Black men who have sex with men (MSM) have disproportionate HIV disease burden in the United States. Black MSM have been underrepresented in biomedical research, including HIV clinical trials, due to a myriad of socio-structural, socio-cultural, and psychosocial factors. The HIV Prevention Trials Network (HPTN) 061, a feasibility study of a multi-component HIV prevention intervention for Black MSM in six US cities, incorporated the development and implementation of a Black Caucus as a culturally grounded model for the integration of Black MSM in clinical trials and research in HPTN. Based on a qualitative methodological approach, we describe the formation and implementation of the Black Caucus from the perspective of Black MSM key community stakeholders. Three major themes emerged from the qualitative narratives: (1) the role of the Black Caucus in shaping the HPTN, (2) how the Black Caucus addresses the needs of Black MSM communities pertaining to the influence of race and sexual identity, and (3) socio-cultural needs of Black MSM. These findings have implications for the provision of culturally congruent expertise, community engagement, cultural mistrust, recruitment and retention of Black MSM in HIV clinical trials, culturally-relevant study design and implementation, and the role of developing Black MSM prevention researchers.  Black men who have sex with men (MSM) have experienced substantial disproportionate HIV infection burden as compared to other populations in the United States (US) ,3,4,5,6.Clinical factors have been shown to affect HIV incidence and prevalence rates among Black MSM, such as infrequent HIV/STI testing patterns, undiagnosed and untreated HIV/STI infections, higher frequencies of HIV/STIs within sexual networks, and suboptimal viral load suppression rates ,20,21,22Black MSM have been under-represented in biomedical research, including HIV clinical trials, due to a constellation of socio-historical, socio-cultural, and psychosocial factors . ResearcHistorically, limited research involving clinical trials has focused on Black MSM in the US . The NatThe HPTN 061 Black Caucus was composed of representatives from each of the study’s eight clinical research sites as well as several consultants who were not directly affiliated with a study site. The majority of the members were highly influential in Black MSM communities and successful in their chosen professions, which ranged from research to advocacy. Sites were able to nominate two members of any race, ethnicity, sexual orientation, or gender as long as they possessed relevant knowledge of racial and sexual identities of the men participating in HPTN 061. Based on a series of semi-structured, in-depth interviews with Black MSM key community stakeholders, we developed a better understanding of the importance of the HPTN 061 Black Caucus and its role in the development and implementation of this clinical trial.Community-based Participatory Research (CBPR) is a collaborative approach that involves the systematic and equitable integration of the community into the research process ,45. CBPRThis multi-tiered partnership between researchers and Black MSM communities is designed to facilitate cooperation, collaboration, and participation during each phase of the research process . An inteThe HPTN 061 protocol was the first large-scale prospective cohort study among Black MSM in the US that the Division of AIDS within the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) funded. A protocol team was formed to conceptualize HPTN 061. As the study protocol was evolving, one of the Protocol Co-Chairs sought out selected members of the Black Gay Research Group (BGRG) and the National Black Gay Men’s Advocacy Coalition (NBGMAC) for feedback. Both groups identified significant gaps in the proposed study concept. These limitations were counterintuitive to the implementation of the proposed study. The gaps identified by our interviews can be categorized into three main dimensions: (1) lack of collaboration with Black MSM communities, (2) lack of Black MSM representation in leadership roles, and (3) lack of culturally-relevant data collection methods. HPTN 061 was a feasibility study of a multi-component HIV prevention intervention of 1553 Black MSM in six US cities . The methods for the study have been described in detail elsewhere  research ethnic minority populations. And that’s because of our tradition and our history of passing down oral traditions and we’re talking about researching a group of men who have sex with men who had never been researched at this level, and you’re trying to do this at the community level.” Another respondent captured the sentiments of most of the group around the issue of the role of the HPTN Black Caucus in helping shape HPTN 061:“In terms of the development of the [Black] Caucus, I think there was some feeling at some point that some of the original investigators haven’t had as much depth in dealing with that community, and some wanted to have a [Black] Caucus pulled together to make sure that all of the issues concerning Black gay men were understood and were part of it.” The following respondent eloquently captured the perspectives of most of the participants regarding the lack of inclusion of Black gay men’s voices in the research process. This respondent considers this the main problem and powerfully states that finding out why something is the way it is requires more than just a set of numbers. He states: “I’m not so much interested in the numbers. And I feel that studies like these are problematic because they don’t want to know why. They just want to know who and how and where. Because why is more complicated. Why is not necessarily easy. There are multitudes of reasons why they are situated in a wide range of contexts. So that was the main problem.” These considerations identified the need for the continued integration of race and sexuality components to develop a relevant protocol among Black MSM in the US. As the launch of HPTN 061 came closer, conversations began around convening a formal body that would provide ongoing culturally congruent expertise to the protocol that addressed the current concerns of Black MSM communities. The HPTN Black Caucus formed and began offering the HPTN more internal accountability for the study. The qualitative narratives of the early HPTN Black Caucus members reflect a deep concern about race and the lack of inclusion of Black MSM at the planning table and in key decision-making positions and processes. The following two respondents enthusiastically illustrate this point: “And, of course, we were like, who do they think is going to do this research? At that point, they were all White researchers. We were like that’s not going to work. And how do they propose to do this going into our communities on this grand scale and they aren’t even including us.” “And, then, we started talking with the [current Co-Chairs] about [HPTN] 061, and there were issues that were being raised about the implementation and the [formation] of the investigative team, and were especially concerned about the lack of participation from the Black gay men themselves, including as investigators. And so out of that process, the HPTN leadership committed to forming this Black Caucus that would meet regularly. Because of the advocacy of the two groups [BGRG and NBGMAC] collectively that was used to push the agenda forward with NIH and the protocol co-chair.” Two powerful points made by other respondents really made salient the importance of acknowledging the cultural significance of “Blackness”; they assertively noted the following:“First of all, I think we have to own the fact that this is a race-based study and that it is a study that is focusing on Black men. So at its inception and heart is that we acknowledge this box called Black. And I’m always conflicted by that because I don’t think science has done a good job at saying what that means.” “Racial and cultural identity struggles were imminent with developing HPTN 061—addressing these factors became top priority over time and was captured and addressed mostly on behalf of the Black Caucus. It was explicitly clear acknowledgement of such identities in order to press the agenda forward in addressing the public health concern of Black MSM. Due to the salient factors around Black MSM contracting HIV, it was imperative to keep these identities in the forefront while developing the protocol.”  Based on these qualitative narratives, the HPTN 061 protocol team began to incorporate suggestions to establish collaboration between the HPTN 061 protocol team, Black Gay Research Group, and the National Black Gay Men’s Advocacy Coalition. The first recommendation that both BGRG and NBGMAC strongly endorsed was that the HPTN 061 research team diversify its leadership. Using its rich networks, the new trilogy not only identified a new co-chair to join the HPTN 061 research team leadership, but also found highly qualified Black MSM and other people of color to work at each of the clinical research sites. These new individuals became official members of the HPTN 061 protocol team.The HPTN formed the HPTN 061 Black Caucus to provide community input, scientific expertise, and support to all aspects of this study. Through a wide range of activities, the HPTN 061 Black Caucus ensured that the design, implementation, analysis, and interpretation of the study were rooted in the appropriate racial and cultural contexts and that the study was responsive to the needs of Black MSM communities. The group also acted as a liaison between the study sites and HPTN 061 leadership, providing a safe venue through which study participants and Black MSM who were working at the study’s clinical research sites could voice their ideas and concerns about the study. Through its work plan, the HPTN 061 Black Caucus was able to raise awareness about many of the socio-cultural nuances of conducting research among Black MSM and within Black MSM communities that helped shape HPTN 061 in several important ways. Our key findings illuminate the contributions of the Black Caucus into two distinct areas: cultural responsiveness and study design and implementation.One of the themes that emerged in the qualitative narratives related to incorporating the provision of cultural responsiveness into the research process. One participant commented:“The [Black] Caucus helped the peer health navigators shift from a purely health focus model to a more community-centered focus based on the beginning interaction with the participants. This change recognizes the comprehensive needs that lie within the Black MSM community including housing, employment, and skills development. Working with clinical research sites to establish clinics in convenient locations, ensuring the clinics were open at times that were best for the participants, and establishing a respect continuum for the participants within those settings.” Another respondent’s comment illustrates the community benefits of providing cultural responsiveness: “Winning over the community’s goodwill. We didn’t have that before. It provided the attention to population issues and culture to ensure that this trial was able to move forward.” Many of the respondents noted the importance of having representation at the top level, so the inclusion of a Black gay man as a Principal Investigator (PI) or co-chair was a very important and strategic move. One respondent noted: “I think it changed the direction of the study as it was initially developed. I say that because [name of investigator] is a PI now, the qualitative working group, and other things have been developed. You can’t do the study without Black gay men, and I think they realized that.” Another respondent’s comment supported this assertion noting that having a Black PI was probably the most important contribution of the Black Caucus, when asked “What are the most important contributions the Black Caucus has made?” He states unequivocally: “We certainly think that really pushing to get a Black PI on it. I don’t think that can be underestimated…but this also goes to the larger issue of NIH…. NIH can’t have it both ways. They basically say that they want to support more minority investigators, but they don’t get a lot of applications….So how are Black boys and girls ever going to be inspired to believe that is a welcoming place to exist and to invest themselves in science?” Along with incorporating the qualitative component, the HPTN Black Caucus was instrumental in helping to shape the recruitment and retention methods of HPTN 061. Research suggests that innovative, culturally-relevant methods are more successful than traditional methods for recruiting and retaining members of communities of color in research studies ,55. In t“You can’t recruit in only traditional ways. We work in social networks using index patients. It’s not necessarily always commonly done, but I also think the way it is done in Black communities is different because it’s not just a Black gay social network.” “As a collective, we have brought awareness on how to work and recruit participants in our community. The [Black] Caucus has been able to provide suggestions and recommendations to leadership, and those recommendations have been able to shape the study differently.” The wide range of expertise among HPTN Black Caucus members facilitated the building of relationships and sharing of ideas to help push a Black MSM agenda forward. To support the career development of historically-underrepresented researchers, the HPTN Black Caucus endorsed the establishment of the HPTN Scholars Program, which was supported by funding from the National Institute of Allergy and Infectious Diseases (NIAID) . The HPTOur research explored the development and implementation of the HPTN Black Caucus. The purpose of the HPTN Black Caucus was to provide a mechanism for the HPTN leadership to garner increased insight into the specific socio-cultural, socio-structural, and psychosocial complexities of the lived experiences of Black MSM. Three primary themes emerged from the qualitative narratives of Black MSM community stakeholders that related to the formation and implementation of the HPTN Black Caucus. The role of the HPTN Black Caucus in shaping the HPTN was prominent in providing culturally-congruent scientific and community contributions to the richness of the study protocol. The HPTN Black Caucus provided an innovative, interdisciplinary framework for understanding and addressing the needs and concerns of Black MSM communities pertaining to the multi-layered intersections of race and sexual identity. The HPTN Black Caucus served a critical role in facilitating accountability for the incorporation of a cultural responsiveness framework into the study protocol as a basis to facilitate community input, as well as to ensure that the design, implementation, analysis, and interpretation of the study were situated in the racial and cultural contexts of Black MSM communities.Connected to the role of the HPTN Black Caucus in shaping the HPTN, engaging culturally-responsive members in the protocol development process enriched the HPTN 061 research protocol. The HPTN Black Caucus members strengthened the original HPTN 061 concept in relation to theoretical and methodological rigor as well as cultural responsiveness and relevance for Black MSM communities. Community members served as vital resources for the research investigation related to an incorporation of Black sexuality frameworks involving the socio-historical and socio-cultural contexts of Black MSM communities ,57. The Based on the theme of how the HPTN Black Caucus addressed the needs of Black MSM communities, it is imperative to maintain the nexus of race and sexual identity as a conceptual framework in relation to addressing persistent HIV-related inequalities among Black MSM. Having researchers who are aware of the nuances of these socio-cultural frameworks within Black MSM communities became important in developing a protocol that was inclusive of such a diverse population ,60,61. MCultural responsiveness often had various meanings for HPTN 061 in that it was defined as addressing the socio-cultural contexts of Black MSM culture and implementing a clinical trial within this population as a basis to determine its feasibility. Adapting the model of including culturally responsive staff in clinical trials and prevention research will begin to improve future protocols and ensure acceptability and adaptability within historically-underserved communities ,41. ThesNotably, there were lessons learned about the key struggles or obstacles that were experienced within the HPTN Black Caucus in facilitating a paradigm shift for biomedical and prevention science studies focused on Black MSM communities. First, much of the formative work that was conducted by the HPTN Black Caucus was facilitated without guaranteed funding. Since HPTN Black Caucus members were affiliated with a study site or part of the protocol team, the majority of our work was conducted altruistically in the benefit of Black MSM communities. It would be important for groups who are forming a similar body for historically-underrepresented communities to incorporate a funding component into the design of the study based on relevant resources. Second, as a strategy to develop a research model for groups who are forming a similar body based on a holistic understanding of the life-contexts of historically-underrepresented communities and a commitment to exploring issues of equity and social justice, an integral component would involve articulating a vision for research that incorporates guiding principles  .Further, based on the considerable efforts that were put forth in the formation of the HPTN Black Caucus, it would be imperative to develop a formative work plan during the initial phase of the process. For example, as a new initiative, substantial engagement occurred between Black MSM researchers/advocates/staff and non-Black protocol team members in that the HPTN was accustomed to a specific framework. In this context, another lesson learned relates to the importance of garnering stakeholder buy-in early within the process. Buy-in coupled with advocacy allowed the HPTN Black Caucus to have a clear runway or path for the development and implementation of programmatic efforts. This advocacy was needed throughout the project to support involvement from managers and senior-level research investigators for the staff members involved. It took proof of concepts  to help shift the mindset of those persons who did not believe that the HPTN Black Caucus was an essential component for a HIV prevention protocol study. Parallel to this process, advocacy from members of the BGRG and NBGMAC was instrumental in providing leadership and accountability to ensure that the needs of Black MSM communities were addressed within a culturally-relevant framework. In this context, the formidable changes that emerged have had a long-lasting positive impact on the HPTN. Our findings from this study should be interpreted within the context of a number of limitations. First, the cross-sectional nature of qualitative data collection based on in-depth interviews posed a limitation in that data were captured at one point in time. The use of multiple in-depth interviews may have provided additional insights into the multifaceted complexities of the research process during the full duration of the study. For example, an array of complex domains  were explored in the study and a longitudinal qualitative design may have allowed for further exploration of these domains. Second, the incorporation of a multi-method approach, using the combination of in-depth interviews and focus groups, may have yielded richer data. Third, based on our CBPR framework, qualitative data collection with members of Black MSM communities reflective of the cities where the study took place may have yielded another layer of community perspectives about the development of the HPTN Black Caucus. Fourth, while we collected standard demographic characteristics about study participants, additional information would have been helpful in understanding the professional interests of the Black MSM community stakeholders who participated in this study. However, this study provided a rich, multi-faceted understanding of an understudied area of clinical research based on the narratives of Black MSM within a culturally relevant framework. Black Caucus members were influential and essential in informing the HPTN leadership and research agenda about Black MSM communities and cultural expectations.The Black Caucus strategically and systematically addressed the core needs of Black MSM communities by acting as a liaison between the study sites and HPTN 061 leadership.Black Caucus members eloquently described the conception, evolution, and function of the Black Caucus as a salient component of research involving Black MSM.Through the provision of cultural expertise in such areas as cultural relevancy, community engagement, participant recruitment, retention, data analysis, and data interpretation, the HPTN Black Caucus provided strong leadership to move the science forward for future research studies involving Black MSM and other marginalized communities . Most reOur findings suggest that the development and implementation of the HPTN Black Caucus into the HIV Prevention Trials Network served a critical role in integrating the voices of Black MSM and creating a framework of sustained community engagement, leadership, and commitment to addressing persistent HIV-related inequities within Black MSM communities. For example, standards for community stakeholder engagement changed in HPTN. For any study enrolling a population or using a novel approach, HPTN began incorporating community stakeholder meetings to collect information on the study design. In this context, the HPTN Black Caucus created a guidance document that the HPTN has used when developing efficacy studies to mandate enrollment quotas for studies in the US to ensure adequate representation for Black MSM. Our findings suggest that the research model of the HPTN Black Caucus could be utilized as a framework for other historically-underrepresented communities. Elements of the HPTN Black Caucus model have been used in engaging and soliciting cultural expertise from key community stakeholders  for other HPTN studies in an effort to strengthen study design and community engagement. While the initial iteration of the HPTN Black Caucus focused specifically on HPTN 061, later formulations provided guidance to any research study enrolling or engaging Black MSM and other historically-underrepresented communities. The HPTN Black Caucus developed a cultural responsiveness training that sought to increase understanding of the perspectives of researchers, stakeholders, and research participants from different backgrounds to improve multicultural, multi-racial, -ethnic, -gendered, and -sexuality-oriented clinical research environments. While this training was initially developed to improve rapport with Black MSM participants and community stakeholders, it was later revised to address the needs of transgender people as well. HPTN Black Caucus members identified several representatives from the transgender community to assist in training clinical research sites across biomedical HIV prevention studies. The HPTN Black Caucus has provided ongoing leadership with consultations for HPTN studies with priority populations reflective of historically underrepresented communities.In the face of what seemed like insurmountable odds, the resilience and tenacious resolve of the HPTN Black Caucus helped communicate to the researchers the benefits of including Black MSM voices in the process. These early warriors challenged and held accountable those persons who would espouse traditional methodologies of conducting research, not only among Black MSM, but in Black communities overall. By insisting that the needs of Black MSM be clearly identified, acknowledged, and supported, the HPTN Black Caucus ushered in a community-based participatory research model that has become the hallmark of the HPTN 061 project and set the standards for future research among this population within all the NIH clinical trial networks. Future research projects should consider the inclusion of priority populations using the tenets of community-based participatory research as a culturally-relevant framework for designing research studies for such populations. Examples like the HPTN Black Caucus can be utilized to ensure that cultural voices are included in research studies which can help to eliminate potential barriers to engaging relevant communities.


Scientific Reports 10.1038/s41598-018-36453-6, published online 24 January 2019Retraction of: The authors are retracting this Article.We were alerted to the possibility that our designed structure is a broadband cross-polarization metamaterial converter, rather than a broadband metamaterial absorber.We have now performed additional simulations and calculations, confirming that we did not account for cross-polarized reflection in the absorption calculation.As such, we are unable to support the conclusions regarding design of a broadband metamaterial absorber, because the designed structure exhibits broadband cross-polarization.All authors agree to the retraction of the Article.


Cisplatin is one of the most commonly used antineoplastic drugs; nonetheless, its ototoxic dose-limiting side effects have remained a significant challenge in clinical practice. The recognition of the exact template of hearing loss induced by multiple low doses of cisplatin could be of great help in managing the treatment process. The present study aimed to investigate the effects of multiple doses of this drug on the auditory system.The present study was performed using an experimental guinea pig model in four groups as follows:1- 0.9% sodium chloride solution, 2- total dose of 7.5 mg/kg Cisplatin, 3- total dose of 10 mg/kg Cisplatin, and 4- total dose of 12.5 mg/kg cisplatin. The drugs were injected as 2.5 mg/kg/daily IP access in all groups. The auditory brainstem response (ABR) test was performed before the treatment and after every injection on a daily basis up to 72 h after the last injection.There was dose-dependent significant hearing loss in all evaluated frequencies in three cisplatin groups. The general template of induced hearing loss during experimental days was almost the same in groups Cis7.5 and Cis10. In Cis 12.5 group, there was a jump in the threshold shift on the 5th day of the experiment and an upward trend in the function.As evidenced by the obtained results, the monitoring of hearing loss after every injection in patients who receive the drug and detecting the exact dose-dependent pattern of the induced hearing loss is of great help in controlling its undesirable destructive side effects on the auditory system. American Chemist, Barnett Rosenberg, in 1965 proved that certain platinum-containing compounds inhibit cell division and make considerable changes to bacterial morphology . NowadayOtotoxicity is manifested as bilateral progressive and irreversible sensory neural hearing loss in most cases . More thFurthermore, the economic burden of hearing loss on society is the leading cause of the increasing necessity for finding an applicable method for the protection of cochlea against cisplatin ototoxicity. One of the principal considerations in the development of conservative methods is preventing cisplatin ototoxicity without disturbing its anticancer function . The preAccordingly, the audiological monitoring of patients and exerting limitations on drug dosage is considered the most appropriate method for the protection of cochlea against cisplatin-induced ototoxicity. The physician must be aware of early signs of ototoxicity and its progression and could manage to change the treatment method if necessary. Therefore, the importance of dose-dependent changes in the hearing system by cisplatin treatment has been highlighted in the literature. With this background in mind, the present study aimed to daily check the impact of multiple dosages of cisplatin on the hearing system of guinea pigs by auditory brainstem response.In the present study, albino guinea pigs were selected as the laboratory animals for a few reasons. They are easy to handle and anesthetic infusion and the intraperitoneal (IP) injection of the drug are effortless in these animals; therefore, they are more sensitive to the effects of cisplatin. Animals were obtained from Razi Vaccine and Serum Research Institute consisting of 24 albino male guinea pigs within the weight range of 250-300 grams. Animals were kept under standardized housing and feeding conditions in separate cages. All of them had free access to commercial food and water and were maintained in an environment with a controlled temperature of 20-25 degrees Celsius and 12 h light-dark cycle. The protocol was approved by the Institutional Review Board, and the animals were handled in accordance with the guidelines proposed by the Animal Use Committee of the institution. After a 24-hour rest and confirming the positive Preyer’s reflex in all animals, guinea pigs were assigned to four treatment groups (n=6 each) as follows:1. The daily IP administration of 0.9% sodium chloride solution 2.5 ml/kg 2. The daily IP administration of cisplatin (1mg/ml) 2.5 mg/kg for three consecutive days  (Cis7.5)3. The daily administration of cisplatin (1mg/ml) 2.5 mg/kg for four consecutive days  (Cis10)4. The daily IP administration of cisplatin (1mg/ml) 2.5 mg/kg for five consecutive days  (Cis12.5)For the controlled application of drugs, we used 1 cc disposable insulin syringe for each animal.Auditory Brainstem Response (ABR) test was performed using an electrophysiologic system . The test was carried out before the treatment (baseline measurement) and after every injection on a daily basis. The injections were daily performed at 7 am and the hearing assessments were carried out at 7 pm. The hearing evaluation was continued up to 72 h after the last injection. The guinea pigs were anesthetized by intraperitoneal injection of a mixture of ketamine 40 mg/kg and xylazine 4 mg/kg. While ABR tests were performed, the body temperature was maintained at 36°C with an electric blanket controlled by a rectal thermistor. In the pilot study, it was confirmed that cisplatin induces bilateral and symmetrical hearing loss. Consequently, it was required to avoid data duplication and consider inter-subject variability factors. In this regard, the test was performed using a far-field technique from the right ears of all animals, and the left ear was blocked by an earplug during the test. The reference electrode was subcutaneously inserted into the ipsilateral pinna, the ground electrode into the contralateral pinna, and the active electrode into the top of the head. Before the commencement of the test, the impedance of electrodes was confirmed to be below 5 k Ohms. Acoustic stimuli were delivered by a far-field speaker in a sound-proof box, and the sound levels within the animal hearing range were calibrated with a sound level meter. Acoustic stimuli consisted of tone-bursts of 4, 8, 12, and 16 kHz . The result of the time window analysis for each response was 10.66 ms. The evoked potentials were filtered with a band-pass filter between 100 and 3000 Hz. The stimulus presentation rate was 23.1 bursts of alternating polarity per second. The minimum number of sweeps was reported as 1024, and the gain was adjusted at 100k.In evaluating the hearing thresholds, the stimuli levels were lowered from 80 in 5-dB steps until the identification of a visible repeatable wave III. The recordings were repeated twice at the threshold level for confirming the reproducibility of the waves. Wave III was used for the detection of thresholds.The obtained data were analyzed in SPSS software (version 17) using repeated measures. The quantitative data were presented as mean±SD. A p-value less than 0.05 was considered statistically significant.The total change in ABR thresholds (last measurement-baseline measurement) in four different groups are displayed in The mean of ABR thresholds(dB) within 8 experiment days for all four groups is depicted in rd day of experiment. In group Cis7.5, which received the drug up to the 3rd day, the diagram of threshold elevation grows by a gradual slope in the next days. In group Cis10, there is another rising step almost identical with the recent increases; thereafter, the graph rises almost as slightly as the Cis7.5 group at the 4th day of experiment. In group Cis12.5, following the last injection on the 5th day, there is a sharp surge in the graph demonstrating an upward trend during the next days. This trend leads to a total threshold change of 56.16 dB in mean ABR thresholds in 72 h after the last injection on the 8th day. From a clinical perspective, finding such a momentous dose could be of great help in governing the ototoxicity symptoms. Previous studies have reported that above a certain critical cumulative dose of the drug in the blood, the destruction increases very rapidly. This occurs as the result of permanent renal damage leading to the accumulation of the drug in the blood (A wide spectrum of unpleasant effects on the inner ear, including hearing loss, tinnitus and vertigo have been reported for some drugs, such as cisplatin . For manhe blood .The results of the current study highlighted the importance of daily evaluation of the auditory system in patients who receive this drug. The recognition of the template of this degeneration could be of significant importance for the avoidance of undesirable side effects of this drug. On the other hand, daily monitoring of the hearing system is definitely an inexpensive and cost-effective strategy, as compared to the adverse effects of hearing loss on patients who receive the drug, especially children. It is worth mentioning that in humans, the exact pattern of induced hearing loss and the dose of the drug which causes ototoxicity is not as identical as it is in experimental animals. On a final note, in the light of the obtained findings of the present study and the results reported in the literature, it can be concluded that the recognition of the template of this degeneration could be of significant importance for the avoidance of undesirable side effects of this drug on hearing system, especially the hearing loss.


Mycoplasma hyopneumoniae: is the etiological agent of porcine enzootic pneumonia (EP), a disease that impacts the swine industry worldwide. Pathogen-induced damage, as well as the elicited host-response, contribute to disease. Here, we provide an overview of EP epidemiology, control and prevention, and a more in-depth review of M. hyopneumoniae pathogenicity determinants, highlighting some molecular mechanisms of pathogen-host interactions relevant for pathogenesis. Based on recent functional, immunological, and comparative “omics” results, we discuss the roles of many known or putative M. hyopneumoniae virulence factors, along with host molecules involved in EP. Moreover, the known molecular bases of pathogenicity mechanisms, including M. hyopneumoniae adhesion to host respiratory epithelium, protein secretion, cell damage, host microbicidal response and its modulation, and maintenance of M. hyopneumoniae homeostasis during infection are described. Recent findings regarding M. hyopneumoniae pathogenicity determinants also contribute to the development of novel diagnostic tests, vaccines, and treatments for EP. Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease that affects pigs [M. hyopneumoniae are highly prevalent worldwide, causing major economic losses to the swine industry [M. hyopneumoniae infections and vaccination against EP are also challenging due to the still limited repertoire of well-characterized antigens. Some diagnostic and/or vaccinal antigens have been characterized immunologically, i.e., with an assessment of their antigenicity and immunogenicity [M. hyopneumoniae physiology and pathogen-host interactions [cts pigs . Infectiindustry  due to cindustry . Immunodgenicity , and funractions .M. hyopneumoniae and the swine host. It is known that M. hyopneumoniae infection leads to epithelial damage of the swine respiratory tract, either directly, considering possible bacterial cytotoxicity [M. hyopneumoniae, however, are not clear. It is reasonable to assume that the onset of EP in swine depends on M. hyopneumoniae pathogenicity determinants, classically described as virulence factors, as well as on the triggered host responses, immunological or otherwise. M. hyopneumoniae pathogenicity determinants are considered the bacterial factors that allow the pathogen to override the host defense mechanisms and comprehend molecules that mediate processes such as cell adhesion to the host, response to host environment stress, and immunomodulation. On the other hand, host factors contributing to EP include molecules and processes that mediate innate and adaptive defenses against the pathogen, but also cause damage host tissues, such as lung lesions resulting from exacerbated inflammatory responses.In the context of the damage-response framework of microbial pathogenesis , EP pathtoxicity , or inditoxicity . The exaM. hyopneumoniae strains, and between M. hyopneumoniae and the closely related species Mycoplasma flocculare, have considerably improved the knowledge of M. hyopneumoniae pathogenesis. M. hyopneumoniae strains may differ in pathogenicity or virulence, ranging from the nonpathogenic type strain J  to virulent strains isolated from EP outbreaks, such as the Brazilian strain 7448 or the American strain 232 [M. flocculare, in turn, is genetically closely related to M. hyopneumoniae but is a commensal species, with its presence in the host being virtually asymptomatic. The “omics” prospective and comparative studies with these strains and species, discussed in a later section, have improved our knowledge on the factors that determine M. hyopneumoniae pathogenicity.In the last decade, comparative analyses between pathogenic and nonpathogenic rain 232 . M. flocM. hyopneumoniae or M. flocculare and the swine host demonstrated differences in intracellular and secreted protein repertoires of swine cells infected by these mycoplasma species [Host EP determinants, on the other hand, have been less assessed, aside from some punctual studies regarding innate and adaptive immunological response and cell death . Neverth species ,26.M. hyopneumoniae and swine molecular mechanisms and cellular processes underlying the disease. Moreover, it also surveys recent advances toward the identification of both the bacterial and host repertoires of EP determinants, including the most recent efforts involving functional and comparative “omics” approaches to elucidate why some M. hyopneumoniae strains are more virulent than others and why M. flocculare is nonpathogenic despite of sharing most of the virulence factors with M. hyopneumoniae. Interactions between M. hyopneumoniae and swine cells are discussed in the context of their intimate contact during infection, emphasizing molecular/cellular mechanisms related to cell adhesion, biofilm formation, host cell invasion, secretion and signaling, cytotoxicity and apoptosis, immunomodulation and stress response.This review includes recent data regarding EP epidemiology, prevention, and control of this disease, and a comprehensive overview of the main M. hyopneumoniae is a specific pathogen of domestic pigs (Sus scrofa domesticus) and wild boars (Sus scrofa scrofa), and has a worldwide geographical distribution [M. hyopneumoniae prevalence by country are scarce in the literature, as EP does not require mandatory notification in many countries and does not limit commercial trade [M. hyopneumoniae prevalence of 30–80% has been reported in domestic pig herds worldwide [M. hyopneumoniae prevalence was 48% for pigs in the Mendoza province, Argentina, based on molecular diagnosis [M. hyopneumoniae prevalence, near 10%, was reported for pigs in Africa (Uganda) [M. hyopneumoniae of 24.8% and 21.12%, respectively [M. hyopneumoniae and other pathogens [As far as is known, ribution . Specifial trade . An averorldwide . In Soutiagnosis , and preiagnosis . A lower(Uganda) . For wilectively ,34. In tMycoplasma hyopneumoniae transmission dynamics within swine herds depend on the intensity of the production system used, as recently reviewed by Maes et al. [M. hyopneumoniae is important in segregated production systems, since pigs are transferred to clean facilities for the growing and finishing phases [M. hyopneumoniae among pen-mates is slow, and a clear understanding of M. hyopneumoniae transmission in the field is still needed to improve infection models used in experimental researches [M. hyopneumoniae is its long pathogen persistence [s et al. . The firs et al. ,36. At wg phases ,38. Subssearches . A critisistence ,43, but M. hyopneumoniae epidemiology is the occurrence of distinct strains, with different degrees of virulence, circulating in the field [M. hyopneumoniae strains circulating within a herd or geographical region are of utmost importance. Discrimination among M. hyopneumoniae strains usually rely on partial sequencing of the P146 gene [M. hyopneumoniae variability in pathogenicity and at the antigenic, proteomic, transcriptomic, and genomic levels, and proposed the variable number of tandem repeats (VNTR)-based common terminology and classification. This VNTR-based system is expected to avoid discrepancies and allow to make inferences across the literature.An additional complicating factor regarding he field . Therefo146 gene , multilo146 gene . More re146 gene  reviewedM. hyopneumoniae infections can be achieved with strategies involving management practices such as all-in/all-out pig flows, medicated and segregated early weaning, and multisite operations [M. hyopneumoniae are also important. Garza-Moreno et al. [M. hyopneumoniae monitoring strategies of incoming gilts and recipient herds and proposed a farm classification based on their health status. According to clinical signs, lung lesions, and ELISA and PCR results, farms and incoming replacements can then be classified into negative, provisional negative and positive.Enzootic pneumonia control and prevention are based on the optimization of management conditions, vaccination and treatment with antibiotics. Factors such as management, biosecurity practices and housing conditions should be optimized within the herd . Decreaserations . Prevento et al.  presenteM. hyopneumoniae infections. Gilt replacement acclimation procedures against M. hyopneumoniae in positive farms in Europe and North America showed that vaccination is the main strategy to avoid EP [M. hyopneumoniae vaccines are used worldwide and consist mainly of inactivated, adjuvanted whole-cell preparations that are administered intramuscularly [M. hyopneumoniae infection [Vaccination is today still regarded as the most effective way to control avoid EP . Anti-M.scularly . Presentnfection .M. hyopneumoniae strains, and unknown aspects of the induced immune responses, along with technical issues, such as improper vaccine storage conditions and administration, and lack of vaccination compliance [M. hyopneumoniae vaccines have conferred only a limited reduction in the transmission ratios [Vaccinated animals present reduced clinical signs and lung lesions, improved performance and reduced number of microorganisms in the respiratory tract ,38,52,53mpliance ,57. Furtn ratios ,58.M. hyopneumoniae infection are not yet fully understood, but constant efforts are invested in the development of new vaccines that may confer better protection. The latest efforts toward the development of more efficient vaccines against EP have been recently reviewed by Tao et al. [The exact mechanisms of protection needed to avoid o et al. . These eo et al. ,59–62. HM. hyopneumoniae infection can predispose swine to secondary infections, combined vaccines have gained attention, as they prevent multiple diseases at the same time. Combined vaccines developed thus far for EP and other swine respiratory infections consist of a mixture of M. hyopneumoniae bacterin and live attenuated viruses, such as porcine reproductive and respiratory syndrome virus and porcine circovirus 2 (PCV2) [M. hyopneumoniae and other porcine pathogens [M. hyopneumoniae and PCV2 [As 2 (PCV2) , or geneathogens . There hand PCV2 ,67, and M. hyopneumoniae infections can be accomplished using medication with antibiotics against M. hyopneumoniae and major secondary invading bacteria [M. hyopneumoniae include tetracyclines, macrolides, lincosamides, pleuromutilins, amphenicols, aminoglycosides, aminocyclitols and fluoroquinolones [M. hyopneumoniae strains with high minimal inhibitory concentration (MIC) values for some antibiotics have been reported, along with a description of resistance mechanisms [When EP control by the improvement of management and biosecurity and the implementation of vaccination fails, the clinical disease occurs. Then, the treatment of affected animals with antibiotics is required to maintain animal health and welfare. Treatment of bacteria . Potentiinolones ,69. M. hchanisms ,71. TherM. hyopneumoniae in 2004, when the first strains  had their whole genomes sequenced [M. hyopneumoniae genomes are available, 10 already assembled and annotated, and 11 still not fully assembled (https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/190/). Apart that of M. hyopneumoniae J strain, the available sequenced genomes are from American, Brazilian, European, Korean, and Chinese pathogenic strains. These genomes are 0.86–0.96 Mb in size, and in each of them there are 528 to 691 protein-encoding genes. Interestingly, despite their small sizes, up to 30% of their gene contents are still of unknown function [M. hyopneumoniae genes code for surface proteins  [The “omics” era started for equenced ,19. Sincfunction . Moreoveunction) , therefoM. flocculare and have provided insights into M. hyopneumoniae pathogenicity determinants. M. hyopneumoniae strains and M. flocculare share most (>85%) of the genes that code for known (or predicted as such) pathogenicity determinants, including most adhesins, proteases and antioxidant proteins [M. hyopneumoniae strains, and between them and M. flocculare. Therefore, comparative studies between M. hyopneumoniae and M. flocculare have focused on possible differences in gene expression and protein abundances. Transcriptomics, proteomics, secretomics, and metabolomics approaches have been carried out as attempts to correlate differential M. hyopneumoniae transcripts, proteins or metabolites to pathogenicity or virulence [M. hyopneumoniae virulence factors and both bacterial and host EP determinants.More recently, functional “omics” studies, including transcriptomic, proteomic, and metabolomic, have followed the pioneering descriptive and comparative genomics research. These studies were enriched by data generated for the close relative species proteins ,72. Moreirulence . In the M. hyopneumoniae adhesion to ciliated epithelial cells of the respiratory tract is the initial event in host colonization. This adhesion process is complex, dynamic, and not fully understood, but it is known to involve several surface-displayed molecules of both the pathogen and the host cells [M. hyopneumoniae and host cell adhesion determinants are discussed in the next sub-sections and are schematically represented in In EP, st cells ,78. The M. hyopneumoniae must cope with the mucociliary apparatus in the swine respiratory tract, as the so-called lung mucociliary clearance (MCC) is the primary defense mechanism against respiratory pathogens [The interaction between pathogenic bacteria and host cells during colonization is a critical process for pathogen survival and disease development . Upon inathogens . It is tMycoplasma hyopneumoniae attachment to the cilia of the respiratory epithelium allows the pathogen to overcome MCC. It has been long known that the pathogen adhesion causes ciliostasis and subsequent cilium loss and epithelial cell death [in vitro infection assays provided evidence that M. hyopneumoniae infection disrupts the mucociliary function of respiratory epithelial cells by transiently reducing the amounts of mucin 5B secreted in the respiratory epithelium [M. hyopneumoniae adhesion to ciliated respiratory cells.ll death ,82. Moreithelium . This reM. hyopneumoniae proteins have been previously associated with cell adhesion, including several related to the P97/P102 paralog families and other surface proteins that moonlight as adhesins [M. hyopneumoniae adhesins can be higher, considering that its surfaceome includes more than 290 proteins [M. hyopneumoniae pathogenic and nonpathogenic strains, and the nonpathogenic M. flocculare, which differ in pathogenicity and were classically described as having differential adhesion capacities [A repertoire of at least 35 adhesins ,84,85  [Regarding proteolytic processing, it has been described that many adhesins may be targets of endoproteolytic post-translational processing ,78,88–93s (GAGs) ,89,90, os (GAGs) .M. hyopneumoniae and M. flocculare adhesins [M. hyopneumoniae pathogenic strain 7448 are differentially processed compared to their corresponding orthologs in the nonpathogenic M. hyopneumoniae J strain and M. flocculare. Most of the analyzed surface-displayed adhesins from the pathogenic strain were more proteolytically processed  than the orthologs from the nonpathogenic counterparts, which is consistent with the observed enrichment of several aminopeptidases and endoproteases at the surface of this pathogenic M. hyopneumoniae strain [A recent comparative analysis of endoproteolytic processing between adhesins  demonstre strain . There ie strain . MoreoveM. hyopneumoniae infection). Therefore, the mechanism of post-translational proteolytic processing of adhesins (including proteases and their target adhesins) would be a main M. hyopneumoniae pathogenicity determinant, of upmost relevance for EP determination. It would lead to differential presentations of adhesin proteoforms at the cell surface of M. hyopneumoniae strains (and M. flocculare), resulting in, and explaining, at least in part, the observed differential adhesion capacities of these strains and species.The differential adhesin proteoforms generated by proteolytic processing may play differential roles. Those retaining adhesive domains, may retain adhesion properties  and contribute to the overall mycoplasma adhesion capacity. Alternatively, proteoforms devoid of adhesive domains may exert moonlight functions. As many of the generated proteoforms are potentially antigenic , and mayM. hyopneumoniae [M. hyopneumoniae strain 7448 in comparison to the nonpathogenic J strain and M. flocculare [Besides the canonical adhesins and derived proteoforms, other proteins, with different primary function, may moonlight as adhesins. For instance, some typical cytosolic proteins that are also consistently displayed at the cell surface likely exert alternative functions in this ectopic compartment. They may exert moonlight functions by interacting with host components and contributing to host colonization. Among these surface-displayed cytosolic proteins, there are glycolytic enzymes, proteases, chaperones and translation factors that have been characterized as adhesins in different mycoplasma species, including eumoniae ,85,95,96occulare . This ovM. hyopneumoniae adhesins (or adhesin proteoforms) and host cells also depends on the corresponding host ligands on the cell surface or in the extracellular matrix (ECM). Preliminary studies demonstrated that some swine ciliary glycolipids could act as receptors for M. hyopneumoniae attachment [M. hyopneumoniae P97/P102 adhesin family can bind to GAGs from proteoglycans exposed on the cilia surface, which thereby could act as receptors [The interaction between tachment , but theeceptors ,90. Indeeceptors ,98–100M. hyopneumoniae, contributing to host colonization [Mycoplasma pneumoniae, Mycoplasma gallisepticum and Mycoplasma bovis, among others [M. hyopneumoniae may mediate the adherence to swine respiratory cilia and can provide a mechanism for host cytoskeleton rearrangements, which may facilitate bacterial internalization [M. hyopneumoniae may facilitate its traffic via the circulatory system and penetration into host organs, such as liver, kidneys and spleen, from which it has already been isolated [Apart from the cilia-exposed glycans, some swine ECM molecules, such as fibronectin and plasminogen, also provide binding sites for surface adhesins of nization ,101,102.g others . Intereslization . Additioisolated ,110.M. hyopneumoniae P97 adhesin and another 143 proteins, including lipoproteins, glycolytic enzymes, chaperones and translation factors, among others [M. hyopneumoniae to adhere and colonize PK-15 swine cells, indicating that extracellular actin is an important receptor for M. hyopneumoniae infection. Surface proteins of M. hyopneumoniae also interact with other cytoskeletal proteins besides extracellular actin, such as vimentin, keratin, tubulin, myosin, and tropomyosin [More recently, it was also demonstrated that extracellular actin is used as a surface receptor by different proteoforms of g others . Interespomyosin ,111.M. hyopneumoniae cells with each other and with host cells or ECM molecules may render the pathogen capable of forming biofilms. Biofilm formation is a strategy used by many bacteria, including other mycoplasma species [M. hyopneumoniae strains are indeed capable of forming biofilms, in experimental in vitro conditions, on abiotic surfaces or on host cell monolayers, and within the respiratory tract of experimentally infected swine [M. hyopneumoniae more resistant to antibiotics, at least in vitro [M. hyopneumoniae biofilm formation are thus far mostly unknown. However, at least in vitro, biofilm formation involves the generation of a subpopulation of unstable large cell variants that may contribute to the release of extracellular DNA, essential for forming biofilms on abiotic surfaces [Direct contact of  species , to copeed swine ,116. Biosurfaces .Mycoplasma penetrans and M. pneumoniae, for example [M. hyopneumoniae is usually regarded as an extracellular mycoplasma. However, the demonstration that it binds to fibronectin and plasmin at the site of infection [M. hyopneumoniae could invade in vitro-infected host-derived epithelial cells [M. hyopneumoniae. Moreover, infected host porcine cells could function as a source of pathogen cells for re-infection, as internalized, dormant M. hyopneumoniae could eventually leave to the extracellular environment. In line with that, evidence that M. hyopneumoniae may influence the endosomal trafficking and modulate the maturation of early endosomes was provided by proteomic analyses of swine epithelial cells infected with this pathogen [M. hyopneumoniae within the respiratory tract, thereby contributing to the chronic infection status.Along with biofilm formation capacity, another valuable strategy for mycoplasma survival and pathogenesis is the ability to invade host cells, which is well described for  example ,118. M. nfection ,119,120,nfection ,122, sugal cells . Host cepathogen . In suchM. hyopneumoniae has been considered an exclusive respiratory pathogen [M. hyopneumoniae cells have been re-isolated from inner organs, such as liver, spleen, brain, kidneys, and lymph nodes, although at frequencies lower that of the respiratory tract [M. hyopneumoniae is able to disseminate to extrapulmonary sites within the swine host, but the mechanisms that allow the systemic trafficking of the bacterium across the respiratory epithelial barrier remain elusive. One possible mechanism mediating this M. hyopneumoniae trafficking might involve its cell invasion capacity, discussed above. As it has been recently demonstrated that M. hyopneumoniae could also, at least in vitro, invade porcine macrophages and avoid phagocytosis [As it is mostly recovered from trachea and lung lesions from infected pigs, pathogen . Howeverry tract ,123,124.ocytosis , it can M. hyopneumoniae trafficking to alternative host sites was revelead more recently by experiments using an in vitro air-liquid culture system of porcine bronchial epithelial cells [M. hyopneumoniae cells could migrate across the epithelial barrier by the paracellular route, but not by the transcellular route. In this in vitro model, M. hyopneumoniae reversibly disrupts the tight junctions between epithelial cells, increasing the permeability and damaging the integrity of the epithelial barrier. In line with that, several studies have demonstrated that M. hyopneumoniae infection facilitates the activation of plasminogen to plasmin, which contributes to the degradation of several ECM and cellular junction components [M. hyopneumoniae and to the persistence of infection. Overall, further studies are needed to elucidate how and to what extant M. hyopneumoniae disseminates to different extrapulmonary niches in natural infections.Evidence of another possible mechanism that could mediate al cells . With thmponents ,110,120.M. hyopneumoniae genome, only the genes coding for SecY, SecG, SecD/F, YidC and SecA proteins were identified, and the absence of the SecE protein may indicate an incomplete SecYEG transmembrane channel [M. hyopneumoniae provided evidences that the Sec secretory pathway is functional, as several detected proteins in the soluble secretome fraction were predicted to be secreted by this secretion pathway [M. hyopneumoniae genome has thus far no functional annotation.Protein secretion is a vital process for all organisms and has a particular role in the pathogenesis of bacterial infections. Gram-negative and Gram-positive bacteria have at least 6 reported secretion/translocation systems . The gen channel ,19,72. H pathway . This evM. hyopneumoniae soluble secretome were predicted as secreted by Sec-independent secretion pathways [M. hyopneumoniae may present alternative secretion mechanisms not yet identified due to their lack of conservation with other well-characterized secretion pathways. In line with that, protein secretion in extracellular vesicles was observed in several mycoplasma species [M. hyopneumoniae may alternatively use a vesicle system to secrete proteins.In contrast, most of the detected proteins in the pathways . This fi species ,129, sugM. hyopneumoniae can be considered a reservoir of virulence factors (M. hyopneumoniae cell surface (discussed in M. hyopneumoniae adhesion determinants), eventually resulting in the release of some adhesin proteoforms in the extracellular milieu. These adhesin proteoforms may act as extracellular antigens that could trigger the host immunological defenses (discussed in Host immune response and immunomodulation during M. hyopneumoniae infection). The potential relevance of the M. hyopneumoniae secretome for pathogenesis is evident by comparative analyses with M. flocculare, the secretome of which is less complex and presents few orthologs of known M. hyopneumoniae virulence factors.The soluble secretome of  factors , being cM. hyopneumoniae and M. flocculare secretomes are differential. Both species possess the genes coding for the Sec secretory pathway components, although the partial Sec secretory machinery may be supplemented by unknown proteins not shared by these mycoplasma species, leading to differential secretion efficiencies. Moreover, M. hyopneumoniae and M. flocculare may use differential secretion signals and secretion pathways, which resulted in the observed differences in secretome contents. More studies are needed to elucidate the secretion mechanisms used by these and other mycoplasma species.In addition to the differences in the virulence factor content, the overall protein content and abundance in M. hyopneumoniae also acts as a reservoir of virulence factors (The insoluble secretome (or surfaceome) of  factors , which s factors , lipoproM. hyopneumoniae adherence in the swine respiratory tract  revealing that M. flocculare and M. hyorhinis induce the secretion of higher levels of TNF-α and IL-12, respectively, than M. hyopneumoniae [M. hyopneumoniae infection also induces the expression of high levels of IL-10 but decreases the levels of IL-12 and interferon-γ (IFN-γ) expression in dendritic cells [M. hyopneumoniae elicited the secretion of high levels of IL-10 in murine splenocytes [M. hyopneumoniae infection modulates the host defenses to favor the Th2 immune response, which was also reported in dendritic cells against mycoplasma infection [M. hyopneumoniae [M. hyopneumoniae [M. hyopneumoniae can induce mixed Th1/Th2 responses, but more studies are necessary to confirm this dual response and to understand how it is elicited.Despite of the acute host inflammatory immune response elicited by defenses . This iseumoniae . Moreoveic cells . In lineenocytes . These fnfection , promotieumoniae . In facteumoniae ,135–137.M. hyopneumoniae infections [M. hyopneumoniae down-regulates the expression of the CD1a protein in porcine dendritic cells. Since this protein is responsible for displaying antigenic lipids to T cell receptors, its down-regulation by the pathogen (by a still unknown mechanism), decreases the host dendritic cell capacity for antigen presentation. Moreover, M. hyopneumoniae also reduces the overall populations of dendritic and T cells in the porcine nasal cavity in long-term infections, weakening the immune function in the upper respiratory tract [The functional modulation of porcine antigen-presenting cells, such as dendritic cells, was also reported in fections . It was ry tract .M. hyopneumoniae to reduce the populations of immune cells may be associated with the cytotoxic potential of some bacterial proteins displayed at its surface  from M. hyopneumoniae are known to be crucial for M. hyopneumoniae cytotoxicity to host cells, as they induce cell death by apoptosis or necrosis and modulate the inflammatory response [in vitro by increasing the levels of nitric oxide (NO) and reactive oxygen species (ROS) and by caspase-3 activation. Besides LAMPs, the M. hyopneumoniae P68 surface lipoprotein was also identified as an inflammatory and pro-apoptotic mediator to swine immune cells [The ability of  surface . M. hyopresponse ,22,138. ne cells . In thisM. hyopneumoniae LAMPs presented high levels of NO and superoxide anion radicals, which form peroxynitrite, and lead to oxidative stress and activation of the apoptotic cascade [M. hyorhinis, Mycoplasma synoviae, and M. pneumoniae [Nitric oxide and ROS are important players in host immune response, contributing to inflammation and microbicidal response. NO is used as a signaling molecule involved in the regulation of vascular hemodynamics and mediates interaction and recruitment of immune cells during infection . ROS are cascade ,22,138. eumoniae .M. hyopneumoniae, no evidence of such a mechanism has been found. Instead, the differential proteolytic processing resulting in the presence of differential proteoforms on the M. hyopneumoniae surface (discussed in M. hyopneumoniae adhesion determinants) may contribute to antigenic variation, which can be associated with immune response modulation and/or evasion. In this sense, M. hyopneumoniae surface-associated proteases would be indirectly involved in the modulation of the host immune response, by generating proteoforms of adhesins (and possibly of other surface proteins) presenting different epitope sets.The success of host colonization by several mycoplasma species depends on their ability to rapidly alter the antigenic repertoire of their surface, using different genetic systems, such as those of phase- or antigenic variation caused by DNA slippage . HoweverM. hyopneumoniae surface proteases have also been associated with immunomodulation through the proteolytic degradation of pro-inflammatory peptides, such as bradykinin, kininogen, substance P, neurokinin A, and neuropeptide Y [Furthermore, eptide Y ,151. It eptide Y . Moreoveeptide Y ,154. TheMycoplasma hyopneumoniae also displays the ability to evade cellular immune responses. Recently, it was demonstrated that M. hyopneumoniae evades the phagocytic uptake by porcine alveolar macrophages in vitro, although the mechanism underlying this resistance to phagocytosis is still unclear [M. hyopneumoniae phagocytosis by cell. This result indicates that, although M. hyopneumoniae can induce the production of antibodies in the host [M. hyopneumoniae may be the inhibition of the complement pathway. Indeed, it has recently shown that several M. hyopneumoniae surface-displayed proteins bind complement factor H, namely elongation factor thermo unstable (EF-Tu), P146, pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha (PdhA), P46, pyruvate dehydrogenase E1 component subunit beta (PdhB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and three different hypothetical proteins [M. hyopneumoniae cell surface can confer to the bacterium the ability to inactivate the C3b eventually deposited on the M. hyopneumoniae surface, thereby avoiding the consequences of complement activation [M. hyopneumoniae surface and increased M. hyopneumoniae adhesion to epithelial cells [M. hyopneumoniae, these mechanisms contribute to immune evasion and allow more efficient colonization of the respiratory epithelium by the pathogen. unclear . The prethe host , they arproteins . Factor tivation . The binal cells . By counM. hyopneumoniae is also able to evade neutrophil/macrophage extracellular traps , at least in vitro [M. hyopneumoniae to escape the host immune defense [Moreover, it has been shown that in vitro ,158. In  defense .M. hyopneumoniae can persist for several months in the swine host [M. hyopneumoniae to modulate the host immune responses is an important feature that impacts virulence and disease progression. The entire set of immunomodulation mechanisms used by M. hyopneumoniae, however, remains elusive and is worthy of future investigation.Overall, despite fostering a strong immune response, ine host ,43. In tM. hyopneumoniae pathogenesis described thus far are bacterium-induced loss of cilia and cell death, which can also be correlated to the damage caused by the intense host microbicidal response against this pathogen [M. hyopneumoniae virulence mechanism causing damage in swine tissues as will be discussed below.The mechanisms underlying pathogen . On the M. hyopneumoniae infection, the cytotoxicity of proteins displayed at the M. hyopneumoniae cell surface has been associated with immunosuppression because, at least in some cases, these proteins induce apoptosis in host immune cells. In addition, they have also been associated with epithelial cell death, causing physical damage to the swine respiratory epithelium . The putative SPase I, in turn, is likely involved in the secretion of many proteins, including at least some adhesins, to the cell surface or extracellular milieu (as discussed in Protein secretion of M. hyopneumoniae and its impact on pathogen-host interactions). The additional roles of M. hyopneumoniae MnuA and SPase I in M. hyopneumoniae biology may be the result of the specific evolutionary history of Mollicutes, which might have led to the acquisition of additional functions by some proteins to compensate in part for the observed genome reduction events [As discussed in Host immune response and immunomodulation during ithelium , which mpathways . Besidesal cells ,14. MnuAn events .Figure 3M. hyopneumoniae can produce hydrogen peroxide (H2O2) when glycerol is available as a carbon source, while M. flocculare is unable to produce detectable amounts of this toxic molecule [glpO), one of the enzymes responsible for H2O2 production in other mycoplasmas, is present in the genomes of multiple M. hyopneumoniae strains, but is absent in the M. flocculare genome [2O2 production in M. hyopneumoniae strains is still unknown. Moreover, the production of H2O2 by M. hyopneumoniae has the potential to cause oxidative stress to host cells and, at least in vitro, was associated with the antioxidant response elicited in swine respiratory epithelial cells [Mycoplasmas can also produce ROS, by which they can induce cell death and tissue damage ,161. In molecule . Notablye genome . Howeveral cells .In vitro experimental infections of a swine respiratory epithelial cell line with M. hyopneumoniae have also shown that several genes related to ciliary motility, ciliogenesis and ciliary polarization were down-regulated in infected host cells [M. hyopneumoniae infection could modulate the expression of genes related to the mucociliary apparatus, explaining, at least in part, the ciliostasis and loss of cilia observed in infected host tissues.st cells . This evM. hyopneumoniae infection causes apoptosis-related cell events involving endoplasmic reticulum (ER) stress , counteracting at least in part the pro-apoptotic effects and favoring the survival of infected cells [M. hyopneumoniae infection on UPR may represent alternative mechanisms favorable to the pathogen at different times of infection. An anti-apoptotic effect may be interesting in the initial steps of infection, to allow bacterial adherence and colonization of the host respiratory tract. The pro-apoptotic effect would be more prominent later on, contributing to tissue damage.Besides the cilia damage, ) stress . ProteomR stress . It was ed cells . These aM. hyopneumoniae infection were also provided by secretome analyses of cells infected in vitro with pathogenic and nonpathogenic strains and with M. flocculare [M. hyopneumoniae pathogenic strain, suggesting a specific host response to pathogenic M. hyopneumoniae. Among these secreted cell death-related proteins, there were danger-associated molecule patterns (DAMPs), known to be secreted by host cells undergoing apoptosis during pathogen infections to alert the immune system and trigger a pro-inflammatory immune response.Additional evidences of swine epithelial cell death triggered by occulare . Cell deM. hyopneumoniae infection relies not only on its ability to modulate and evade the host immune response, but also on its arsenal of protective mechanisms to cope with different stress conditions imposed by the swine host. Moreover, M. hyopneumoniae survival also depends on the uptake of nutrients provided by host cells. In this final section, these aspects, which are essential for M. hyopneumoniae cell homeostasis, will be presented.The persistence of M. hyopneumoniae infection and Cell damage induced by M. hyopneumoniae infection, M. hyopneumoniae infection is marked by the production of ROS by both host and mycoplasma cells. However, it is not yet clear how this pathogen can protect itself from endogenously and host produced toxic metabolites. The M. hyopneumoniae anti-toxic metabolites arsenal is limited, as its genome lacks genes encoding important antioxidant proteins [M. hyopneumoniae 7448 pathogenic strain in comparison to the nonpathogenic strain J and M. flocculare [M. hyopneumoniae pathogenicity. Furthermore, the M. hyopneumoniae peroxiredoxin (MhPrx) was functionally characterized and shown to protect DNA from ROS-mediated damage in vitro [As discussed in Host immune response and immunomodulation during proteins ,72. Thusocculare . This evin vitro ,165. TheM. hyopneumoniae genes/proteins classically involved in oxidative stress between stress and control conditions [M. hyopneumoniae proteins involved in the oxidative stress response  were among the top 20 most abundant proteins, even in the absence of oxidative stress conditions [M. hyopneumoniae is constitutively expressed, enabling the pathogen being always prompt to respond to this kind of stress. Interestingly, pathogenic and nonpathogenic strains of M. hyopneumoniae presented similar abundances of antioxidant proteins under oxidative stress [M. hyopneumoniae pathogenic strain 7448 was enriched with potential virulence factors, as adhesins, nucleases and lipoproteins, upon exposure to oxidative stress in comparison to control conditions (absence of oxidative stress) and to the M. hyopneumoniae nonpathogenic J strain.In functional “omics” studies, virtually no changes in transcript or protein levels were observed for nditions . At the nditions . Altogete stress . On the Mycoplasma hyopneumoniae must also cope with temperature shifts in the host environment due to the release of pyrogenic cytokines as a mechanism of the immune response [M. hyopneumoniae pathogenic 7448 strain under heat stress.response ,132. A tresponse . Howeverresponse . As discM. hyopneumoniae may require the constitutive presence of proteins involved with stress protection. These findings also suggest that the synthesis of at least some virulence factors, including adhesins, nucleases, and lipoproteins, can be triggered by oxidative and temperature stresses, which would imply increased virulence in infection (stress) conditions. Further studies will be required to improve our understanding of this relationship between stress response and virulence, and also to identify, among the non-annotated genes of M. hyopneumoniae, those possibly encoding so far unknown stress-response proteins.Taken together, the transcriptomic and the proteomic findings suggest that M. hyopneumoniae capacity to respond to stress conditions, its metabolic capacity is essential for survival in the host environment and may contribute to virulence. The M. hyopneumoniae transcription unit coding for myo-inositol catabolism proteins provides a good example of that (and thus far the only one characterized regarding this aspect). In silico metabolic reconstructions for swine respiratory mycoplasmas demonstrated that the metabolic capacities of M. hyopneumoniae, M. hyorhinis and M. flocculare are similar [M. hyopneumoniae is the only one with a transcription unit coding for myo-inositol catabolism proteins in its genome [M. hyopneumoniae strains uptake myo-inositol from culture medium, while M. hyorhinis and M. flocculare are unable to do so [M. hyopneumoniae to persist longer than M. hyorhinis and M. flocculare in the host respiratory tract [Along with the  similar . Howevers genome ,73, suggs genome . Indeed,to do so . This cary tract ,177.M. hyopneumoniae utilizes several pathogenicity mechanisms, some of which are not fully understood. These mechanisms include the adherence to host ciliated epithelial cells, modulation of immune response and other host defenses, and induction of cell damage by both pathogen and host components. Moreover, comparative “omics” studies between M. hyopneumoniae and a nonpathogenic closely related species, M. flocculare, have provided evidence of proteins and functions associated with pathogenicity, as the production of differential adhesin proteoforms presented in the cell surface, the secretion of differential repertoires of proteins, and the overrepresentation of proteases, antioxidant proteins, and adhesion-related proteins in pathogenic M. hyopneumoniae strains. A schematic representation of the M. hyopneumoniae pathogenicity determinants discussed in this review is presented in M. hyopneumoniae pathogenicity, especially those related to the in vivo infection process. Overall, the identification of M. hyopneumoniae pathogenicity determinants and their mechanisms of action are of utmost relevance to discover new and efficient targets for the development of novel diagnostic methods, therapeutic drugs, and preventive vaccines against EP.Regardless of its inherent simplicity, as a small, wall-less bacterium with reduced genome,


Hearing loss and tinnitus are global concerns that can be reduced through hearing protection behaviors . Little is known about the effectiveness of interventions to increase hearing protection use in recreational domains. For the first time we review systematically the effectiveness of such interventions.Systematic searches of nine databases, as well as grey literature and hand-searching, were conducted. Any study design was included if it assessed quantitatively a purposeful attempt to increase hearing protection in recreational settings. Studies were excluded if they assessed noise exposure from occupational sources and headphones/earphones, as these have been reviewed elsewhere. PROSPERO protocol: CRD42018098573.d ≥ 0·3 ≤ 0·5), one a small effect (d ~ =0·2) and two no real effect. Three posttest experimental studies detected small to medium effects (d ≥ 0·3 ≤ 0·5). Studies were rated as “poor quality” and 17 out of a possible 93 behavior change techniques were coded, with the majority targeting the intervention function ‘education’.Eight studies were retrieved following the screening of 1908 articles. Two pretest-posttest studies detected a small to medium effect (Hearing loss and tinnitus due to recreational noise exposure are major public health concerns yet very few studies have examined preventive interventions. The present systematic review sets the agenda for the future development and testing of evidence-based interventions designed to prevent future hearing loss and tinnitus caused by noise in recreational settings, by recommending systematic approaches to intervention design, and implementation of intervention functions beyond education, such as incentivization, enablement and modeling. Approximately one billion teenagers and young adults 12–35 years) are at risk of noise-induced hearing loss and tinnitus due to hazardous recreational noise exposure –35 years contribuPrevious hearing protection systematic and narrative reviews have investigated occupational settings,  recreatiThe use of health psychology theories and models have been discussed as a means to improving hearing health behavior change interventions,  with CouThe 93 techniques in the ‘Behavior Change Technique Taxonomy Version 1’ (BCTTv1)  are the The present systematic review looks beyond occupational settings, includes hearing protection behaviors beyond personal listening device use, considers intervention functions in addition to education , and coThe primary objectives of the current review are to: 1) quantify the effectiveness of hearing protection interventions in recreational noise domains, and 2) identify the active ingredients (behavior change techniques) of such interventions to help future designers of interventions to increase uptake and use of hearing protection behaviors in recreational settings.https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=98573.This is a systematic review that followed the ‘Preferred Reporting Items for Systematic Reviews and Meta-Analyses’ guidance,  which waClinicalTrials.gov, International Clinical Trials Registry. Hand-searching was conducted using the reference lists of papers that were included for full review. Authors of 12 studies were contacted for further clarification before inclusion or exclusion could be determined. The most recent full search was performed on 1st May 2020. All age groups, years and languages were considered for review.Searches were carried out on electronic databases: Cochrane Central Register of Controlled Trials; PubMED; EMBASE; MEDLINE; PsycINFO; Web of Science Core Collection; ComDisDome; Database of Abstracts of Reviews of Effects and Centre for Reviews and Dissemination. Grey literature was searched via: Grey Literature Report, Prospero, Open Grey, A broad search strategy was developed alongside a research librarian to capture the large variety of hearing protection interventions . Keywords and structure to each search were altered depending on each electronic database  and ‘single group pretest-posttest designs’ . Secondary outcomes included hearing health outcomes such as hearing loss or tinnitus as well as perceptions of capabilities, opportunities and motivations to engage in hearing protective behaviors. Interventions were also coded for the presence of the active ingredients aimed at delivering the desired change, namely, the behavior change techniques. The 93 techniques are clustered into 16 groups  within the behavior change technique taxonomy (BCTTv1), each technique has its own identifiable number for coding purposes, with the first digit identifying the cluster group, and the second the order of the technique within said group  . These tThe screening of abstracts and titles commenced with the 1st reviewer (ML) screening all articles and 2nd reviewer (SL) screening 10% of these reports for comparison. The 1st reviewer (ML) read all the fully eligible studies, with the 2nd and 3rd reviewers (SL and CJA) reading a proportion of the eligible studies, as well as discussing any query papers for full inclusion. Several authors were contacted directly to clarify information, leading to both inclusion and exclusion of studies for full review.Data were extracted using a pre-designed and piloted data extraction form, including general study information, study characteristics, participant characteristics, intervention design (including behavior change techniques), and outcome measures (see Table Outcome measures were assessed in numerous different formats (often with multiple categories) and so the main outcome was recoded as ‘never-performers’ (never performed a hearing protection behavior) and ‘ever-performers’ (performed a hearing protection behavior at least some of the time). Quantitative data were extracted by the 1st and 2nd reviewer, and compared for agreement. The 3rd reviewer checked over the extracted quantitative data for agreement and calculation errors. One paper written in German was translated and included in the analysis . Where sd was calculated using Z statistic and sample size (N) [d could be inferred from the presented adjusted means and sample sizes provided [For dichotomous data, risk ratios and Z statistics were extracted; Cohen’s size (N) . In termsize (N) . One stusize (N) ; Cohen’sprovided ..The 1st and 2nd reviewers, trained via the University College London online behavior change technique taxonomy v1 program,  coded al1n = 708). Following screening, a total of 59 reports were eligible for full review, eight of which were suitable for inclusion according to our criteria ; measurement of ‘intended behavior’ and not actual behavior (n = 13); no recognizable intervention (n = 11); occupational noise exposure (n = 6); unretrievable data (n = 5); acoustic intervention (n = 1); and contained historic data superseded by a subsequent paper (n = 1).Initial searching recovered 2616 articles, of which 1908 (73%) were eligible for screening after the removal of duplicates , a range of follow up periods (16 weeks to 16 years) and a large population range (39 participants up to 1535 participants). All studies examined use of earplugs, one study additionally measured regeneration breaks alongside earplugs,  and a fuSeven of the included interventions were described as hearing conservation/education programs, , 36–41 wd = 0·14) during loud events with German school children in the 5–6 weeks following the intervention. No secondary outcomes were reported. The study had high risk of allocation concealment bias as there was no randomization, and detection bias (blinding of outcomes) as blinding was not mentioned.When examining the five single pretest-posttest designs we compared baseline data with final follow-up. Neyen’s  hearing d = 0·21) with increased use of earplugs at discotheques within Austrian high school children, 1 year post intervention. The children received four teaching sessions that included information on the risks of noise exposure  alongside multimedia/listening examples , and presentation of ear protection devices . No secondary outcomes were reported. The study had high risk of allocation concealment bias, performance bias, and detection bias (blinding of outcomes), due to no randomization or mention of blinding.Weichbold and Zorowka’s  hearing d = 0·14) in earplug use 1 year post intervention, but no real effect (d = 0.06) of increased regeneration breaks. There are no reported secondary outcomes within this paper. Similarly, as within Weichbold and Zorowka [Weichbold and Zorowka  continue Zorowka  this stud = 0·34) in increased use of hearing protection devices from baseline  to 6 months post intervention  within Belgian young adults. Secondary outcomes assessed the audiometric thresholds of the participants at baseline and 6 months post intervention, but no significant effects were found. Participant self-reports between sessions indicated that 28·2% of participants perceived their hearing loss to have increased, with 20·5% reporting that their tinnitus increased. However, the study had high risk of allocation concealment bias, performance bias, and detection bias, due to no randomization or mention of blinding.Keppler et al.’s  hearing d = 0·34) in increased use of devices while in noisy recreational environments. There are no reported secondary outcomes within this paper. The study was high risk of allocation concealment bias, performance bias, and detection bias, due to no randomization or mention of blinding.The aim of Gilles and Van de Heyning’s  hearing d = 0·30) in the difference between groups for hearing protection use during gunfire. However, no effect was found for ‘all recreational activities’ (d = 0·07) or ‘personal stereos’ (d = 0·03). The program consisted of information on the ear and hearing delivered by a study educator, alongside videotape examples , and demonstrations/practice of how to fit hearing protection devices ; concluding with the provision of free devices that continued over the course of the intervention, alongside additional information . Secondary outcomes assessed changes in audiometric threshold frequencies from baseline across five categories, however no significant differences between groups were found for any of the categories. The study had high risk of detection bias as there is no mention of blinding, attrition bias due to dropouts, and reporting bias due to collection methods not known to be valid or reliable. There was also a high risk of selection bias due to a low agreement rate at the 16 year follow up, with an overall low risk of allocation concealment bias through initial randomization.When examining the three experimental post-test designs we compared control and intervention data at the final follow-up. Marlenga et al.  completed = 0·30) in differences of earplug use at live music events at 16 weeks post intervention within Australian young adults who regular attended live events. However, it is worth noting for this study that 82% of participants had used earplugs previously. This is unusually high and not representative of the general population, which is ∼5% for these types of events [Beach et al.’s  hearing f events , 47. Secd = 0·31) with greater use of earplugs being observed when earplugs were freely available at rock concerts : BCTTv1: 10.1). However, no secondary outcomes were measured meaning that it is not clear what was the mechanism of action. We considered this to be a quasi-study as it did not randomize participants but instead had control/interventions groups defined by venues, and was therefore high risk of allocation concealment bias; with large discrepancies in sample size between comparison groups. The study also had high risk of detection bias, attrition bias, and reporting bias, due to no mention of blinding or dropouts/withdrawals, and collection methods not known to be valid or reliable.When comparing control venues (three concerts) to experimental venues (three concerts) Cha et al.  detectedd) and behavior change techniques were extracted. Throughout screening it was apparent that interventions tended to measure people’s intentions to protect their hearing, as opposed to their actual hearing protection behaviors, [This review set out to assess, the effectiveness of recreational hearing protection interventions, and for the first time, to identify the active ingredients of these interventions. Considering hearing symptoms through recreational noise exposure is a global concern that is highly preventable,  only eighaviors, –52 with haviors,  and thisd), measuring an increase, or difference, in earplug use across a range of recreational contexts. However, those five studies had strong risk of bias, particularly detection bias (blinding of outcome assessment), with poorly reported methodologies affecting replication credentials.The most common hearing protection behavior reported in each study was the use of earplugs. This supports previous evidence that hearing protection devices are the most accessible preventive method against overexposure to recreational noise , 48, 54.The overall increase in ‘ever-performers’ of earplugs across all eight studies ranged from 3% – 14·6%; few studies indicate people always or often use earplugs, indicating many people are at risk of hearing symptoms in a short space of time, , 14 whenUnique to the present systematic review was the coding of behavior change techniques deployed within hearing protection interventions, with a total of 17 coded as involving behavior change from a possible 93 techniques, the majority of which link to the intervention function ‘education’ . With so few techniques being identified it leaves a large scope to deploy previously untested techniques to bring about changes in hearing protection behaviors. Coding did reveal other less frequently used intervention functions, such as ‘environmental restructuring’ (adding objects to the environment: BCTTv1:12.5), that yielded consistent effect sizes when implemented. This particular deployment provided the most reliable relationship throughout the review; it was seen in all three experimental post-test studies, which provided earplugs within the environment, and all three measured a small to medium effect, for at least one recreational context. ‘Environmental restructuring’ should thus be considered for future recreational hearing protection interventions. Based on coding it would also be valuable to try other approaches in conjunction with environmental restructuring, such as incentivization : BCTTv1.10.1), enablement (prompts and cues: BCTTv1:7.1) and modeling .Studies indicate a lack of theory applied during the design process, or at least a lack of a description of theory within the methodologies. These issues have been raised by health psychologists previously, in that researchers face an uphill battle to replicate interventions to further test outcomes, , 55 due Limitations of this review include the inability to retrieve all data from the authors who were contacted directly. The provision of results that were missing or unclear in the original publications may have enabled a meta-analysis to be performed.The present systematic review found very few hearing protection interventions addressing recreational noise exposure, a global hearing health concern. However, ‘environmental restructuring’ through the provision of earplugs (adding objects to the environment: BCTTv1:12.5), showed promise and might be considered a starting point for future interventions. Further hearing protection intervention studies should be conducted that employ randomized controlled designs, use systematic approaches to intervention development , considAdditional file 1. Search Strategy Supplement.Additional file 2. Supplement Table.