This report presents data on the acoustic environments in which older adults with age-related hearing loss wear their hearing aids.This is an observational study providing descriptive data from 2 primary datasets: (a) 128 older adults wearing hearing aids for an average of 6 weeks and (b) 65 older adults wearing hearing aids for an average of 13 months. Acoustic environments were automatically and continuously classified about every 4 s, using the hearing aids' signal processing, into 1 of 7 acoustic environment categories.For both groups, older adults wore their hearing aids about 60% of the time in quiet or speech-only conditions. The automatic classification of sound environments was shown to be reliable over relatively short (6-week) and long (13-month) durations. Moreover, the results were shown to have some validity in that the obtained acoustic environment profiles matched a self-reported measure of social activity administered prior to hearing aid usage. For a subset of 56 older adults with data from both the 6-week and 13-month wear times, the daily amount of hearing aid usage diminished but the profile of sound environments frequented by the wearers remained stable.Examination of the results from the automatic classification of sound environments by the hearing aids of older adults provides reliable and valid environment classifications. The present data indicate that most such wearers choose generally favorable acoustic environments for hearing aid use. According to the U.S. Census Bureau, 43.1 million Americans were aged 65 years and over in 2012, and by 2050, this population is expected to nearly double in size to 83.7 million . This reHearing loss is the third most prevalent chronic health problem experienced by Americans aged 65 years and over . VariousThe everyday benefits provided by hearing aids to those with ARHL will depend critically on the acoustical environments in which the older adult is immersed daily. Both self-report outcomes and objective measures of speech recognition demonstrate greater hearing aid benefit in quieter environments e.g., . Thus, wThere has been a long-standing interest in better establishing the typical acoustical environments experienced by older adults, including those with ARHL. M = 74 years) with moderate hearing loss who were experienced hearing aid users. For the 606 diary entries obtained, 26% were for “speech in quiet,” and an additional 15% were for “mostly quiet,” whereas 24% were for “speech in noise.” Using an approach somewhat similar to Rather than have the participants record key acoustical features of daily experiences, N = 27) all had mild–moderate bilaterally symmetrical sensorineural hearing loss and a mean age of 66 years, with 20 of the 27 being hearing aid users. Across the 1,267 entries covering 2,032 hr of dosimeter recordings, 61.2% involved speech, including conversations and listening to media, such as television. The three most common situation/environment combinations noted by the participants were little or no speech listening at home (24%), speech listening via media at home (21%), and small group conversation at home (10%). A subgroup of adults older than 65 years (n = 14) tended to experience quieter listening situations and was less active socially than a subgroup younger than 65 years (n = 13) with comparable hearing loss. The differences in acoustic environments between these two subgroups of older adults, however, tended to be small.One of the limitations of the acoustical data from Although the study by Many contemporary hearing aids include sophisticated sound environment classification systems making use of the digital signal processing resources available. It has long been thought that such sound environment classification systems could be used to automatically guide the application of various signal-processing algorithms in the hearing aid, including noise reduction and directional systems e.g., . An advaMdn = 13 months) representing more than 240,000 hr of acoustic input classified. Not only are these data much more extensive than those published in prior studies but they are also exclusively from new hearing aid wearers. In this approach, complementary to those pursued previously as reviewed above, we obtain continuous analysis of the acoustic environment experienced by older adults with ARHL wearing hearing aids over extended periods of time, but the device is automatically classifying the acoustic environment rather than the participant doing so periodically with pencil and paper or via smartphone (as in EMA).There were two study samples from which the environmental sound classification data were obtained. The larger group provided such data over a shorter period, on average for about 6 weeks of hearing aid usage, and was composed of 128 adults, 67 males and 61 females, having a mean age of 69.4 years (ranging from 54 to 80 years). The median education level was a master's degree, and all 128 participants had at least completed high school. About two thirds (68%) of the participants were retired at the time of study enrollment. All participants were from southcentral Indiana. The median audiograms for the left and right ears are provided in Mdn = 13 months) after the original hearing aid fitting. This subgroup was composed of 37 males and 28 females. The educational level and retirement status of this subgroup were identical to those of the larger group described above. Additional demographic information for this subgroup also appears in The second study sample was composed of 65 older adults, 56 of whom were also among the 128 participants described above. These 65 participants had agreed to return for a follow-up visit from 1 to 3 years  is not logical, and this combination is omitted. In this report, we have designated levels < 75 dB SPL as “moderate” and those ≥ 75 dB SPL as “high.” In the end, each of the seven acoustic environment classifications is assigned a probability, and the set of seven probabilities adds to 1. As an example, a high probability of speech from the speech detector and a low probability of noise from the noise detector, together with a low probability of the level exceeding 75 dB SPL from the level detector, would indicate that this environment likely was composed of “moderate level speech only” with the other six possible environments all having relatively low probabilities. In this way, the environment classification with the highest probability is logged automatically into the hearing aid's datalogger with updates to the classification about every 4 s. In the datalogger, the values logged are in cumulative time (hours) for each of the seven acoustic environments. For example, given updates every 4 s, 15 classifications of “moderate level speech only” would amount to 1 min in that environment, and 900 such classifications would register as 1 hr in that environment. These logs are generated separately for each of the four programs to which the devices could be set  and left (Mdn = 265 hr) ears and average daily usage of about 6.5 hr/day (t(127) = −0.47, p = .64, and the correlation was strong .The distribution of total hours of hearing aid usage, summed across right and left ears, is shown in 5 hr/day , the medr = .85). For the environmental classifications, data from the clinical trial of r < .70) between right and left device environmental classification proportions were all for those who had low usage during the 6-week trial. Based on these correlations between the classification proportions made by the right and left devices in 60 device pairs, about half of the sample, the results for the sample of 128 wearers are considered reliable.The reliability of the datalogging can be assessed by correlating the results for the devices worn on the right and left ears. The devices are independent, but the environments in which they are worn are not, at least in most typical situations. That is, in most cases, the acoustic environment of the left device should be very similar, if not identical, to that of the right device. Thus, if the hours and the classifications are reliable, we would expect strong correlations between the results of the right and left ears. As noted above, this was the case for the total hours of usage for right and left devices .Given that the data are reliable and we have such classification data from 128 wearers, we wondered whether there were individual differences in the patterns of acoustical environments in which hearing aid wearers wore their devices, and if so, what variables or factors might predict those different usage patterns. To examine differences in patterns among the 128 wearers, we made use of a two-step cluster analysis with a log-likelihood distance clustering metric, a Schwarz Bayesian criterion for clustering, and automatic identification of clusters (up to 15). The seven input variables into this cluster analysis were the proportions of usage in each of the seven acoustical environments. Three clusters were identified, and the silhouette measure of cohesion and separation for the three clusters was 0.4, considered to be Quiet (black bars), Speech (red bars), and Noise (green bars) groups. The Quiet group, representing about 35% of the wearers, spent almost half of their time in environments classified as quiet, close to twice the proportion of time spent in such environments as either of the other two groups. The Speech group, representing 41% of the wearers, had the highest proportions of usage in environments involving speech only, both moderate and loud speech. Finally, the Noise group (24% of wearers) had the highest proportions of usage in each of the four environments involving noise. Seven univariate analyses of variance were performed, one for each of the seven sound environments, with the classified cluster membership serving as the sole independent variable and the proportion of time in that environment as the dependent variable. This was followed by three independent-samples pairwise t tests, with Bonferroni corrections, to examine group effects in more detail. All seven analyses of variance were significant, smallest F = 24.831, p < .001, all post hoc pairwise t tests were significant (p < .05), except the Quiet versus Speech group differences for speech + noise high, noise moderate, and noise high environments. In these three environments, all of which included noise, the Noise group spent significantly greater proportions of time in such environments than the other two groups. This pattern of usage across environments led to the designation of these 31 wearers as the “Noise group.”F = 2.09, p = .13. In similar fashion, a series of Pearson chi-square analyses were conducted for a variety of categorical data from case histories for the three groups. The variables in these analyses were gender, highest level of education completed, total household income, living arrangements at home, frequency of social activities, and frequency of attending outside events. Here, one group comparison emerged as significant, χ2(4) = 11.5, p < .05: the frequency of attending social activities. Social activities on the case history form were defined as “visiting with a friend or in a group, entertaining in your home, eating in a restaurant, playing cards, etc.” There were three response alternatives for this item: “regularly participate…,” “occasionally participate…(weekly),” and “rarely participate…(monthly or less).” About 49% of the Quiet group participated in such activities regularly, whereas a strong majority of the other two groups did so (75% of the Speech group and 81% of the Noise group). Importantly, the case histories were obtained prior to being fit for the first time with the hearing aids in this study. Thus, the acoustic environment profile that emerged for each group  hearing loss in both ears, duration of hearing loss in years, years retired, score on a dementia screen, the Mini-Mental State Examination , unaidedroup see  for aideF = 4.89, p < .01, with post hoc pairwise comparisons revealing that the Noise group had significantly less  daily usage than both the Quiet (M = 7.7 h) and Speech (M = 7.2 h) groups. Those who wore their hearing aids in noisier conditions tended to wear their hearing aids less on a daily basis.We also examined the influence of sound environment classification pattern on overall usage of hearing aids. The mean daily usage  for the 128 wearers was 6.9 hr. The effect of environmental classification pattern on daily usage was significant, r = .98 (p < .001) supporting the reliability of these data and their pooling as total hours logged. The total hours logged for the entire group over time is 242,272 hr with that approximately evenly divided between the right  and left  devices. In terms of calendar months lapsed for the period of usage examined here, the median value was 13.3 months with a range of 4.5 to 39.4 months. For the participant with 20,112 total hours, about 10,000 per device, represented at the far right in t tests, t(55), were calculated. Using a Bonferroni-corrected significance level of p = .007 (or 0.05÷7), only the two environments with speech and noise changed significantly, although the proportion of time in quiet approached significance (p = .009). Over time, it appears that these 56 wearers increased the proportion of time spent in moderate and high levels of speech-and-noise, with this increase largely offset by less time spent in quiet conditions. Overall, however, the picture that emerges from the data in r correlations for the proportions of time spent in each environment over each period of usage. Only one of the seven correlations was weak (r = .26) and nonsignificant (p = .06). The other six were between 0.45 and 0.71 and statistically significant (p < .001). Thus, on both a group and individual basis, the acoustical environments in which older adults with ARHL wear their hearing aids appear to be quite stable through the first year of usage.To examine the possible changes in listening environments over time by older adults with ARHL who wear hearing aids, we selected the 56 participants who had provided datalogging information for both the 6-week and 13-month periods. M) of 7.0 hr per day (SD = 3.7 hr per day). A paired t test showed that the decline to a mean of 4.4 hr per day (SD = 3.7 hr per day) over the first year or so of usage was statistically significant, t(64) = −5.8, p < .001. The Pearson correlation between the average usage over the first 6 weeks and the subsequent year was r = .54 (p < .001), indicating that the general trend for the 65 participants was for daily usage to decrease over time. Significant declines over a year of hearing aid usage have been observed previously in older adults with ARHL  or high (≥ 75 dB SPL) levels, were presented from tens of thousands of hours of usage by older adults with ARHL wearing hearing aids for the first time. The general picture that emerged was that hearing aids were worn in favorable listening conditions most of the time by these older adults. The measurements appeared to be reliable in that two independent devices, one on each ear, provided highly correlated datalogging results. Moreover, three distinct patterns of exposure to acoustic environments emerged, with participants following each pattern labeled here as the


The effect size was 1.5 for the EI total score. The 6-week resilience training program was very effective at improving emotional intelligence and could be readily used to help students with hearing loss improve their emotional abilities.Adolescents with hearing loss have been shown to have some emotional difficulties. This study investigated the effectiveness of a resilience training program on the emotional intelligence of mainstreamed adolescent students with hearing loss. In this experimental study, a pre-test, post-test, follow up and control group design was implemented. After receiving informed consents, 122 students with hearing loss in mainstreaming settings were randomly assigned to intervention and control groups . The interventional group received training in groups of 3 to 5, for 6 weeks (two times per week for 75 min). The intervention focused on feelings, thinking  and outcomes of negative thinking, coping strategies, strengths and weakness, problem solving, communication skills, social skills, negotiation, despising and ridiculing, intra- and interpersonal skills. The Connor-Davidson resilience scale (CD-RISC) and the Schutte Emotional Intelligence Scale were used to measure the resilience and the emotional intelligence (EI) of participants respectively just before intervention, as well as at the 6- and 14-week marks. The Friedman Test was used to compare changes in emotional intelligence between interventional and control groups. The intervention increased the resilience scores by 20 points. Although the EI of both groups were similar at the beginning of the research, there was a significant difference between the control and interventional groups in emotional intelligence and its aspects after intervention, at the 6-week and 14-week measurements ( Emotional Intelligence (EI), consisting of a group of related mental and cognitive abilities, offers solutions to education, work and health problems. It plays a major role in developing an individual’s personal and social capabilities. Mayer and Salovy described emotional intelligence as the ability to perceive, integrate, understand, and regulate emotions to promote personal growth . EmotionThere has been a growing interest in EI among adolescents over recent years. Adolescents may face adjustment difficulties and have potential for alienation . This agThe emotional abilities of individuals with hearing loss are influenced by their unique developmental path, and they usually experience more socio-emotional risks than their hearing peers . HearingResilience has been described in various disciplines, mostly in psychology, psychiatry, education, and pediatrics. Based on Developmental Systems Theory, resilience is determined as the capacity of a system to adapt successfully to challenges that intimidate the function, durability, and development of the system ,12,13. FResilience and emotional intelligence influence each other and are tools for defeating stressful and difficult situations. People with higher emotional intelligence show less distress in stressful situations ,16. ResiThis was an experimental study with a pre-test, post-test, follow up and control group design.298 students were originally introduced by the Education Ministry to be considered for participation in the study, all in 6th to 9th grades. Of the 298 students, 34 were not included because the schools, parents or students themselves did not agree to participation in the study. Of the 264 who agreed to participate, 125 were randomly selected to include in the study. After obtaining informed parental and student consent, the participants were randomly assigned to experimental and control groups. This is presented in The ethics committee of the University of Social Welfare and Rehabilitation Sciences approved this study .The Connor-Davidson resilience scale (CD-RISC)  used in The Schutte Emotional Intelligence Scale  used in A brief questionnaire was used to gather demographic data on age, gender, grade, severity of hearing loss and using hearing assistive devices.Pre-test measurements were administered just before the intervention, the post-tests were completed at the end of the intervention—that is, 6 weeks later—and finally, the follow-up tests were completed 8 weeks after the post-test.The interventional program emphasized promoting the developmental integration of emotions, feelings, as well as cognitive and behavioral skills. Due to the spread of students across multiple schools in the intervention group, cohorts of 3–5 students were created based on school proximity and were provided with training.The lessons in the program were about: feelings, thinking , outcomes of negative thinking, coping strategies, strengths and weakness, problem solving, communication skills, social skills, negotiation, despising and ridiculing, and intra- and interpersonal skills. A review of resilience programs in the literature helped us to develop and adapt the program. Input included the Penn Resiliency Program (PRP) , ResourcThe lessons were delivered by experienced itinerant teachers. They had several hours of training by the researcher. Students in groups of 3, 4, and 5 (intervention group) received the instructions in 12 sessions of 75 minutes over 6 weeks. In each session, the lessons followed a common format: an introduction from the teacher (in which the lesson topic and objectives were stated), group activities , individual activities such as drawing, and finally a brief plenary/closure (in which learned points were reviewed).The Klomogorov-Smirnove Test was used to examine the normality of EI scores’ distribution. The Non-parametric Friedman Test was used to compare the changes taking place between three stages of measurements. The Cohen’s d effect size was calculated and classified as small (0.2–0.49), medium (0.5–0.79), or large (0.8 or more) .The participants consisted of 122 students with hearing loss; the M/F ratio was 3/2 (74 boys and 48 girls). The age range was 12–15, and the mean and standard deviation for the age of participants was 13.65 ± 1.00. There were no significant differences between mean age and sex ratio of intervention and control groups. Of all participants, 78 (51.6%) used hearing aids in one ear, 44 (36.1%) in both ears, and 8 (6.6%) students used cochlear implants. With using hearing aid, the corrected hearing thresholds in the better ear of 83 (68%) participants were within the mild, 24 (19.7%) in the moderate, and 15 (12.3%) in the severe range of hearing loss. The cause of hearing loss for 106 (86.89%) students was congenital and for 16 (13.11%) was acquired. Two students had a parent with hearing loss (1.6%).The resilience intervention increased the resilience scores by 20 points in a score range of 0–100.As The follow-up emotional intelligence scores in the intervention and control groups are demonstrated in As This study investigated the effectiveness of a resiliency intervention program on the emotional intelligence of adolescent students with hearing loss. The results showed that the resilience training program enhanced the emotional intelligence of the students. There are reports of the success of such programs in promoting emotional intelligence in adolescents with and without hearing loss. The effectiveness of the program (PATHS) was examined for students with hearing loss. The results showed significant progress in emotional recognition skills, social problem-solving skills, and teacher- and parent-rated social competence .The findings of this study indicated that the effect sizes were large for EI, Emotion Regulation, and Emotion Appraisal and Expression and medium for Emotion Utilization after intervention. There are studies on emotion focused interventions with different results. Claro et al. studied the effectiveness of ‘Cognitive Emotion Regulation’, a school intervention for high school students, aiming at emotional regulation skills . The proAs the results indicate, for the intervention group, the scores for EI and its domains  increased from pre intervention to post intervention. Scores for the Emotion Appraisal and Expression domain increased more than other domains of emotional intelligence. It is believed that children with hearing loss can be taught to identify and express emotions through training  ,36, and Our results show a slight drop in follow-ups of Emotion Utilization scores for both groups. This could be related to the timing of the follow-up test, which was close to the midterm school exams. Therefore, in that stressful time, they might experience some difficulties in the processing of emotional information. It is known that poor emotion information processing deforms emotion knowledge and degrades emotion utilization .In this study, parents were informed about the aim and the activities done in each session by sending them an information sheet, and they were asked to follow them at home, but no assessments were scheduled for parental competence in following instructions. Further research could consider some structured practices and assessments in this regard, or the management of training sessions for parents along with adolescents.Emotional intelligence is one of the top topics among mental health professionals for adolescents. Since one of the causes of mental health problems is the inability to manage emotional states, some research in this area is addressing the effects of EI interventional programs ,39. SchoA limitation of the study was the use of self-reporting to measure emotional intelligence, and this could have affected the results. Future studies could be done using assessment instruments completed by parents/teachers and/or a professional observer. In this study, only students in mainstream schools were included, so the findings may not be generalizable to adolescents with hearing loss who study in special schools. Hopefully, future research will address this issue.This study offers valuable information regarding the benefits of providing programs that target resiliency for emotional intelligence skills in adolescents with hearing loss. Children with hearing loss are at risk of emotional problems. Hopefully, the interventional programs in schools such as this program which includes interesting activities and practices adapted to the adolescents with hearing loss, would promote their emotional intelligence and resilience. These kinds of interventions might also stimulate the teacher’s sense for what excites students and captures their attention and curiosity for learning.


Mycoplasma hyopneumoniae, the agent of porcine enzootic pneumonia (EP), is able to persist in the lung tissue and evade destruction by the host for several weeks. To understand the mechanism of pathogen survival, phagocytic uptake of M. hyopneumoniae by primary porcine alveolar macrophages was investigated. Intracellular location and survival of the pathogen were explored using gentamicin survival assays, flow cytometry and confocal microscopy of M. hyopneumoniae 232 labelled with green fluorescent protein (GFP). Following 1 h and 16 h of co-incubation, few viable M. hyopneumoniae were recovered from inside macrophages. Flow cytometric analysis of macrophages incubated with M. hyopneumoniae expressing GFP indicated that the mycoplasmas became associated with macrophages, but were shown to be extracellular when actin-dependent phagocytosis was blocked with cytochalasin D. Confocal microscopy detected GFP-labelled M. hyopneumoniae inside macrophages and the numbers increased modestly with time of incubation. Neither the addition of porcine serum complement or convalescent serum from EP-recovered pigs was able to enhance engulfment of M. hyopneumoniae. This investigation suggests that M. hyopneumoniae evades significant uptake by porcine alveolar macrophages and this may be a mechanism of immune escape by M. hyopneumoniae in the porcine respiratory tract. Mycoplasma hyopneumoniae is considered to be the primary agent of porcine enzootic pneumonia  at 37 °C with agitation at 210 rpm, and on LB agar [LB broth supplemented with 1.5% (w/v) bacteriological agar no. 1 (Oxoid)].M. hyopneumoniae, Actinobacillus pleuropneumoniae or porcine reproductive and respiratory syndrome virus. The lavage fluid was centrifuged at 200 × g for 10 min at room temperature. The cell pellet was resuspended in RPMI 1640 1 × medium , supplemented with 10% heat-treated foetal bovine serum (FBS) (GIBCO). Viability counts were performed by trypan blue exclusion. If the number of viable cells was ≥ 90%, the cells were frozen on the day of procurement in FBS supplemented with 10% dimethyl sulfoxide (DMSO). These cells were identified as macrophages based on cellular morphology under the microscope and positive staining with anti-porcine CD-163 mouse antibody [Two conventional large white specific-pathogen free pigs were sourced from a herd which did not demonstrate viral-, enzootic pneumonia-, nor pleuropneumonia-like lung pathology at abattoir surveillance. The animals were euthanised by sedation with a ketamine and xylazine mixture, followed by overdose of sodium pentobarbital via ear vein injection. Primary alveolar macrophages were collected as done by Cullen, Rycroft . Brieflyantibody . Two bat2 at 37 °C for no more than 32 h.The alveolar macrophages were routinely cultured in RPMI 1640 Glutamax supplemented with HEPES (GIBCO) and 10% heat-treated FBS (referred to as tissue culture medium). The macrophages were maintained in vitro in 24-well plates in a humidified atmosphere of 5% COM. hyopneumoniae strain 232 P97 promoter sequence from pMHC9-1 [gfpmut2, under control of the spiralin promoter sequence of Spiroplasma citri [gfpmut2 was amplified from the pKENmut2 vector [spiralin promoter sequence was digested from pMHGFP2, and replaced with the P97 promoter sequence at restriction sites MluI and BamHI to give rise to plasmid pMHGFP-P97.Polymerase chain reaction (PCR) was performed using Phusion High Fidelity DNA polymerase  according to the manufacturers instructions. DNA oligonucleotides used in PCR amplification reactions are listed in Table  pMHC9-1 , using p pMHC9-1 , containma citri ; gfpmut22 vector  using prE. coli DH5α were transformed with ligation reactions by heat shock transformation. Briefly, ice-cold competent E. coli DH5α (100 μL) were incubated on ice for 30 min with ligation reactions (4 µL). E. coli were heat-shock transformed at 42 °C for 45 s and then cooled on ice for 2 min, followed by recovery in SOC medium (2% (w/v) tryptone (Oxoid), 0.5% (w/v) yeast extract (Oxoid), 0.05% (w/v) NaCl, 12.5 mM KCl, 10 mM MgCl2 and 20 mM glucose) (900 μL) for 1 h at 37 °C and 210 rpm. Transformed E. coli (100 μL) were spread on LB agar supplemented with 50 μg/mL ampicillin and incubated overnight at 37 °C.M. hyopneumoniae strain 232 was transformed with plasmid DNA by electroporation as described previously [M. hyopneumoniae stock culture was grown static for approximately 41 h at 37 °C. The M. hyopneumoniae were washed and incubated on ice for 30 min with 10 μg of plasmid DNA. M. hyopneumoniae were transformed by electroporation at 2.5 kV, 100 Ω and 25 μF. Afterwards, 900 μL ice-cold Friis medium was added and the transformations were incubated on ice for 15 min. Transformed M. hyopneumoniae were allowed to recover for 3 h in static incubation, at 37 °C, and were then cultured on Friis agar supplemented with 0.2 μg/mL tetracycline at 37 °C in 5% CO2 for 7–14 days. Transformed M. hyopneumoniae colonies were transferred to 1 mL Friis medium supplemented with 0.5 μg/mL tetracycline, and incubated at 37 °C until the culture became peach/orange in colour. Liquid cultures at this stage were stored at −70 °C.eviously . Briefly6 in 1 mL) that had been frozen (batch A) were seeded into 24-well cell-culture treated plates  and allowed to adhere in a humidified atmosphere of 5% CO2 at 37 °C, for at least 1 h.Primary porcine alveolar macrophages (1 × 10M. hyopneumoniae culture (20 mL) grown for approximately 32 h was centrifuged, and then briefly washed in sterile PBS. The M. hyopneumoniae cell pellet was resuspended in 10 mL tissue culture medium. E. coli DH5α was grown to 1 × 108 colony forming units (CFU)/mL, and 1 mL was centrifuged and washed in sterile PBS. The E. coli cell pellet was resuspended in 1 mL sterile PBS and then diluted 1 in 100 in tissue culture medium (10 mL).A M. hyopneumoniae or E. coli inoculum, or tissue culture medium. The experiment was performed in duplicate and the intended multiplicity of infection (MOI) of bacteria/macrophage was 1:1. The inoculated PAMs were incubated in a humidified atmosphere of 5% CO2 at 37 °C for 1 h, and then the medium was removed and wells were washed very gently three times with sterile PBS. Tissue culture medium supplemented with or without 400 μg/mL gentamicin [g for 2 min. Pellets were washed once in PBS, and then resuspended in 40 μL PBS and the entire volume was spread onto solid microbiological medium. The culture supernatants of wells without gentamicin were serially diluted 1 in 10 in PBS (1 mL) and 10 μL spots were inoculated onto solid microbiological medium.Medium was removed from PAMs and replaced with either 1 mL of ntamicin , was addM. hyopneumoniae 400 μL of Friis medium was added, and 400 μL of LB broth to wells inoculated with E. coli. The well surfaces were scraped with sterile mini cell scrapers  and the contents were transferred to 2 mL microcentrifuge tubes containing a single tungsten carbide 3 mm bead . The tube contents were homogenised for 2 min at 30 cycles per second in a Retsch MM300 homogeniser . It was previously confirmed that this method of homogenisation did not reduce the viability of M. hyopneumoniae. The homogenised samples that had not been treated with gentamicin, were serially diluted 1 in 10 in PBS (1 mL) and 10 μL spots were spotted onto solid microbiological medium. The entire volume (400 μL) of gentamicin-treated samples was spread onto 9 cm plates. Samples from uninoculated controls were plated to check for contaminating microorganisms.After the culture supernatants had been removed, the wells were gently washed three times with warm sterile PBS. To wells inoculated with 6 in 1 mL) that had been frozen (batch A), were seeded into 24-well cell-culture treated plates and were incubated for at least 1 h.Primary porcine alveolar macrophages  to gate bacterial cells. GFP-transformed bacteria were also diluted 1 in 10 in PBS and analysed. Green fluorescent bacteria were detected by the FL1 detector. To distinguish fluorescent and non-fluorescent bacterial cells, a marker was drawn on the FL1 histogram plot of untransformed bacteria. This marker was then applied to FL1 histogram plots of GFP-transformed bacteria to establish the percentage of fluorescent bacteria.5 in 0.5 mL per reaction) that had been frozen (batch A) were supplemented with or without 20 μg/mL cytochalasin D (CCD) . The macrophages (0.5 mL) were seeded into wells of a 24-well plate and incubated for 15 min at 37 °C prior to the addition of bacteria.Alveolar macrophages (5 × 10M. hyopneumoniae (pMHGFP-P97) culture was prepared by inoculating 0.5 mL stock culture into 200 mL Friis medium. This culture was grown for 122 h until the medium was orange in colour. The M. hyopneumoniae (pMHGFP-P97) culture was centrifuged at 9000 × g for 10 min at room temperature. The mycoplasma pellet was briefly washed in sterile PBS, and then resuspended in 4 mL tissue culture medium such that 200 μL was equal to 25 mL original culture. E. coli (pKENmut2) was grown until optical density600nm (OD600nm) 0.6, centrifuged at 2500 × g for 5 min, washed briefly in PBS and then diluted to 1.3 × 107 CFU/200 μL. The intended MOI of bacteria/PAM was 25:1. The macrophages, with and without CCD, were inoculated with 0.5 mL M. hyopneumoniae (pMHGFP-P97) or E. coli (pKENmut2); this halved the CCD concentration to 10 μg/mL. Some of the wells containing macrophages, with and without CDD, were mock-inoculated with tissue culture medium as the negative controls. The experiment was performed in triplicate. The macrophages were incubated with the bacteria for 1 h, static, in a humidified atmosphere of 5% CO2 at 37 °C. After incubation, the macrophages were gently scrapped from the well surface with mini cell scrappers, and then centrifuged at 400 × g for 5 min at room temperature in microcentrifuge tubes. The supernatants were removed and the macrophage cells pellets were resuspended in 500 μL FACSFlow. The macrophages were analysed immediately on a FACSCalibur flow cytometer (BD) using the FL1 detector. Uninoculated macrophages were used to gate non-fluorescent cells. The flow cytometric data were analysed in FlowJo version 10 software .A 200 mL M. hyopneumoniae were grown until orange (72 h) in colour. The cultures were centrifuged at 9000 × g for 10 min at room temperature, and washed once in 1 mL PBS. M. hyopneumoniae cells were mounted onto glass coverslips as previously described [M. hyopneumoniae cells were resuspended in 100 μL PBS for every 1 mL of original culture. Glass coverslips  were coated with 0.01% poly-l-lysine (w/v) (Sigma Aldrich) and air-dried at room temperature. M. hyopneumoniae suspensions (100 μL) were spotted onto the poly-l-lysine coated coverslips and allowed to adhere for 30 min at room temperature. The coverslips were washed twice in PBS, then M. hyopneumoniae were fixed in 2% formaldehyde in PBS overnight at 4–8 °C. Formaldehyde was removed by three brief washes in PBS. M. hyopneumoniae cells were stained with 1 μg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)  in PBS for 30 min at room temperature in the dark, followed by two brief washes with PBS. The coverslips were air-dried, and then 5 μL of Vectashield  was spotted on the coverslip and a glass slide was placed on top. The edges of the coverslip were sealed with transparent nail varnish .Liquid cultures of GFP-transformed escribed . BrieflyM. hyopneumoniae mounted onto glass coverslips were viewed on a confocal scanning laser Leica SP5 microscope . The samples were viewed by 63× objective with 4× digital zoom. Blue light (405 nm) was used to excite DAPI to visualise nucleic acid, and blue light (488 nm) to excite and visualise GFP. Untransformed M. hyopneumoniae were used as the negative control.GFP-transformed 5 cells per well) into an 8-chamber glass Lab-Tek Chamber Slide System (Nunc). The macrophages were incubated for at least 1 h prior to use.Primary porcine alveolar macrophages that had been frozen (batch B) were seeded (2.5 × 10M. hyopneumoniae (pMHGFP-P97) culture that was orange in colour was centrifuged at 9000 × g for 10 min at room temperature. The mycoplasma pellet was washed briefly in PBS and then resuspended in tissue culture medium (supplemented with 0.5 μg/mL tetracycline hydrochloride) such that 200 μL was equal to 12.5 mL of original culture. When M. hyopneumoniae wild type strain 232 were used, the mycoplasma pellet was suspended in tissue culture medium such that 200 μL was equal to 5 mL of original culture. E. coli (pKENmut2) were grown until OD600nm 0.6. The E. coli were centrifuged at 2500 × g for 5 min at room temperature, and briefly washed in PBS. The E. coli cell pellet was resuspended and diluted in tissue culture medium (supplemented with 50 μg/mL ampicillin) such that 200 μL contained 2.5 × 106 CFU.A M. hyopneumoniae (pMHGFP-P97), or M. hyopneumoniae wild type, or E. coli (pKENmut2). The macrophages and bacteria were incubated for the specified time in a humidified atmosphere of 5% CO2 at 37 °C. After incubation, the culture medium was removed from wells and each well was gently washed with warm PBS. The samples were fixed with 2% paraformaldehyde in PBS (Sigma Aldrich) overnight at 4–8 °C in the dark.The tissue culture medium was removed from macrophages, and replaced with either 200 μL of Following fixation, the wells were washed with PBS twice, and then 100 μL 0.5% Triton-X-100 in PBS was added, and the samples were incubated at room temperature for 15 min. The wells were then washed with PBS three times, and 5% bovine serum albumin in PBS was added and the samples were incubated at 4–8 °C for 30 min. The wells were then washed with PBS twice, and 200 μL of 1 μg/mL DAPI and 5 U/mL phalloidin-CF568 (Biotium) in PBS was added, and the samples were incubated at room temperature for 30 min in the dark. The wells were washed with PBS twice and then 200 μL of 1 μg/mL mouse IgG1 anti-GFP  was applied to each well and incubated at 4–8 °C for 1 h in the dark. After this incubation, the wells were washed with PBS three times, and the chamber and gasket were removed from the slide. The slide was air-dried and 30 μL of Vectashield was spotted onto the slide. A glass coverslip was placed on top and the edges were sealed with transparent nail varnish.The samples were viewed on a confocal scanning laser Leica SP5 microscope. The samples were viewed by 63× objective with 4× digital zoom. Blue light (405 nm) was used to excite DAPI to visualise nucleic acid, blue light (488 nm) to excite and visualise GFP, and yellow light (594 nm) to excite phalloidin-CF568 to visualise macrophage actin. Micrographs were captured at increments of 0.13 μm along the z-axis to produce z-stack images of macrophages. To determine if bacteria were inside macrophages, the z-stack micrographs were viewed in x, y and z-planes simultaneously using the computer software Volocity version 6.3 .M. hyopneumoniae (pMHGFP-P97) and E. coli (pKENmut2) was performed using preimmune porcine serum and convalescent porcine sera A, B and C. The preimmune and convalescent sera A were derived from the same pig at different times; the preimmune serum was collected before M. hyopneumoniae infection and serum A was collected 5 weeks after infection with M. hyopneumoniae strain 232 (supplied by Jessica Beddow). Serum B was derived from a pig infected with M. hyopneumoniae strain 325 (known as 9954 serum), and serum C was derived from a pig infected with M. hyopneumoniae strain 89 (known as 9965 serum) (supplied by Andrew Rycroft). The presence of M. hyopneumoniae-specific antibody in the convalescent sera was confirmed by Western Blot (data not shown). Heat-treated FBS was the negative control serum.Opsonisation of M. hyopneumoniae (pMHGFP-P97) cell pellets, each equivalent to 12.5 mL original culture, were resuspended in 100 μL of tissue culture medium supplemented with 1 μg/mL tetracycline hydrochloride. To separate M. hyopneumoniae (pMHGFP-P97) suspensions (100 μL), 100 μL of the different sera were added and incubated at 37 °C for 15 min. E. coli (pKENmut2) pellets, each equivalent to 2.5 × 106 CFU, were resuspended in 100 μL of tissue culture medium supplemented with 100 μg/mL ampicillin sodium salt. These E. coli (pKENmut2) suspensions were treated the same as M. hyopneumoniae (pMHGFP-P97). Macrophages (batch B) were inoculated with these 200 μL suspensions of bacteria and incubated for 2 h. All other experiment details were identical to the time-dependent experiments.M. hyopneumoniae, cells were prepared by lavage of the lungs from a freshly killed pig known to be free of M. hyopneumoniae and other respiratory pathogens [M. hyopneumoniae 232 or E. coli DH5α for 1 h before addition of gentamicin for 3 h to kill extracellular bacteria. Enumeration of bacteria in the cell supernatants showed complete destruction of the extracellular E. coli. However, a small proportion of M. hyopneumoniae (0.0045%) survived the treatment with gentamicin  was then applied for 6 h. Extended exposure to gentamicin was intended to eliminate all extracellular M. hyopneumoniae. No M. hyopneumoniae were recovered from the culture supernatants following extended exposure to gentamicin. This indicated that all extracellular M. hyopneumoniae had been eliminated  cells fluoresced green, enabling visualization of individual mycoplasma cells  was analysed further analysis by flow cytometry. The effect of culture time on fluorescence intensity was determined. At each time point the fluorescent population was depicted by a broad peak suggesting that cells differed in fluorescence intensity. At 24 h post-inoculation, 17% of the M. hyopneumoniae (pMHGFP-P97) population were fluorescing green, this increased in intensity with incubation to 96 h  was considered sufficient as a label in further investigations.h Figure . The intM. hyopneumoniae 232 (pMHGFP-P97) was used to investigate phagocytic uptake by porcine alveolar macrophages [M. hyopneumoniae (pMHGFP-P97) for 1 h. Green fluorescent E. coli (pKENmut2) was used as the control organism.A flow cytometric phagocytosis assay using GFP-labelled rophages . The assE. coli had significantly higher fluorescence intensity than CCD-treated macrophages , and that phagocytosis was inhibited by CCD. In contrast, the mean fluorescence intensity of macrophages incubated with M. hyopneumoniae (pMHGFP-P97), with and without CCD, were very similar  were bound to CCD-treated macrophages than untreated macrophages. It was therefore concluded that M. hyopneumoniae (pMHGFP-P97) had not been engulfed by primary macrophages.Untreated macrophages incubated with the positive control s Figure . This rer Figure . The fluM. hyopneumoniae (pMHGFP-P97) was utilised in confocal microscopy experiments to look for evidence of uptake by porcine alveolar macrophages. Cells were incubated with M. hyopneumoniae (pMHGFP-P97) for 2, 4 and 6 h, and were examined for green-labelled mycoplasma localised in the cytoplasm. E. coli (pKENmut2) was again used as the positive control organism.E. coli was observed inside macrophages at each time point, demonstrating the macrophages were phagocytic  exhibited numerous externally-bound mycoplasma cells, many of which appeared flattened against the macrophage cell-surface  were also observed inside the macrophages. An average of 2 M. hyopneumoniae (pMHGFP-P97) cells were found inside macrophages after 2 and 4 h of incubation (Table M. hyopneumoniae (pMHGFP-P97) after 6 h of incubation. Untransformed M. hyopneumoniae, visualised by nucleic acid staining, behaved similarly to GFP-labelled M. hyopneumoniae (data not shown).c Figure . MacrophpMHGFP-P9 exhibiteM. hyopneumoniae (pMHGFP-P97) were exposed to three different convalescent porcine sera  or preimmune serum prior to incubation with alveolar macrophages for 2 h. The convalescent sera were sources of complement and specific antibody; preimmune serum provided complement only. Heat-inactivated foetal bovine serum was used as the negative control.To assess whether opsonisation improved engulfment, M. hyopneumoniae (pMHGFP-P97) did not differ between samples containing the different porcine sera or heat-inactivated foetal bovine serum  can be found intracellularly in the porcine kidney epithelial cell line, PK-15.In the present study, gentamicin survival experiments were conducted to ascertain whether viable s et al.  for exam killing , 36. Resd et al.  also useM. hyopneumoniae to inform on phagocytic uptake, an additional approach was taken that did not measure mycoplasma viability. Flow cytometric and confocal microscopy experiments were set up to provide more information about the physical interactions between M. hyopneumoniae and macrophages. To perform these experiments M. hyopneumoniae was transformed with green fluorescent protein using the extrachromosomal plasmid pMHGFP-P97, comprising gfpmut2 controlled by the P97 gene promoter. Two additional plasmids were investigated to express GFP in M. hyopneumoniae; plasmid pMHGFP2, comprising gfpmut2 controlled by the spiralin gene promoter; and pMHGFP-tuf which contained gfpmut2 controlled by the tuf gene promoter of M. hyopneumoniae strain 232. Plasmid pMHGFP-tuf failed to transform M. hyopneumoniae and M. hyopneumoniae (pMHGFP2) did not fluoresce as brightly as M. hyopneumoniae (pMHGFP-P97) (data not shown).Since the gentamicin survival experiments relied on the viability of M. hyopneumoniae was first reported by Ishag et al. [gfp gene under control of the P97 gene promoter sequence, and a tetM gene under control of an additional copy of the P97 gene promoter. In contrast, pMHGFP-P97 produced in this study only possessed one copy of the P97 promoter sequence to control expression of gfpmut2. Hence, pMHGFP-P97 may offer more stability than pMD18-TOgfp. The fluorescence intensity of M. hyopneumoniae (pMHGF-P97) cells was found to be dependent on time, at least 72 h of culture was required for maximal numbers of M. hyopneumoniae (pMHGFP-P97) cells to fluoresce. In addition, flow cytometric analysis of M. hyopneumoniae (pMHGFP-P97) revealed the fluorescence intensity of mycoplasma cells was heterogeneous, as indicated by the broad histogram peak. This may be related to different levels of intracellular GFP in differently aged M. hyopneumoniae (pMHGFP-P97) cells. Neither Ishag et al. [M. hyopneumoniae (pMHGFP-P97) fluorescence, it was still suitable to use in downstream flow cytometry and confocal microscopy experiments.Expression of GFP in g et al.  who usedg et al.  nor inveg et al. –41. DespM. hyopneumoniae (pMHGFP-P97) became associated with macrophages within 1 h of co-incubation. Since the fluorescence intensities of M. hyopneumoniae (pMHGFP-P97)-infected macrophages treated with and without CCD were similar, it was inferred that all M. hyopneumoniae (pMHGFP-P97) were attached to the external surface of macrophages. An unexpected finding was that more M. hyopneumoniae (pMHGFP-P97) appeared bound to CCD-treated macrophages than untreated macrophages. This might be explained by an unanticipated side effect where CCD caused the macrophages to detach from the surface of the culture dish. Hence, while in suspension the CCD-treated macrophages may have encountered M. hyopneumoniae (pMHGFP-P97) more frequently than the adhered untreated macrophages. The confocal microscopy experiments also confirmed M. hyopneumoniae (pMHGFP-P97) adhered to the surface of macrophages, some mycoplasmas even appeared flattened to the macrophage cell surface. In contrast to results from the flow cytometry experiments, analysis of confocal micrographs revealed a small number of M. hyopneumoniae (pMHGFP-P97) were in-fact located in the cytoplasm of porcine alveolar macrophages. The slightly differing results of the confocal microscopy and flow cytometer experiments may have been due to lower sensitivity of the flow cytometer to detect internalisation of such a low number of M. hyopneumoniae (pMHGFP-P97). It is also noted that the gentamicin survival and flow cytometry experiments utilised macrophages originating from the same pig, whereas macrophages from another animal were used in the confocal microscopy experiments . Consequently, variability in the activity of macrophages obtained from different animals should be considered. However, the phagocytic capability of both macrophage batches appeared comparable throughout this investigation, as demonstrated by engulfment of the control organism, E. coli, via three different methods.Results from the flow cytometry experiments showed M. hyopneumoniae by porcine alveolar macrophages was limited and that this mycoplasma may even resist significant uptake. Powell and Clyde [M. pneumoniae also readily associated with the membrane of guinea pig alveolar macrophages, but were rarely found inside macrophages. The addition of hyperimmune rabbit antiserum however led to uptake of M. pneumoniae [M. hyopneumoniae (pMHGFP-P97) were opsonised with porcine serum containing complement, or convalescent serum containing complement and anti-M. hyopneumoniae antibody. M. hyopneumoniae (pMHGFP-P97) were not susceptible to the bactericidal activity of serum complement, unlike the control E. coli (pKENmut2) which was killed by exposure to serum (data not shown). Analysis of confocal micrographs suggested serum complement did not enhance phagocytic uptake of M. hyopneumoniae (pMHGFP-P97), and contrary to expectation neither did the convalescent sera. It was not entirely surprising that a pathogenic organism like M. hyopneumoniae exhibited resistance to the bactericidal activity of complement. Although M. hyopneumoniae appeared resistant to complement killing, deposition of complement onto the surface of M. hyopneumoniae cells was not determined in this study and could not be ruled out.It appeared that uptake of nd Clyde  found theumoniae . In the M. hyopneumoniae convalescent serum had been purposefully excluded from some experiments to mimic the in vivo environment of the naïve host, and provide more insight into the initial interactions between M. hyopneumoniae and alveolar macrophages. It was surprising to find that antibody from three different convalescent sera did not promote engulfment of M. hyopneumoniae. Other investigators have reported swift uptake of mycoplasmas such as M. dispar, M. pulmonis, M. ovipneumoniae, M. pneumoniae and M. bovis by macrophages following antibody opsonisation [M. bovis by monocyte-derived macrophages using confocal microscopy. The fluorescent M. bovis were opsonised with pooled bovine convalescent serum , and within 30 min of co-incubation green fluorescent clumps of M. bovis were observed inside the macrophages. In light of these findings for M. bovis it was expected that convalescent serum would promote uptake of M. hyopneumoniae (pMHGFP-P97), but this was not the case. Although M. hyopneumoniae (pMHGFP-P97) were present inside macrophages, the number of intracellular mycoplasmas did not differ in the presence of naïve or convalescent serum; and intracellular M. hyopneumoniae (pMHGFP-P97) were located discreetly in the macrophage cytoplasm. It remains to be determined the mechanism by which the mycoplasmas entered macrophages. It was assumed that intracellular M. hyopneumoniae resulted from phagocytic uptake, yet a recent publication has offered a potential alternative mechanism. Raymond et al. [M. hyopneumoniae appears to enter PK-15 cells by exploiting clathrin- and caveolae-mediated endocytosis pathways; these pathways are available in porcine alveolar macrophages [M. hyopneumoniae were apparently able to survive fusion of endocytic vesicles with lysosomes and escape into the cell cytoplasm. So far, the authors provide only limited evidence that M. hyopneumoniae might invade respiratory epithelial cells in vivo [M. hyopneumoniae is a strict extracellular pathogen.nisation , 22, 42.nisation  investigd et al.  have sugrophages , 44. The in vivo . However in vivo  do challM. hyopneumoniae (pMHGFP-P97) in the present investigation. Two of the convalescent sera, sera B and C, were raised against heterologous strains of M. hyopneumoniae compared to M. hyopneumoniae (pMHGFP-P97) used in the present study. Hence, it is possible that strain differences prevented efficient opsonisation. However, convalescent serum A was produced following infection with M. hyopneumoniae strain 232, so should have contained antibody recognising surface antigens of M. hyopneumoniae (pMHGFP-P97). It is conceivable that the porcine host did not produce effective opsonising antibody isotypes IgG1 and IgG3 in this instance. This raises questions about the efficacy of the porcine serum response to M. hyopneumoniae infection. Anecdotally, production of antibody following M. hyopneumoniae infection can take many weeks and does not correlate well with resolution of disease. It must be noted that in this investigation the opsonising powers of mucosal antibody isotypes IgA1 and IgA2 were not examined. Experimental evidence has suggested that humoral immunity alone is not enough to overcome M. hyopneumoniae infection, a cell-mediated immune response is also required [M. hyopneumoniae is preventing antibody opsonophagocytosis in some way. An antibody capture and cleavage system, designated MIB-MIP, has been identified in M. mycoides subsp. capri which may function as a virulence mechanism [Mycoplasma spp. which included M. hyopneumoniae. The expression and functionality of a putative M. hyopneumoniae MIB-MIP system requires further investigation to determine whether this potential virulence mechanism aides M. hyopneumoniae in the evasion of humoral effector functions such as opsonophagocytosis.It is not clear why convalescent sera from experimentally-infected pigs failed to promote phagocytosis of required , 45, 46.echanism . The autM. hyopneumoniae is not readily engulfed by primary porcine alveolar macrophages, even in the presence of convalescent porcine serum. To strengthen further this observation of an antiphagocytic property, different strains of M. hyopneumoniae could be evaluated following exposure to a larger panel of convalescent serum, including serum from vaccinated animals; this would also aid analysis of the opsonising power of the humoral response against M. hyopneumoniae. Overall, this investigation has shed more light on the interactions between M. hyopneumoniae and porcine alveolar macrophages, in particular it has identified a possible mechanism of immune evasion by this important pig pathogen.In conclusion, results indicate that


Hematologic diseases are rarely present with sudden hearing loss as an initial symptom. Although the precise cause of sudden sensorineural hearing loss has not been identified, several pathophysiological mechanisms have been proposed. However, a variety of hematologic diseases are among the causes of sudden onset deafness. This article represents the first reported case of Hairy cell leukemia (HCL) which presented with acute unilateral profound sensorineural hearing loss as an inital mainfestation.A 41-year-old man presented with unilateral sudden hearing loss for one day’s duration was found to have HCL during a worked up for his hearing loss. The disease worsened, and there was no improvement in his hearing.Sudden sensorineural hearing loss can rarely be seen as a paraneoplastic occurrence.Very few cases have been reported in the literature. It has been reported as a presenting symptom of leukemia in numerous clinical and histopathological reports concerning the hearing loss in hematologic diseases. Our case is an example of such a rare incidence.The purpose of this study is to draw physicians’ attention to the possible association between acute sensorineural hearing loss to HCL, and to highlights permanent deafness as a complication secondary to HCL. Furthermore, it is important to increase awareness on early diagnosis and treatment that may improve treatment outcomes. HCL, wh2A 41-year-old Egyptian man who moved to the Saudi Arabia approxmately 10 years ago presented with left hearing loss of sudden onset in the left ear for a duration of one day, accompanied by persistent non-pulsatile tinnitus, spinning sensation vertigo lasting for seconds which was aggravated by head movement to the left side, and fullness in the left ear for one day’s duration, along with abdominal pain and nausea. His past medical and surgical history were unremarkable except for bronchial asthma that was well controlled without medication. No history of ototoxic medication, trauma, recent infection, or family history was noted. His examination revealed vitally stable and not on distress, normal external auditory canals with intact tympanic membranes bilaterally. No nystagmus was detected. There was no lymph node enlargement in the neck, supraclavicular fossa, axillary, inguinal, or femoral regions. The spleen was enlarged on percussion. Cranial nerves were intact and motor strength was equal in all four extremities. Weber radiated to the right ear. Rinne + ve right side with no response on the affected side. The Pure tone audiometry revealed good hearing in her right ear but severe to profound sensorineural hearing loss at mid and high frequencies in the left ear , Fig. 2.The patient was transfused with 18 unites of platelets. Radiological evaluation of the temporal bone and the brain, including computer tomography scan and magnetic resonance imaging revealed no particular abnormality, no cerebellopontine tumor or, any other pathology. The patient was treated with chemotherapy consisting of vincristine sulfate, daunorubicin hydrochloride, methotrexate, and prednisolone. Immediate treatment with the a higher dose of prednisone 60 mg taper courses of oral steroid was started. The patient was scheduled for an audiological follow up; however, but he was unable to tolerate the test and no subjective improvement of hearing was noted. However, an Intratympanic injection was not adminstered because the control of the disease was poor, and he subsequently developed severe thrombocytopenia, anal fistula and a rash. These complications aggravated his status, and further testing was difficult to perform.3The precise cause of SSNHL has not been identified; however, several pathophysiological mechanisms have been proposed. The most common neoplastic disease associated with SSNHL is acoustic neuroma (vestibular schwannoma), which usually presents with unilateral tinnitus and progressive sensorineural hearing loss, although it can also present with sudden loss in a few cases . SSNHL iPopulation studies of SSNHL show a wide age distribution, with an average of 50–60 years with no sex preference. Our patient was in his early forties age which makes this case different, due to different reasons such as the presence of leukemia. The evidence of SSNHL in the literature suggests: Vascular and Infectious causes. Furthermore, Studies have investigated several possible mechanisms, including atherosclerosis, hypotension, thrombophilia, vasospasm, hyperviscosity, and paradoxical embolism as risk factor . Many vi4This article represents a rare case in which acute sensorineural hearing loss occurred as an initial manifestation of HCL and not during the clinical course of the disease. To our knowledge, this is the onset sensorineural hearing loss as an initial-symptoms in HCL. The purpose of this study is to increase awareness regarding the development of acute sensorineural hearing secondary to HCL and the importance of early diagnosis and treatment, which may result in an improved hearing outcome. Hematological disorders such as HCL, should be considered to be among the causes of sudden onset deafness, despite the rarity of this presentation. In addition, further studies are also needed on this topic.There are no conflicts of interest.The author(s) received no specific funding for this work.Case reports do not require ethical approval by our institution if no patient identification and case is anonymous.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal upon request.Abdulaziz Alenzi: Study concept, writing the paper.Riyadh Alhedaithy, Study concept and review the paper.Abdulrhman Alfayez Study concept and review the paper.Yazeed Alghonaim Study concept and review the paper.NA.Abdulaziz Saud Alenazi.Not commissioned, externally peer-reviewed.


Noise exposure can lead to hearing loss and multiple system dysfunctions. As various forms of noise exist in our living environments, and our auditory organs are very sensitive to acoustic stimuli, it is a challenge to protect our hearing system in certain noisy environments.Herein, we propose that our hearing organ could serve as a noise eliminator for high intensity noise and enhance acoustic signal processing abilities by increasing the signal-noise ratio. For suprathreshold signals, the hearing system is capable of regulating the middle ear muscles and other structures to actively suppress the sound level to a safe range.To test our hypothesis, both mathematic model analyses and animal model studies are needed. Based on a digital 3D reconstructed model, every structure in the auditory system can be analyzed and tested for its contribution to the process of noise reduction. Products manufactured by this bionic method could be used and verified in animal models and volunteers.By mimicking the noise-reduction effect of the sophisticated structures in the hearing system, we may be able to provide a model that establishes a new active-sound-suppression mode. This innovative method may overcome the limited capabilities of current noise protection options and become a promising possibility for noise prevention. Various types of noise have emerged and increased in our surroundings, e.g., traffic noise, industrial noise, construction noise and loud noise from civilian lives. Noise exposure can lead to not only hearing problem but also multiple system dysfunctions –5. ProteThe traditional method for preventing noise-induced damage is to block noise transmission with walls, sound-absorbent materials or sound-deadening materials. However, the most fundamental method is to decrease the sound energy generated from the sound source. The recent concept of “noise canceling” is based on the generation of a reverse signal by a digital signal processing system , 9. ThisHerein, we are attempting to propose a brand new hypothesis named “bionic active noise reduction” by presenting the innate structures in the auditory system that may contribute to noise suppression. The novelty of our hypothesis is the use of a bionic method to replicate the relevant hearing structures and their noise-reduction functions to achieve better efficiency in noise protection. The application of such active noise-reduction structures may possibly overcome the current limitations in active noise prevention. Once this hypothesis can be verified, it may have significant implications for noise protection in social medical services and national economies.i.Noise reduction in the auditory systemOur acoustic apparatus has evolved into an elaborate organ throughout human history. Within a response range of 20 Hz to 20,000 Hz, it is required to be not only sensitive but also durable to protect itself from injury. Researchers have focused on its precise signal processing ability, but its self-protective noise-reduction mechanism has never been noticed.In normal physiological conditions, the internal noise of our body, such as the sounds of breathing, blood flow, muscle contractions and joint movements, are suppressed by our internal “sound-reduction system”. This system addresses the internal noise made within oneself and prevents it from influencing external sound processing. Vascular murmurs, created by hemodynamic changes in blood flow, could lead to audible pulses, known as pulsatile tinnitus –12.More importantly, for external noise, we think that this innate “sound-reduction system” could actively process external sounds to minimize excessive energy. Sound signals are amplified by the signal amplification system to maintain its sensibility and clarity Furthermore, for suprathreshold signals, the auditory system could actively suppress signals to prevent injury.ii.Features of the active noise-reduction systemNoise-blocking and reducing effect of the Eustachian tube (ET)For patients with hearing loss, both the ability to sense signals and the ability to reduce noise are decreased. Simply amplifying the signal with a hearing aid cannot mimic the denoising effect of the normal system and makes the amplified sound unbearable. The excessive energy has been dissipated via the middle and inner ear in a functioning auditory system, and if it was not, which indicates a loss of the active noise-reduction function, hearing damage would occur. This process prevents disturbances from our internal physical noise and protects our vulnerable hearing system from being damaged by external intense noise.The ET is a key passage between the nasal cavity and the middle ear. The pharyngeal orifice of the ET works as a valve, which when closed prevents internal noise, for example, self-talking and breathing, from being transmitting to the tympanic cavity. The dysfunction of the ET invariably leads to middle ear problems, such as aural fullness, pain, tinnitus and otitis media –15. If tb)Function of middle ear skeletal muscles in the protection from noise-induced injuryThe external 1/3 of the ET, the tympanic section, is an open, funnel-shaped tube covered with mucosal folds . These fThe middle ear muscles, the tensor tympani and stapedius muscles, also have important functions in noise protection. The stapedius reflex is a significant protective reflex when the ear is stimulated by intense sounds. This mechanism reduces the excessive energy that passes into the inner ear. The amplitude of the stapedius reflex is decreased in patients with noise-induced hearing abnormities even when their thresholds are normal at 1000 Hz, which confirms that their noise-reduction system is affected . A reseac)Function of inner-ear-related structures in noise protectionThe oval window, blocked by the stapes footplate and ligament, transfers sound energy from middle ear to the inner ear. The structure of the footplate and ligament ensures the efficient and precise conductive movement of the oval window. It is notable that the surrounding ligament could limit the vibration of the oval window in response to loud sound stimuli within an appropriate range. This prevents hair cell injury caused by excessive basilar membrane movement.The moderate transmission of sound wave energy in the inner ear from liquid (perilymph) to solid (basilar membrane) can largely avoid direct mechanical injury to hair cells. On the other hand, the existence of endolymph and perilymph could facilitate the later attenuation of sound when waves dissipate in the fluid. The vibration of the perilymph causes the round window to vibrate, and energy is buffered and reduced through vibration and deformation.The endolymph and perilymph are connected with the endolymphatic sac and the cochlear aqueduct. We presume that these structures also facilitate the energy dissipation. When hydrolabyrinth and aqueduct obstructions emerge, lymphatic discharge abnormities will cause vestibular dysfunction and hearing loss, such as Meniere’s disease. Large vestibular aqueduct dysfunction also leads to severe hearing loss.The noise-reduction system we proposed involves the mentioned structures in both the middle ear and the inner ear, and it works as a cooperative system to protect the hearing system from intense noise injury. When the system fails to limit the sound signal within an appropriate range, inner ear damage occurs and may eventually develop into noise-induced hearing loss.iii.Progress in research on the pathogenesis and therapy of noise-induced hearing lossPathogenesis of noise-induced hearing loss damageThe reflexes of both the stapedius and tensor tympani are important innate mechanisms that prevent noise damage by effectively decreasing the intense noise rushing into the inner ear.b)Progress in the therapy of noise-induced hearing lossSurgical therapy of noise-induced hearing loss promoted the development of otologyUnderstanding the mechanisms of noise-induced hearing loss is still a challenge in otolaryngology. Nordmann proposed the theory of cilium damage in the year 2000 . The fir2)Breakthrough in gene therapy of noise-induced hearing lossBased on almost 1000 cases of cochlear implants, we performed the first auditory implantation in a noise-induced hearing loss patient, and it restored the patient’s hearing –26. In a3)Studies on stem cells make progressWe found that the expression of the Math1 gene increased hair cell numbers in guinea pigs. It could induce the regeneration of hair cells and improve hearing , 28. TheStudies on stem cells indicate a promising prospect for the treatment of noise-induced hearing loss . We founThrough the understanding and verification of this active system, we are proposing a creative method to mimic the noise-suppression function of our hearing system. To test our hypothesis, model analyses and animal model studies are needed.Using micro-CT scanning and three-dimensional reconstructions, a digital model of our hearing system  can be rebuilt and used for acoustic analysis. Through a preliminary mathematic model analysis, we found that the energy passing from the tympanic membrane to the closed tympanic foramen of the ET is less than that to an open ET (data not shown). This suggests that the opened tympanic foramen of the ET receives part of the acoustic energy and plays a specific role in energy dissipation. Every structure in the auditory system can be analyzed and tested for its particular contribution to the process of noise reduction mentioned above.Then, we could imitate the structure with a bionic method and apply it in the manufacture of protective devices or muffling devices. For example, a bionic ET could be created through 3D printing, and its function could be verified in animal models or volunteers with ET dysfunctions. Once proven, these structures or products could be widely used in both disease treatment and industrial manufacturing. Similar mechanisms to those mentioned above, such as the stapedial reflex, are also candidates. Products designed based on this theory will overcome the limitations of traditional reverse-wave elimination and damping absorbers.The hearing system is a complex neurobiological feedback control system, and researchers have discovered the active cochlear amplifier, which is important in evolution. We think the hearing organ has equipped itself with a self-protection mechanism, which we summarize as a function of active noise reduction. We should realize that under different frequency or intensity conditions, different effects may be present in the same structure.Bionic structures manufactured based on this theory would have a noise reduction function and could be used in the treatment of patients with noise reduction problems, for which there is no current medical treatment. For example, 3D–printed ETs could be implanted to improve dysfunctions in the original ETs.On the other hand, it is very important especially for the military industry and military noise prevention. The application of this type of bionic auditory structure in weaponry or military equipment will greatly decrease military noise-induced damages.These promotions may lead to favorable directions for the prevention and treatment of hearing loss and facilitate the development of the military industry and social health.


Congenital hearing loss affects approximately 1–2 infants out of every 1000, with 50% of the cases resulting from genetic factors. Targeted gene panels have been widely used for genetic diagnosis of hearing loss. This study aims to reveal new diagnoses via reanalyzing historical data of a multigene panel, and exam the reasons for new diagnoses.A total of 210 samples were enlisted, including clinical reports and sequencing data of patients with congenital/prelingual hearing loss who were referred to clinical genetic testing from October 2014 to June 2017. All variants listed on the original clinical reports were reinterpreted according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). Expanded analysis of raw data were performed in undiagnosed cases.Re-analysis resulted in nine new diagnoses, improving the overall diagnostic rate from 39 to 43%. New diagnoses were attributed to newly published clinical evidence in the literature, adoption of new interpretation guidelines and expanded analysis range.This work demonstrates benefits of reanalysis of targeted gene panel data, indicating that periodical reanalysis should be performed in clinical practice.The online version of this article (10.1186/s12920-019-0531-6) contains supplementary material, which is available to authorized users. GJB2 in hearing loss), resulting in a low detection rate [Congenital hearing loss affects approximately 1–2 infants out of every 1000, with 50% of the cases resulting from genetic factors [ion rate ; well-esion rate  makes seWith the advance of next generation sequencing (NGS) techniques, targeted genomic capture and massively parallel sequencing has become an important diagnostic tool for hereditary hearing loss . This meIt was recently reported that reanalysis of genome-wide NGS data with updated knowledge can improve the diagnostic rate , 8. For In this study, we recruited patients with congenital/prelingual hearing loss. We sought to reveal new diagnoses via reanalyzing their targeted gene panel data, and exam the reasons for new diagnoses.A total of 210 patients with congenital or prelingual hearing loss, defined as detection before three years of age, were retrospectively studied. They had been referred to clinical genetic testing from October 2014 to June 2017 and consented to anonymous use of their data for scientific research. Healthy relatives of the patients were not included. The Institutional Review Board of the BGI approved this study.After a clinical diagnosis of hearing loss, peripheral blood samples were submitted for testing with commercial exome sequencing panels, either HearingCare_127 or HearingCare_81. The two tests use the same gene panel but involve analysis of a different number of genes, with 127 genes analyzed in HearingCare_127 and 81 in HearingCare_81. The gene list is presented in Additional file GJB2):c.109G > A. Then, the functional consequences of the remaining variants were predicted by Condel [Variant filtering and prioritization were first based on population databases (the Exome Aggregation Consortium (ExAC), the Genome Aggregation Database (gnomAD), Exome Sequencing Project v. 6500 (ESP6500), 1000 Genomes and local databases). Variants at a minor allele frequency of > 1% in either one of the databases were excluded except for hotspot variants, such as NM_004004.5.For categorical data, summary data were reported as frequencies and percentages, and chi-square tests were used for between-group comparisons. A Out of 210 patients, 49% were male. A total of 52 patients (25%) have a self-reported family history of hearing loss. The majority of cases had been tested with HearingCare_127 , while the remainder used HearingCare_81  (Table p = 0.128). The diagnostic rate was not significantly different in patients with or without a family history  or in patients from different calendar years (p = 0.220). In diagnosed patients, GJB2 and SLC26A4 were the most significant contributors, present in 45 and 35% of cases, respectively. As expected, autosomal recessive inheritance was the most common inheritance pattern in congenital/prelingual hearing loss patients . The diagnostic yields of HearingCare_127 and HearingCare_81 were 41 and 28%, respectively :c.2858G > T) from likely pathogenic to likely benign .In addition, one initial diagnosed case was reclassified to undiagnosed due to downgrade of a X-linked dominant variant :c.10245_10247delCTC resulted in two probands getting diagnosed (patient 1 and 2). This variant was first reported in 2015 as a compound heterozygote with MYO15A c.8198A > C [Variants that were reclassified from VUS to likely pathogenic in patients 1–5. The upgrade of NM_016239.3:c.1021C > T (p.Arg341Cys) was discovered from original file of an unreported VUS in patient 5, who presented only congenital profound hearing loss when they were referred for genetic testing.In patients 4 and 5, two missense amino acid changes occurred in ril 2016 . The recril 2016 , and in ril 2016 , furtherTMC1 p.Gly417Glu and CDH23 p.Pro346Arg were detected in patient 6 and patient 7, respectively. Pathogenic missense changes at the positions of these two variants have been established before [Two patients were reclassified due to the application of standards and guidelines for the interpretation of sequence variants recommend by ACMG/AMP . Specifid before , 26, proPAX3):c.870_871insC, was discovered and is known to be the genetic cause of Waardenburg Syndrome [Patient 8 was clinically diagnosed with nonsyndromic hearing loss, and their sample was tested using HearingCare_81 panel. The number of genes analyzed was increased to 127 during reanalysis. An autosomal dominant frameshift variant, NM_001127366.2:c.10245_10247delCTC, from VUS to pathogenic in light of evidence that it cosegregates with the disease [Consistent with previous publications, 9-2A > G , 32. In  disease . This va disease . TogetheMITF):c.1021C > G in patient 6, whereas it was reported as NM_000248.3(MITF):c.718C > G [Many challenges remain for variant interpretation in practice. First, transcript discrepancy can lead to inaccurate variant interpretation . For exa718C > G , creatin718C > G . Recentl718C > G , offerinGJB2):c.109G > A was detected and reported in a heterozygous state. Our expanded analysis revealed a frameshift autosomal dominant variant, NM_001127366.2(PAX3):c.870_871insC, in the original unfiltered files. Since PAX3 is linked with Waardenburg syndrome with variable clinical features [Second, genetic heterogeneity  is notable in inherited diseases, especially hearing loss, making it harder to identify causal variants. Allelic heterogeneity sometimes leads to heterogeneity in the clinical phenotype. In our study cohort, patient 8 presented with congenital nonsyndromic hearing loss, leading to the use of HearingCare_81 panel, which mainly targets nonsyndromic hearing loss genes. As a result, the Chinese hotspot variant NM_004004.5 . SimilarAlthough we did not recontact the patients for the updated results yet, we noted that a number of genetic centers are recontacting patients occasionally or periodically for modified results , which mIn conclusion, this work demonstrates benefits of reanalysis of targeted gene panel data from congenital/prelingual hearing loss patients. A total of 9 previously undiagnosed case obtained diagnosis, improving the overall diagnostic rate from 39 to 43%. New diagnoses are attributed to newly published clinical evidence in the literature, adoption of new interpretation guidelines and expanded analysis range. In spite of the fixed design of targeted gene panels, reanalysis of such data is still beneficial due to the improvements in variant interpretation. We propose that periodical reanalysis should be performed in clinical practice.Additional file 1:Table S1. Gene list of diagnostic hearing loss panel (HearingCare). (DOCX 42 kb)Additional file 2:Table S2. Molecular characteristics of diagnosed patients. (DOCX 26 kb)


Dear Editor,1 The prevalence of ASD has rapidly increased worldwide, rising from 1/149 in 2000, 1/68 in 2012, and to ~1/40 in 2016 of children at the age of 3–17 in the United States.2 ASD prevalence was found to be associated with socioeconomic status (SES) as overpresented in high-income households,3 and to be significantly enriched in certain regions, e.g., reaching up to 4.88% in Florida.2 In the last decades, advances in genetic studies have led to unprecedented understanding of the genetic factors that were associated with < 50% of human ASD. Tens of thousands of genomic events or gene mutations were found in ASD patients. Among these genetic factors, many seemed to indeed play causal roles in ASD etiology as animals bearing some of ASD-associated mutations manifested autistic-like behaviors.7 Since genetic factors are usually SES independent and unlikely to change in every few years, non-genetic factors including changes in life styles, environmental factors and their complex interaction with genetic factors are believed to prominently contribute to the recent rapidly increasing ASD prevalence.6 However, their nature and mechanisms of action remain largely undetermined.Autism spectrum disorders (ASD) are complex neurodevelopmental disorders characteristic of core behavioral traits like restricted, repetitive behaviors and deficiency in social interactions.8 TCS is also widely employed as preservative in food or products such as kitchen utensils and furniture, which could be particularly important to prevent the proliferation of microbes in tropical and stormy states like Florida. TCS goes into water and soils, and significantly accumulates within food chains. By penetrating human skin or being swallowed through mouth and digestive duct, TCS can enter human body efficiently and circulate in body fluids, remain in plasma and breast milk, and reach up to ~3000 µg/L in urines of high-income individuals.9 Therefore, the bioaccumulation of TCS exhibits a SES-dependent pattern, similar to that of ASD.3 Despite multiple alerting safety concerns,9 the rising demand for better hygiene is to drive fast expansion of TCS consumption worldwide.Triclosan phenol: TCS, Fig. 10 This has established a potentially causal role of down-regulated RA signaling in ASD etiology. Later, fragile X mutations were also found to undermine RA signaling,11 and some ASD patients bore mutations in RA receptor A (RARA), which disrupted downstream RA signaling.12Retinoic acid (RA) is both a morphogen and signaling molecule with prominent roles in mediating neuron differentiation, synaptic plasticity and tissue formation. Recently, we reported that ASD-like behaviors could be induced in mice upon downregulation of cellular RA synthesis, through mimicking ASD-associated UBE3A overdosage or chemical inhibition of RA synthesis with disulfiram, and such ASD behaviors could be efficiently reversed by oral supplementation of RA into the male mice.13 As shown in Fig. Renilla reniformis green fluorescent protein (hrGFP) driven by RA-responsive element (RARE), which could be efficiently blocked by addition of TCS  and the NMDA receptor antagonist D(−)-2-Amino-5-phosphonopentanoic acid  in neurons.14 Wild-type pregnant rats were then subjected to oral gavage with 50.0 mg/kg/d TCS (dissolved in corn oil) from day 7.5 of pregnancy (E7.5) until weaning , and the pups of both genders were then subjected to behavior tests 7–8 weeks after birth  and Rarb (retinoic acid receptor beta), both target genes and mediators of RA signaling, was significantly lower in the rat brains of the TCS group in comparison to that of the control group, suggesting that maternal exposure to TCS disrupted at least part of the neuronal RA signaling in the offspring brains. This result also indicated the impairment of RA signaling in PFC after exposure to TCS. Taken together, these data suggest that maternal exposure to TCS might cause autistic-like behaviors in the offspring, most likely through disrupting the expression of the target genes of RA signaling in brain.Previously, the oral ‘No Observed Adverse Effect Level’ (NOAEL) of TCS for rat was found to be 50 mg/kg/d, as TCS at this dose seemed to have no or little adverse effects on the health of dams and pups.rth Fig. . No signrth Fig. . Comparerth Fig. . The offrth Fig. . The timrth Fig. . Furtherrth Fig. , the expSince ASD showed a male bias in prevalence with a ratio of 4:1  in humans, it was intriguing to ask whether there exists gender-specific behavioral traits in the offspring maternally exposed to TCS. The data from the above behavior tests were re-analyzed for gender specificity. It was evident that the maternal exposure to TCS did not cause gender-specific behavioral traits in the offspring rats  highlights potential avenues and mechanisms through which exposure to certain environmental factors during early development of an individual may cause an expanding set of human diseases.9 Our findings presented above have, unexpectedly, revealed a yet unrecognized impact of TCS on behavioral traits of mammalian pups, thus establishing it as a novel environmental factor that could significantly contribute to the etiology of ASD. In the current practice of safety assessment for chemicals, attentions are usually only paid to the physiological health of animals subjected to the agents, with little or no effort taken to examine their potential impact on the mental health of animals, let alone their offspring. To better safeguard the populace from unwanted consequences, it is the right time to re-evaluate whether and how many other routinely used chemicals might impact the mental health of animals and humans, and act accordingly.The use of TCS has been generally believed to be safe, as the oral LD50  for mice was determined as 4530 mg/kg. Despite the fact that Food and Drug Administration of the United States of America banned the use of TCS among 19 active ingredients used in over-the-counter (OTC) consumer antiseptic products, TCS is still included in over 2000 types of products, which is well poised to further significant increase in the near future. Meanwhile, although the dose for daily TCS exposure seemed to be low among general population, TCS could significantly accumulate in human body due to partial excretion and slow turnover, reaching up to 3.79 mg/L in urines.Supplementary information