Hz than those with type IV. Individuals with MPS type I had better auditory thresholds at 6000 Hz and 8000 Hz compared with the other types. The worst auditory thresholds were obtained by individuals with MPS type III compared with types I and VI .The incidence of MPS has been described in the international literature as 1:29,000 live births Regarding the procedures used, conventional tonal audiometry was most often performed in patients with MPS type IV and type VI. All the patients with MPS type IV and 75% of the patients with MPS type VI underwent threshold tonal audiometry, which is the indicated procedure for their age .This result was expected because patients with MPS types IV and VI generally present normal intelligence In the present study, the evaluation of the audiometric results showed no statistically significant difference in auditory thresholds between the ears, indicating bilateral and symmetrical hearing loss in the majority of the patients  and 3.One individual with MPS type IV and another with MPS type VI had auditory thresholds within normal patterns bilaterally and type A tympanometric curves, revealing good mobility of the ossicular-tympanic system. The low incidence of individuals with normal auditory thresholds and without involvement of the ossicular-tympanic system agrees with the literature Of the total number of ears, 102 presented hearing loss, most of them (96%) with conductive impairment  in the fAuthors In the present study, bilateral mixed hearing loss was observed in 38 ears (35.8% of the sample). Five patients (five ears) presented unilateral mixed hearing loss (4.7% of the sample) accompanied by another type of hearing loss in the other ear. Authors Only four ears 3.9%) presented sensorineural hearing loss; one patient had bilateral sensorineural hearing loss, and two patients had unilateral sensorineural hearing loss . These d% presentAn empirical analysis of MPS patients An older study In the present study, sensorineural hearing loss occurred only in patients with MPS types I, II and III and was most predominant in type III, which presented the worst auditory bone conduction thresholds. It is noteworthy that only one MPS type III patient was evaluated and diagnosed with bilateral sensorineural loss. Two other ears (in MPS type I and type II patients) that presented sensorineural hearing loss had mixed and conductive loss in the other ear . RegardiA study It is important to emphasize that otological evaluations of MPS patients should be performed as early as possible to avoid middle ear conditions that interfere with the quality of life and language development of young children. Manifestations of otitis media have been highlighted in several studies with patients with MPS, and a higher prevalence of conductive hearing loss has been reported One author Another procedure that improved auditory thresholds was described by authors This study described the presence of upper airway infection concomitant with otitis media in patients with MPS types I, II and IV. Hearing loss was reported as a symptom by 45% of the patients studied. There was a high frequency of tympanic membrane retraction (33%). As a result of the data findings and the possibility of irreversible auditory impairments, this study suggests early hearing diagnosis by audiometry and the use of brainstem auditory evoked potentials and otoacoustic emissions for follow-up and intervention in this population Authors Therefore, there is the need for early otological intervention by a multidisciplinary team soon after the diagnosis of MPS because studies have reported strong evidence of otitis media episodes, which may lead to auditory changes. Basic hearing assessment  and periodical follow-up must be performed because of the known progression of hearing loss in this population and the recurrence of middle ear conditions.The results obtained the present study show the importance of audiological evaluations for children with MPS when the disease is diagnosed in the first years of life to minimize communication difficulties and delayed language development due to sensory hearing loss caused by the high rate of otological infections or by decreased auditory sensitivity. Multidisciplinary intervention can make the diagnosis as early as possible and initiate the most appropriate treatment, thus promoting a better quality of life for affected individuals.Most individuals with MPS types I, II, III, IV and VI present mixed or conductive hearing loss of mild to moderately severe degree, type B tympanograms, and an absence of contralateral acoustic reflexes.Silveira MR was the main researcher, responsible for the elaboration of the research project, preparation of the schedule, literature survey, data collection and analysis. Buriti AK was responsible for the manuscript writing, revision of the text, data analysis, updating the references, final formatting, submission of the manuscript. Martins AM was responsible for the sample selection and patient referral. Gil D was the co-supervisor, responsible for the elaboration of the research project, correction of the data analysis, correction of the final version of the manuscript. Azevedo MF was the supervisor, responsible for the elaboration of the research project, preparation of the schedule, correction of the manuscript and approval of the final version of the manuscript.