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In the recent millennium, the constant trend of change in the demands of the community as well as transforming the trend of knowledge production has highlighted the necessity for researchers to adopt a more comprehensive approach . Increasingly, many academic disciplines are utilizing qualitative research (qr) as the qualitative method investigating the why and how of the process of a developed concept (1, 2). Qualitative research is sometimes defined as interpretive research, and as interpretations can be incorrect or biased, the findings may be controversial (3). However, qualitative research is not only useful as the first stage of quantitative research, but can also play a key role in validating it or in providing a different viewpoint on the same social phenomena (4). Qualitative studies tend to use methods that result in text production rather than numerical outputs . Given that the researcher is considered to be the research instrument, and the plan of inquiry needs to be developed and altered as the study progresses, a qualitative researcher cannot depend upon traditional approaches to address certain concerns such as bias and credibility . Therefore, learning from a series of mistakes is often considered an integral part of qualitative research (5, 6). In this study, a literature review was carried out in international electronic databases including pubmed, web of sciences, cumulative index to nursing and allied health literature (cinahl), scopus, ebsco, embase and science direct without any time limitation, using the search terms qualitative research, these keywords were also searched on national electronic databases including scientific information database (sid), iran medex and medical articles library (medlib) using the same strategy . Authors of the present article endeavor to shine a light on the ethical issues affecting researchers and propose strategies to face the ethical challenges of qualitative studies, so as to provide applicable and trustworthy outcomes . This could be the basis for the formulation of specific ethical guidelines in this regard . Up to the 1970s, qualitative research was solely employed by anthropologists and sociologists . During the 1970s and 1980s, however, it was favored by various disciplines and experts of different branches of science and humanity such as health care, psychology, nursing, management, political science, education, and communication studies (2, 7). Qualitative research has been conducted in the field of nursing in order to identify, describe and explain related concepts, experiences and phenomena and to develop the nursing knowledge ., qualitative research has been performed to achieve the concepts of patient care and other main perceptions in the nursing profession . Qualitative studies provide nurses with sensitivity to the lived experiences of individuals from different nursing care aspects (4, 8). In the case of nurses who perform qualitative research, ethical issues are raised when the nurse - patient relationship in the research area leads to some degree of therapeutic communication for the participants (9). Thus, nurse researchers must be aware of the impact of the questioning on the participants, and in order to decrease such harmful effects on human subjects, the reflexive approach is recommended (10). In qualitative studies researchers are often required to clarify their role in the research process (11). In the qr procedure the researcher is involved in all stages of the study from defining a concept to design, interview, transcription, analysis, verification and reporting the concepts and themes . Therefore, whenever instruments are involved in qualitative research, a human being will be an integral part of the process (12). It is argued that humans have increasingly become the instrument of choice for naturalistic research due to certain characteristics: they are highly responsive to environmental stimuli, have the ability to interact with the situation, pull together different pieces of information at multiple levels simultaneously, and perceive situations holistically; moreover, they are able to process findings the instant they become available, can present immediate feedback, and feel unusual responses . Nevertheless, researchers need to improve the abilities that make them appropriate human instruments and consequently, their interpersonal skills are of major importance in natural settings and study processes (table 1) (13, 14). The relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns, and qualitative researchers face dilemmas such as respect for privacy, establishment of honest and open interactions, and avoiding misrepresentations (19). Ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises . In such cases, disagreements among different components such as participants, researchers, researchers discipline, the funding body and the society may be inevitable (20, 21). Some important ethical concerns that should be taken into account while carrying out qualitative research are: anonymity, confidentiality and informed consent (22). According to richards and schwartz findings (22), the term confidentiality conveys different meanings for health care practitioners and researchers . For health care practitioners, confidentiality means that no personal information is to be revealed except in certain situations . For researchers, however, the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study (22, 23). The researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means . When highly sensitive issues are concerned, children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study, and ideally, during data gathering sessions . It is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used (24, 25). Informed consent has been recognized as an integral part of ethics in research carried out in different fields . For qualitative researchers, it is of the utmost importance to specify in advance which data will be collected and how they are to be used (26). The principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language . Clarifications need to include the following issues: the nature of the study, the participants potential role, the identity of the researcher and the financing body, the objective of the research, and how the results will be published and used (27). Informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses (26). Many people consider it necessary to participate in research that their peers, community and/or society may benefit from . Therefore, qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy (5). The qualitative method is utilized to explain, clarify and elaborate the meanings of different aspects of the human life experience . Therefore, researchers can interpret people s experiences because they are involved in human activities . The principle of no harm to participants ought to be considered by researchers, who should be aware of the potential harms that might be inflicted upon study subjects . Obviously, sometimes a conflict between the right to know (defended on the basis of benefits to the society) and the right of privacy (advocated based on the rights of the individual) may happen (27, 28). There are several effective strategies to protect personal information, for instance secure data storage methods, removal of identifier components, biographical details amendments and pseudonyms (applicable to names of individuals, places and organizations) (27). Researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation . It is getting increasingly common for research ethics committees to seek documented proof of consent in a written, signed, and ideally, witnessed form . Researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise (29). Furthermore, in investigations of sensitive topics where written consent puts the informants at risk, audio recorded oral consent would be more appropriate (30). Therefore, researchers should seriously consider the potential impact they may have on the participants and vice versa, and details of such interactions should be clearly mentioned in research proposals (23). Overall, the role of the researcher as (a) stranger, (b) visitor, (c) initiator, (d) insider - expert or other should be well defined and explained (3). As brenner quoted kvale state that, preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting (30). In qualitative research, data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences . The researchers, therefore, play the role of a mediator between the experiences of the respondents and the community of concerned people (28, 31). The post - interview comment sheet could assist the researcher to note the feelings of informants, as well as interpretations and comments that occurred during the interview (32). Data collection needs to be as overt as possible, and findings should be recorded . Although there is no guarantee of absolute confidentiality, openly recording field notes assists participants to decide what they wish to have on the record . In health care research, the problem may be even more exaggerated as the researcher is sometimes the health provider as well (33). In comparison with other research methods, ethnography has singular characteristics . When a researcher aims to study the culture of certain people, living amongst them is inevitable . This method of collecting data is a subject of debate from an ethical point of view . Participants should always be aware of the information that has been obtained and is being recorded, and consent to it . Sometimes this cannot be achieved easily and conflicts may happen, as in studies of cultural and ethnic characteristics (18). The physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field . For instance, when criminals or a group of war veterans suffering from a disease are the subject of a study, the risks involved in living amongst them should be considered . Ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run (33, 34). Researchers can benefit from supervision sessions directed at learning, mentoring and skill development, all of which can foster their ability to carry out research without risking their health . Adequate professional supervision (which may be outside of the university) can be of service to researchers in dealing with the potential stress associated with the study (35 37). In order to gain explicit data, these steps include participant observation, ethnographic record, descriptive observation, taxonomic analysis, selected observation, componential analysis, discovering the cultural theme, cultural inventory, and finally writing ethnography (38, 39). Researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues . The probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated . Interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion, while allowing ample time for analysis of the objective and emotional aspects of the research . It is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done (40 42). The relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns, and qualitative researchers face dilemmas such as respect for privacy, establishment of honest and open interactions, and avoiding misrepresentations (19). Ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises . In such cases, disagreements among different components such as participants, researchers, researchers discipline, the funding body and the society may be inevitable (20, 21). Some important ethical concerns that should be taken into account while carrying out qualitative research are: anonymity, confidentiality and informed consent (22). According to richards and schwartz findings (22), the term confidentiality conveys different meanings for health care practitioners and researchers . For health care practitioners, confidentiality means that no personal information is to be revealed except in certain situations . For researchers, however, the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study (22, 23). The researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means . When highly sensitive issues are concerned, children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study, and ideally, during data gathering sessions . It is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used (24, 25). Informed consent has been recognized as an integral part of ethics in research carried out in different fields . For qualitative researchers, it is of the utmost importance to specify in advance which data will be collected and how they are to be used (26). The principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language . Clarifications need to include the following issues: the nature of the study, the participants potential role, the identity of the researcher and the financing body, the objective of the research, and how the results will be published and used (27). Informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses (26). Many people consider it necessary to participate in research that their peers, community and/or society may benefit from . Therefore, qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy (5). The qualitative method is utilized to explain, clarify and elaborate the meanings of different aspects of the human life experience . Therefore, researchers can interpret people s experiences because they are involved in human activities . The principle of no harm to participants ought to be considered by researchers, who should be aware of the potential harms that might be inflicted upon study subjects . Obviously, sometimes a conflict between the right to know (defended on the basis of benefits to the society) and the right of privacy (advocated based on the rights of the individual) may happen (27, 28). There are several effective strategies to protect personal information, for instance secure data storage methods, removal of identifier components, biographical details amendments and pseudonyms (applicable to names of individuals, places and organizations) (27). Researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation . It is getting increasingly common for research ethics committees to seek documented proof of consent in a written, signed, and ideally, witnessed form . Researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise (29). Furthermore, in investigations of sensitive topics where written consent puts the informants at risk, audio recorded oral consent would be more appropriate (30). Therefore, researchers should seriously consider the potential impact they may have on the participants and vice versa, and details of such interactions should be clearly mentioned in research proposals (23). Overall, the role of the researcher as (a) stranger, (b) visitor, (c) initiator, (d) insider - expert or other should be well defined and explained (3). As brenner quoted kvale state that, preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting (30) in qualitative research, data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences . The researchers, therefore, play the role of a mediator between the experiences of the respondents and the community of concerned people (28, 31). The post - interview comment sheet could assist the researcher to note the feelings of informants, as well as interpretations and comments that occurred during the interview (32). Data collection needs to be as overt as possible, and findings should be recorded . Although there is no guarantee of absolute confidentiality, openly recording field notes assists participants to decide what they wish to have on the record . In health care research, the problem may be even more exaggerated as the researcher is sometimes the health provider as well (33). In comparison with other research methods, ethnography has singular characteristics . When a researcher aims to study the culture of certain people, living amongst them is inevitable . This method of collecting data is a subject of debate from an ethical point of view . Participants should always be aware of the information that has been obtained and is being recorded, and consent to it . Sometimes this cannot be achieved easily and conflicts may happen, as in studies of cultural and ethnic characteristics (18). The physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field . For instance, when criminals or a group of war veterans suffering from a disease are the subject of a study, the risks involved in living amongst them should be considered . Ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run (33, 34). Researchers can benefit from supervision sessions directed at learning, mentoring and skill development, all of which can foster their ability to carry out research without risking their health . Adequate professional supervision (which may be outside of the university) can be of service to researchers in dealing with the potential stress associated with the study (35 37). In order to gain explicit data, ethnographers need to know the role of instrument details . These steps include participant observation, ethnographic record, descriptive observation, taxonomic analysis, selected observation, componential analysis, discovering the cultural theme, cultural inventory, and finally writing ethnography (38, 39). Researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues . The probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated . Interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion, while allowing ample time for analysis of the objective and emotional aspects of the research . It is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done (40 42). It is argued that qualitative research that deals with sensitive topics in depth can pose emotional and other risks to both participants and researchers . Clear protocols for dealing with distress should be in place so that both parties involved in research can use them if necessary . It is not usually easy to predict what topics are likely to lead to distress, and researchers should therefore receive sufficient training in predicting traumatic situations . Preventive measures for researchers who carry out sensitive qualitative studies should include official arrangements for a peer support program consisting of a list of researchers who are involved, or a constellation of researcher support activities aiming at improving psychological fitness in the form of a professional confidence building module . Other such measures include offering adequate supervision to provide opportunities for self - development and self - care, and facilitating the process of self - reflection and self - monitoring . Strategies for emotional distancing need to be considered and adopted if the research topic or participants have the potential to be emotionally challenging . An appropriate planning should be in place before the commencement of the fieldwork, and it must be perfectly clear how the study should be conducted and what level of relationship development is necessary . Measures must also be taken so that levels of self - disclosure, objective displays of emotion during the interviews, and strategies to end the relationships are well defined and communicated . One of the most prominent tasks of qualitative researchers is to minimize the flaws in observation and endeavor to gain truthful knowledge . Therefore, it is necessary for researchers to continuously update their investigation skills in terms of methodology and find novel techniques to better carry out studies in the field of health and sociology . As explained before, qualitative research is carried out in natural settings, which requires researchers to work in close collaboration with other members of the team and under direct supervision to discuss and resolve issues as they arise . Therefore, development of practical strategies and communicating them to researchers can be of great benefit and assist them in conducting more perceptive qualitative studies . It is noteworthy that such research should be directed towards making a difference in people s lives, improving care delivery in different settings and at all levels, and providing a framework for health sciences without any ethical disturbances . As a result of the extensive body of research in the field of medical sciences, research ethics committees are formed to provide independent advice to participants, researchers, funders / sponsors and healthcare organizations on the extent to which research proposals comply with universally endorsed ethical standards . In the history of social and medical science, there have been a few research studies that seriously injured people, and many more in which their welfare was not sufficiently protected . To return to the matter of privacy, the researcher should not rely solely on the informant to identify possible intrusion, but needs to work at anticipating it in advance . Confidentiality does not necessarily preclude intrusion, as anonymity by itself is not enough to protect a person s privacy or prevent disclosure of personal issues . Investigators should refrain from soliciting private information that is not closely related to the research question . Considering the aforementioned challenges, it is recommended to conduct further research in order to provide meticulous and explicit ethical protocols, guidelines and codes with respect to qualitative studies.
Since the discovery of penicillin in 1928 by a scottish scientist alexander fleming followed by the release of many other earlier drugs onto the market to treat infection, the development of drug resistance in various sectors including aquaculture has been reported [13]. The misuse and abuse of the antimicrobial drugs are among the important factors that have contributed to the rise of resistant microbes around the world . Antibiotics, which have saved millions of lives and were also known as miracle drug in the past, are no longer the ultimate way for the treatment of infections because bacteria have continued to develop multiple resistance towards many different types or classes of the drugs . Antimicrobial agents have been widely used in fish farming for either therapeutic, prophylactic, or other purposes . The antibiotics are normally used to increase growth as well as feed efficiency in the animals . However, some of the antibiotics have been frequently used in both veterinary and human medicine such as sulfonamides, chloramphenicol, tetracycline, nitrofurans, oxytetracycline, neomycin, erythromycin, streptomycin, prefuran, and enrofloxacin . The evolution of bacteria towards antibiotic resistance has been accelerated distinctly by selective pressure due to inappropriate and overuse of the antibiotics [3, 9]. In the efforts to cope with this problem, scientists have accelerated the search for alternative antimicrobial agents by screening many potential sources including medicinal plants [10, 11] and microbes [12, 13]. Aquaculture is an important sector in the agriculture industry and is rapidly growing to meet the world's demands for protein source . This sector is challenged with the diverse type of diseases and bacterial infections; and antibiotics are an excellent tool to circumvent the problem [14, 15]. The presence of bacteria with multiple antibiotic resistance found in food products has become a threat to public health as there is potential that the carried or acquired genes are transferred to other bacteria of clinical significance [1618]. Some antibiotics which are commonly used in food - producing animals are also used in human medicine, reducing the antibiotic's efficiency when treating infections and increasing the morbidity and mortality associated with diseases . The resistance limits the choice of antibiotics for the disease treatment [4, 16, 19, 20]. The use of antibiotics needs to be monitored from time to time to evaluate the emergence and spread of bacterial resistance towards antimicrobial agents [5, 21]. There is limited data on the antibiotic resistance of bacteria in fish and other cultured organisms sampled directly from fish farms as well as the aquaculture environment . Sampling was carried out at aquaculture farms located at selected districts in sarawak, malaysian borneo, including kuching, bintulu, limbang, miri, and sampadi (lundu). Three types of samples were collected which were the sediment, water, and cultured species . Cultured species refers to fish or shrimp . In kuching, bintulu, and miri, the cultured organisms collected were the fish while in limbang and sampadi (lundu), the cultured organisms collected were shrimps . The isolation, designation, and identification of the isolates were carried out as reported by kathleen and coworkers earlier . Ninety - four bacterial isolates from 17 different genera were assessed for their susceptibility to different antibiotics utilizing the disk diffusion method according to method described by clinical and laboratory standards institutes (clsi) on mueller - hinton agar (mha). The bacterial groups and the antibiotics tested are listed in table 1 . Briefly, fresh bacterial culture with 0.5 mcfarland turbidity was swabbed onto the mha surface using sterile cotton buds . Commercial antimicrobial discs (oxoid, uk) were evenly embedded onto the inoculated agar incubated at 37c for 18 to 24 hours . Escherichia coli strain from american type culture collection (atcc) 25922 was used as control . The diameter of complete inhibition zone formed around the antibiotic discs was measured to the nearest whole millimeter using standardized ruler . The results obtained were analyzed as resistant or susceptible according to standard interpretative table by clsi and bonnet . Multiple antibiotic resistance (mar) index was then determined for each isolate by dividing the number of antibiotics to which an isolate is resistant with the total number of antibiotics tested . The mar index is an indicator to identify the risk contamination that is potentially hazardous to human . Calculated value of more than 0.2 indicates that the isolates were isolated from high risk sources . In this study, commonly used antibiotics in both veterinary and human medicine were selected for the antibiotic susceptibility testing . The antibiotic selection also depends on the bacterial genera because different bacterial genera require different classes of antibiotics for optimal antibacterial activity . Some of the antibiotics were not tested in this study because some bacterial genera are naturally resistant to certain classes of antibiotics; hence the antibiotics were excluded from the analysis . The antibiotic profile of the bacterial isolates from the aquaculture fish and shrimps and their environment is shown in table 1 . The bacterial isolates showing the top five highest percentages of resistant were towards streptomycin (85%, n = 20), followed by ampicillin (56.8%, n = 74), penicillin (47.1%, n = 51), erythromycin (43.1%, n = 51), and cephalotin (42.3%, n = 71). The bacterial isolates showing the top five highest percentages of susceptible were towards gentamicin (1.1%, n = 90), followed by tobramycin (2.2%, n = 90), chloramphenicol (4.0%, n = 75), norfloxacin (5%, n = 80), and amikacin (5.6%, n = 90). The other bacterial isolates and their percentage of resistant are shown in table 1 . In this study, the antibiotic resistant patterns for all isolates were also determined to monitor the spread of antibiotic resistance . Sixty - one different antibiotic resistance patterns were observed among the isolates through this study (data not shown). The resistance patterns were highly variable; 20.2% (n = 94) isolates have no resistance towards any antibiotics tested, 16% (n = 94) isolates were resistant to only one antibiotic, and 63.8% (n = 94) isolates were resistant to multiple antibiotics . . Isolated from water in limbang aquaculture farm was resistant towards 12 out of 19 antibiotics tested, which was the highest amount of antibiotic resistance observed in this study . Most isolates (53.2%, n = 94) possess distinctive pattern . However, there are also patterns (11 patterns) shared by 2 or more bacteria isolates . The pattern shared by most isolates (19 isolates, n = 94) is 0% resistance (mar index equal to 0). There is at least an isolate with 0% resistance towards all antibiotics tested in all sampling locations except for bintulu . There is difference in the resistance pattern for bacteria isolated from water, sediment, and the cultured organisms . Bacteria that were isolated from the same pond and same source of origin (sediment, water, or cultured organisms) possess different antibiotic patterns . The antibiotic resistant patterns of the bacterial genera are more influenced by the location from where the isolates were isolated rather than their genus . This analysis has been used to group the different sources from where the bacteria were recovered using the frequency of antibiotics resistance . Isolates with mar <0.2 were determined as isolates recovered from low risk sources of contamination while isolates with mar> 0.2 were from high risk sources . In this study, analysis on overall isolates regardless of sampling location revealed that 63.1% (n = 94) isolates belong to group mar <0.2 while 36.9% (n = 94) isolates belong to group mar> 0.2 . Bintulu (btl) has the highest percentage (73.3%, n = 16) of bacteria isolated from high antibiotic - contaminated sources while sampadi (spd) has the lowest percentage (11.7%, n = 17). Assessment of the antibiotic resistance among aquaculture bacteria against antimicrobial agents is important for update on the bacterial antibiotic resistance patterns . It is part of a surveillance system aiming at monitoring emerging antibiotic resistant bacteria and their widespread . Isolation of antibiotic resistant bacteria from aquaculture products and aquaculture environment indicates the health risk associated with the aquaculture . There had been reports on detection of antibiotic resistance genes in bacteria isolated from aquaculture products that can be transferred to human microbiota . This matter is becoming critical if the resistance genes are transferrable to human pathogens . Providing effective treatment towards this infection becomes a problem to the medical practitioners as the choice of antibiotics is limited . Thus, any source of antibiotic resistant bacteria must be carefully monitored . In antibiotic resistance analysis, the history of antibiotic application in particular area is reflected by the percentage of bacterial resistance to antibiotics . The frequency of antibiotics usage is related to the level of resistance among bacteria [2, 29]. In this present study, similarly, lim and kasing and hatha et al . In their respective studies observed that almost none of the bacteria tested were resistant to gentamicin and chloramphenicol . Low frequency of antibiotic resistant bacteria may indicate the less activity associated with the contamination of antibiotics in the area . The use of chloramphenicol in aquaculture has been banned in certain countries including malaysia, korea, and japan since 1983 . This is because of the adverse effect of chloramphenicol in humans, even at very low dosage, which can cause other side effects like severe or fatal blood problems . The problems associated with blood are like anemia and grey syndrome, a syndrome of cyanosis and cardiovascular collapse, which occurs particularly in newborn babies . This poses risks to the workers handling the products containing this antibiotic [5, 31]. Banning of antibiotics has aided in reducing the number of antibiotic resistant bacteria in an environment . Reported the significant reduction in the frequency of vancomycin - resistant enterococci from broiler after the banning of vancomycin in denmark in 1995 . High percentage of streptomycin, ampicillin, and penicillin g resistance was observed in this study . Similarly, high ampicillin and streptomycin resistance were also observed by zhang et al . In their study on antibiotic resistance detection in e. coli strains isolated from two different aquaculture systems in south china . Also recorded high resistance of ampicillin . In another study, identified isolates from mangrove soil in malaysia were 100% resistant towards ampicillin and penicillin while 77.8% of the isolates were resistant towards streptomycin . A study carried out by akinbowale et al . Recorded 54.8% ampicillin resistance of aquaculture bacteria in australia . In contrast with the results in this study, low resistance of streptomycin (21.2%) and ampicillin (6.1%) was observed in a study on 33 marine bacteria isolates by you et al . . In this study, farmers in the aquaculture farms where the sampling was carried out stated that there was no history of utilization of antibiotics in their farms . Despite the absence of antibiotics as medicines or in feeds, high resistance was observed to commonly used antibiotics such as streptomycin, ampicillin, and penicillin . High resistance of ampicillin and streptomycin in this study and other researches was not surprising as these antibiotics were among the first antibiotics introduced since the discovery of penicillin . Although antibiotic usage in the studied farms has been stopped decades ago, antibiotic contamination is still possible as there may still be residues of antibiotics left in the environment . Bacteria isolated from the sediment of the aquaculture pond may have acquired antibiotic resistance characteristics through unconsumed foods and the cultured organism's faeces that contain the remaining antibiotics [3941]. The unconsumed foods and faeces will be deposited in the sediment and the composition of sediment microbiota will be altered due to selective pressure . Fish feeds were a possible reservoir for antibiotic resistant genes in the farm sediments . In this present study, bintulu (btl) recorded the highest percentage (73.3%, n = 16) of bacteria isolated from high antibiotic - contaminated sources (mar> 0.2) while sampadi (spd) aquaculture farm recorded the least number (11.7%, n = 17) of mar index> 0.2 isolates . The aquaculture farm is located at an area that has many agriculture activities (e.g., pig farming, duck farming, and dragon fruit cultivation) surrounding it . There is the possibility that antibiotics from the animal feeds or medications were absorbed into the sediment causing bacterial selection in the nearby environment . Multiple antibiotic resistant bacteria might have travelled through water from these agriculture activities to the aquaculture ponds . Buschmann et al . In their study suggested that antibiotic resistant bacteria in mariculture farm may be transported by water current which flows from surrounding farms that utilize antibiotics excessively . Antibiotic resistance pattern may vary depending on the geographical locations and selective pressure [43, 44] and these patterns change rapidly from time to time . The different patterns exhibited by different strains or species suggest how complex is the understanding of the antibiotics resistance in the study area . In this study, the resistance patterns were highly variable; 20.2% (n = 94) isolates have no resistance towards any antibiotics tested, 16% (n = 94) isolates were resistant to only one antibiotic, and 63.8% (n = 94) isolates were resistant to multiple antibiotics . Awareness on antibiotic resistance threat should be instilled in the community regardless of age as precaution and prevention step against dissemination of antibiotic resistant bacteria . Many surveillance programs had also been introduced to monitor the emergence and spread of antibiotic resistant bacteria . It has also been suggested by son et al . That plasmid screening should be considered as an additional procedure in the monitoring programs to trace antibiotic resistance dissemination . Alternatives to treatment using antibiotics like probiotics, vaccines, and antimicrobials from plants should be also considered . However, most of the alternatives could not really effectively replace antibiotics, so they act as additional preventive measures rather than alternatives . The mar indexing has revealed that 63.1% of the isolates came from low antibiotic usage area . Although antibiotic resistance in aquaculture in the malaysian borneo is still in its infancy, the need for continuous monitoring of the antibiotic resistance patterns should not be overlooked and the community should be educated on the awareness of antibiotic resistance and its implication on human health and environment.
With an incidence rate of 0.92%, maternal puerperal lower extremity nerve injuries are rare . Lateral femoral cutaneous neuropathy (meralgia paraesthetica) is the most common, followed by femoral neuropathy . Nerve injuries are more likely to occur in nulliparae, in cases of prolonged stages of labour and assisted vaginal deliveries . The presented case involved a 32-year - old woman with bilateral heel numbness due to bilateral neuropathy of the medial calcaneal nerve due to external compression during vaginal delivery . A 32-year - old woman presented to our neurology outpatient clinic with tingling and numbness in both heels . She continuously complained about these sensations ever since she had given vaginal birth to her first child 3 months earlier in a hospital . Spinal analgesia (lumbar level, bupivacaine / sufentanyl bolus, followed by a continuous administration of 8 ml / h) was used during labour . The first stage of labour was prolonged, and because of fetal compromise during the expulsion phase, a vacuum delivery system (palmpump kiwi), together with episiotomy, was used for fetal extraction . After delivery, the epidural analgesia was continued during the closure of the episiotomy with sutures . The patient did not use any other medication and had no history of neurological disorders or risk factors for neuropathy (diabetes, hypothyroidism or hereditary liability to nerve pressure palsy). On examination, she had hypoaesthesia in the area of both heels . The strength of both the leg and foot muscles was normal, and so were the deep tendon reflexes ., a near - nerve needle conduction study of the medial calcaneal nerve was not performed . A diagnosis of bilateral neuropathy of the medial calcaneal nerve, most likely due to longstanding pressure on both heels with epidural analgesia as a predisposing factor, was made . Three months after presentation (6 months after delivery), her numbness had diminished, but had not completely disappeared . Bilateral neuropathy of the medial calcaneal nerve due to external compression during delivery using epidural analgesia is very rare . Only 1 patient with numb heels has been reported in a large study, describing 2,615 women who received epidural anaesthesia during delivery . However, no details about delivery or analgesia in that patient were mentioned . In epidural analgesia during delivery, a motor block is unwanted, and thus voluntary muscle movements are possible, which makes longstanding external compression of nerves uncommon . In our patient, prolonged labour with spinal analgesia and a continuation of analgesia during episiotomy probably masked signs of external compression on both heels . The medial calcaneal nerve arises from the tibial nerve at the medial side of the ankle, perforates the laciniate ligament, travels downwards, passing the bony projection below on the inner side of the ankle, and supplies the skin over the medial aspect of the heel . Patients with risk factors, such as diabetes, are at a greater risk of developing neuropathy during epidural anaesthesia . However, also without any risk factors, the clinician should be aware of the absence of the normal reaction to longstanding pressure on a nerve during epidural analgesia . Preventative measures like soft gel pads or intermittent posture changes could possibly prevent neuropathy of the medial calcaneal nerve in long - lasting epidural analgesia . Bilateral medial calcaneal nerve neuropathy as a result of external compression is a rare complication of epidural obstetric analgesia.
Haemotropic mycoplasmas (haemoplasmas) are bacterial organisms without cell walls that attach to and grow on the surface of red blood cells . Three feline haemoplasma species are described: mycoplasma haemofelis (mhf), candidatus mycoplasma haemominutum (cmhm), and candidatus mycoplasma turicensis (cmt). These feline haemoplasmas are the causative agents of infectious anemia in cats and in several mammalian species . There is also potential interspecies transmission of some of these agents as recorded from cats to immunocompromised dogs . Their zoonotic potential has recently been substantiated by the molecular identification of a feline haemoplasma isolate (mhf) in an hiv - positive immunocompromised human patient . The three feline haemoplasma species have different pathogenicities, mhf often resulting in haemolysis and severe anaemia in contrast to cmhm and cmt which are less pathogenic . Although several studies worldwide have reported on the epidemiology of feline hemoplasmosis in sick or healthy client - owned pet cats with prevalences ranging from 7.2% to 45.8% [519] and few studies have focused on stray cat (with prevalences from 11.5% to 60% [2024]), there have been no studies investigating stray cats in northern italy . Information on regional prevalence of haemoplasmas could be used to limit the spread of diseases in feline populations and for predicting the likelihood of infection in cats presented with anemia . The vector potential of ctenocephalides felis has been demonstrated in experimental mhf infections, and stray cats may play a role in multiplying the organism in fleas that then infest pet cats and dogs and human beings . Direct transmission, by aggressive interaction of cats, or interspecies transmission might play a role in the epidemiology of these organisms . In addition, haemoplasmas can be directly transmitted through intravenous infusion of infected blood [4, 26] and have been shown to survive for up to 1 week in stored blood products . As administration of fresh or stored whole blood becoming more common in feline medicine, the knowledge of the regional prevalence of blood transmitted pathogens in owned and stray cats that share the same environment and parasites is increasingly important . This would provide useful information for evaluating the risks of transmission of blood - borne infections from potential blood donors and in the development of optimal screening protocols in blood donors . The aim of this study was to evaluate, using a conventional pcr assay, the prevalence of mhf and cmhm infections in stray cats from colonies in milan and to identify possible risk factors for these infections . During a 2-year collection period (january 2008 to january 2010), blood samples were taken from 260 stray cats from urban colonies in milan (northern italy), under a trap - neuter - release (tnr) program approved by the local authority of the city council . Age (estimated based on dentition, animals <6 months of age were considered juvenile, whereas all others were considered adult), gender (male or female), origin (provenance area of colonies), and body condition score (bcs 46, indicating normal weight, 13 underweight) were recorded together with data obtained from physical examination of the cats (healthy or unhealthy). Unhealthy cats were defined as cats with one or more of the following clinical abnormalities: lymph node enlargement, pale mucous membranes, stomatitis, or signs of ocular and respiratory infections . The seasonal analysis based on the date of sample collection was grouped as follows: winter (january, february, and march), spring (april, may, and june), summer (july, august, and september), autumn (october, november, and december). The cats were not systematically examined for the presence of ticks or fleas and so rates of ectoparasitism were not recorded . Blood samples were collected aseptically from the jugular vein while cats were anaesthetized for neutering and placed in edta - treated tubes and in serum separator tubes . Within 24 h of sample collection, a complete blood count (cbc) was performed on whole blood using an advia 120 system (siemens healthcare diagnostics, milan, italy). Cats were categorized in terms of presence or absence of anaemia (hct <24%), leukopenia (wbcs count <10,570/l), leukocytosis (wbc> 14,390/l), and thrombocytopenia (plt <200,670/l). Following separation, serum samples were tested for antibodies to fiv (relative to gp40 and p24 fiv antigens) and for felv p27 antigen with a commercial enzyme - linked immunosorbent assay (elisa) kit (snap felv / fiv combo plus test; idexx laboratories, hoofddorp, the netherlands). Toxoplasma gondii sera igg antibodies were detected using indirect fluorescent antibody tests (ifat) performed with a commercial kit (fuller - laboratories, fullerton, ca, usa). Titres 1: 64 were considered seroreactive and, therefore, indicative of t. gondii exposure . The results of these serological tests have been already published and were reanalyzed with the present results . Conventional pcr was performed on blood samples to amplify dna associated with haemoplasmas (mhf and cmhm). The reaction mixture included 2 l of template dna, 0.25 mm dntps, 0.4 mm of each primer, 1x reaction buffer, and 2.5 u tap dna polymerase (gotaq dna polymerase, promega, madison, wi, usa). Primers were used that target part of the 16s rrna gene, producing a 170 base pair (bp) product from mhf and 193 bp amplicon from mchm (forward primer, 5-acg aaa gtc tga tgg agc aat a-3 and reverse primer 5-acg ccc aat aaa tcc gra taa t-3) [6, 31]. Pcr products were resolved using 2% agarose gels and fragment size was estimated using a dna molecular weight marker (50 bp dna ladder; promega madison, wi, usa). Control reactions were done in the absence of template dna to rule out contaminations during pcr . Association between the three groups of haemoplasma status (haemoplasma positive, mhf positive only, and cmhm positive only) and categorical variables (age, gender, colony of origin, bcs, season of sampling, health status, presence or absence of selected clinical and cbc abnormalities, felv / fiv status, and t. gondii test results) were analysed by univariate analysis using the chi - squared test (cell frequencies of> 5) or fisher's exact test (cell frequencies of 5). Any parameters statistically linked to positive pcr results were used in a logistic regression model to test for independent risk factors associated with infection . Descriptive statistics (including minimum, maximum, mean, median, and standard deviation (sd)) were obtained for the continuous variables rbcs count, hct, hb, wbc count, and plt count values . Distribution of the data for normality was assessed with kolmogorov smirnov test, and a t - test or a mann - whitney u test, respectively, was used to test the differences between the feline haemoplasma positive and negative cats depending on whether data was normally distributed or not . Associations were considered statistically significant when p <0.05 . Both the p value and odds ratio (or) with 95% confidence interval (ci) are reported . Haemoplasmic dna was detected in 33.1% (86/260, 95% ci 26.540.9%) of blood samples . Of the positive samples, 28 cats (10.8%, 95% ci 7.215.6%) were infected with mhf and 58 cats (22.3%, 95% ci 16.928.8%) were infected with cmhm . Characteristics of the population and association between risk factors and haemoplasma pcr status (negative or positive) are reported in table 1 and between mhf alone and cmhm alone positive results in table 2 . None of the risk factors were associated with the pcr results for haemoplasmas (tables 1 and 2), with the exception of negative associations at univariate and multivariate analysis between winter season of sampling and haemoplasma positive status (p = 0.01, or = 0.29, 95% ci = 0.140.61, p = 0.001) and cmhm positive status (p = 0.01, or = 0.29, 95% ci = 0.120.70, p = 0.01). No significant associations were found with cbc abnormalities and haemoplasma pcr - positive results (figure 1) or in the number of anaemic and nonanaemic cats using a t - test . We present the first study investigating the prevalence of haemoplasmas in urban stray colony cats from the city of milan in northern italy . The prevalence of haemoplasma infection in our sample showed a similar pattern to that reported worldwide in client - owned [57, 915, 1719] and stray cats [1923]. Cmhm infection is reported to be the most common infection, ranging from 8% in arizona to 41.6% in portugal; mhf infection was less common ranging from 0.5% in switzerland to 12.8% in portugal and dual infection was absent or rare (no more than 5.4% as found in korea). The prevalence of haemoplasmas in stray cats in our study (33.1%) was higher than that reported in other studies on stray cats performed worldwide (table 4). Differences in prevalence among countries can be explained by geographical variation, such as climate, vector distribution, and the cat population surveyed . Moreover, direct comparisons of prevalence results are of limited value because of the characteristics of sample of cat populations investigated (number of cats included in the study, healthy versus sick cats), inclusion criteria, diagnostic techniques (molecular tools versus microscopical detection), different statistical methods, stage of infection (acute versus chronic), or a combination of all them, resulting in differences between studies . Stray cats in this study had higher rates of infections than those reported for pet cats in northern italy, in which 18.9% of pcr tested cats were reported to be haemoplasma positive . A high incidence of haemoplasmas in stray cats is not surprising as outdoor access is a recognized risk factor for infection . For example, in a study on haemoplasma in client - owned cats from barcelona (spain), outdoor access was found to be a risk factor for infection (or = 3.8). Additionally, in a study involving feline blood donors from the usa, the prevalence of haemoplasmas was 19.7% in domestic cats with outdoor access and only 3.6% in domestic cats not allowed outdoors . Free - ranging cats may have more exposure to bloodsucking arthropods and exhibit a higher fighting activity than owned indoor cats; thus they might experience a higher infection risk for haemoplasmas . In our study there was a negative statistical association between sampling in the winter season and haemoplasma pcr - positive status and cmhm positive status . This may be due to reduced outdoor activity of fleas in winter seasons, although this hypothesis is purely speculative . There were no statistically significant differences or any relationship between the presence of haemoplasma infection and anaemia status or hct levels between positive and negative haemoplasma group . This finding was somewhat surprising and was in agreement with studies undertaken in client - owned cats in switzerland and in italy, which also found no association between haemoplasma infection and anaemia or hct variations . In addition, other studies have failed to demonstrate a significant difference in prevalence rates of haemoplasma between healthy and anaemic cats [10, 32]. These results could be explained by the higher prevalence of cmhm (a less pathogenic species than mhf) in this and previous studies or by the fact that the stage of infection is not known in our cats with a positive pcr result, since chronically infected cats that recover from acute illness may be asymptomatic . Another explanation for the lack of association between infection status and the presence of anaemia in the present study could be that the hct was known only for 150 of the 260 cats tested . Epidemiological data on haemoplasma are particularly important in areas where feline blood donor programmes are active, as in milan where a donor programme has been running since 2010 at university veterinary transfusion unit (rev, reparto di medicina emotrasfusionale veterinaria). Feline haemoplasmas can be directly transmitted by intravenous infusion of fresh edta - anticoagulated blood, heparinized blood, and by infusion of blood stored in cpda-1 solution for 1 h (mhf) and 1 week (cmhm). Cats do not reliably eliminate the organism following infection and most infections with cmhm are chronic and asymptomatic . A significant number of asymptomatic cats are positive for haemoplasma infection and may play a role, along with infected cats, in the maintenance of haemoplasma infection within a population . All these characteristics of feline haemotropic mycoplasma infection need to be considered when choosing potential blood donors . The limitations of this study include the lack of information on candidatus mycoplasma turicensis in our study population . Lastly, statistical analysis was limited in some groups because of the sample size and so some conclusions or associations may be affected by type i errors; that is, no difference between haemoplasma positive and negative groups was observed due to insufficient sample size; for example, no association was recorded between anaemia and a positive haemoplasma result because of the low number of mhf positive cats (28/260). Regardless of these limitations, we believe that this study provides new and useful information on feline haemoplasma infections in stray cats in italy . From this study it can be concluded that feline haemotropic mycoplasma mhf and cmhm are common in the stray cat population of milan . Indeed, pet cats with outdoor access in this region should be regularly monitored and treated for ectoparasites to minimize health risks . Importantly, feline blood donors in this area should undergo pcr screening for these infections before donations and preferably donors should be drawn from exclusively indoor cats that receive regular flea control.
In a case - control study, 140 patients including two equal groups of traumatic due to traffic injuries and non - traumatic patients were evaluated . Using the persian version of the conners adult adhd rating scale (caars) self - report (screening version) the study was performed in two university hospitals in tabriz, iran during a 13- month period - august 2008 to september 2009 . The sample size was calculated using sas software version 9.1 based on proportion comparison tests to ensure 80% statistical power and type 1 error less than 0.05 . The required data for sample size calculation were obtained from a previous study with conservative rounding of figures to come up with possible dissimilarities (11). Inclusion criteria were as follows: physical trauma, motor vehicle traumas (motorcycle and car), being the driver / rider when injured, age range of 18 to 65 years, and ability to complete the conner's questionnaire . The exclusion criteria were as follows: brain trauma resulting in decreased consciousness, non - driver / rider victims of motor vehicle accidents, psychiatric comorbidity, factors affecting the attention and concentration such as drug and alcohol use while driving, and illiteracy . Patients with physical trauma (case group) and those patients admitted due to non - trauma reasons without previous history of trauma hospitalization were recruited during the study period . Adhd diagnosis was performed using caars, and the psychiatric comorbidities were assessed according to dsm - iv - tr using scid, and those with a psychiatric comorbidity were excluded . All of the patients were informed about the design and purpose of the study, and a written consent form was obtained from each volunteer patient . The assessed factors in both groups included age, sex, educational level, job, accident history, daily driving amount, weekly driving amount, adhd index, a subscale (attention deficit symptoms), b subscale (hyperactivity - impulsivity symptoms), and c subscale (total adhd symptoms). In addition, type of vehicle, light intensity at accident time, and the location of the accident were recorded in the case group . The conners adult adhd rating scale (caars) conner's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . Among its good characteristics is having multiple indices including attention deficit index (subscale a), hyperactivity - impulsivity index (subscale b), adhd symptoms total index (subscale c), and adhd index (subscale d). This questionnaire had been translated to persian by icss (institute for cognitive science studies) in tehran . In this study, the overall internal consistency of the conner's adult adhd rating scale was estimated as cronbaches of 0.83 . In case the diagnosis of adhd was for children, a rating scale for diagnosing adhd in children designed by delavar & et al can be used (13).structured clinical interview for dsm - iv (scid) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria . The reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories (kappa> 0.6). Comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution . In addition, the chi - square and fisher exact tests were used for categorical variables . In all cases, the p values less than 0.05 were considered statistically significant . The conners adult adhd rating scale (caars) conner's adult adhd rating scale is used to screen and treatment follow up of patients and its validity and reliability are in accordance with dsm - iv . Among its good characteristics is having multiple indices including attention deficit index (subscale a), hyperactivity - impulsivity index (subscale b), adhd symptoms total index (subscale c), and adhd index (subscale d). This questionnaire had been translated to persian by icss (institute for cognitive science studies) in tehran . In this study, the overall internal consistency of the conner's adult adhd rating scale was estimated as cronbaches of 0.83 . In case the diagnosis of adhd was for children, a rating scale for diagnosing adhd in children designed by delavar & et al can be used (13).structured clinical interview for dsm - iv (scid) scid is a widely - used clinical tool for the classification of psychiatric disorders based on dsm- iv criteria . The reliability and feasibility of the persian version of this diagnostic instrument were already determined as fair to good for most diagnostic categories (kappa> 0.6). Comparisons were made using independent samples t - test and mann - whitney u - test for numerical variables according to data distribution . In addition, the chi - square and fisher exact tests were used for categorical variables . In all cases, the p values less than 0.05 were considered statistically significant . No significant statistical difference was found between case and control groups on age, gender, educational level and occupation . With respect to type of vehicle, 25 cases had a car accident (35.7%) and 45 had a motorcycle accident (64.3%). The light condition was bright in fifty four (77.1%), semi - bright in eight (11.4%) and dark in eight cases (11.4%). Accident location in the case group was within the town in 30 cases (42.9%), side road in 20 (28.6%), main road in 13 (18.6%), and highway in 7 cases (10%). Accordingly, the percent of subjects with accident history was significantly higher in case group (p= 0.24). Furthermore, the mean driving hours in case group was significantly more compared to controls . No significant difference was observed between the two groups with respect to other factors . Comparison of measured variables in two groups * the data are shown as mean standard deviation and frequency (percent). In self - report questionnaire of conner's adult adhd rating scale, subscales' scores higher than 70 were considered positive.adhd index was positive in 3 (4.3%) subjects in both case and control groups . Adhd total symptoms were positive in 7 (10%) and 10 (14.3%) cases in both case and control groups respectively p= 0.438, or= 1.5 (0.5- 4.2), ci: 95% . In a subscale, inattentive symptoms were observed in 5 (7.1%) and 6 (8.6%) participants in case and control groups respectively p= 0.753, or=1.2 (0.4- 4.2), ci: 95% . In b subscale (hyperactive - impulsive symptoms were observed in 7 (10%) and 6 (8.6%) participants in case and control groups respectively pv= 0.771, or= 0.8 (0.3- 2.7), ci: 95% . In this study, the frequency of adult adhd was compared between traumatic adult patients (due to traffic injuries) and a control group . Accordingly, adhd index was positive 4.3% in both case and control groups, adhd symptoms total were positive 10%, 14.3% in case and control groups respectively . A subscale (inattentive symptoms) was 7.1%, 8.6% in case and control groups respectively; and b subscale (hyperactive / impulsive symptoms) was 10%, 8.6% in case and control groups respectively . Hechtman et al (1984) first described the higher rate of traffic injuries in children with adhd (15). The next studies performed by barkley et al ., and jerome et al ., accredited the initial report (1619). The association of adhd and traffic injuries is so important that driving center of canada was listed the uncontrolled adhd as an item for giving certificate to the drivers . The higher frequency of each type of accident in adhd patients was compared to healthy subjects (odds ratio 1.7) and the car accidents are a subgroup of these events (20). Despite of numerous studies about the association of adhd and car accidents, ludolph et al ., reported twofold higher rate of car accidents in these patients compared with the normal population (21). In barkley studies and cox et al (2000, 2006), this ratio has been different from two to six (16, 2224). Sobanski et al ., evaluated 27 patients with adult adhd and 27 age and sex matched subjects as a control group . The frequency of car accidents in first group was 2.6 fold higher than the control group patients, the other related factors in this field were also (25). Besides the higher rate of car accidents in adhd higher . Among these, we can point to the higher frequency of losing driving certificate, driving after alcohol and substance use, exceeding the speed limit, and other abnormal traffic behaviors, car accidents trauma, high rate of traffic punishments and rate of suspension of driving certificate and higher rate of being responsible for the accidents (2630). Besides the epidemiological studies, there are numerous clinical trials showing the strong association between adhd and car accidents . Cox et al ., in two different studies showed that the frequency of car accidents in adults with adhd receiving methylphenidate was lower than the group without treatment (23, 31). Barkley et al ., in a similar study demonstrated that treatment of adult adhd may result in decreased rate of car accidents and related injuries (32). In a study on selegiline in comparison with methylphenidate in adhd children and adolescents showed that selegiline is also effective and well tolerated for adhd (33)., in a study on modafinil as a treatment for adhd in children and adolescents found that this drug significantly improves symptoms of adhd and is well tolerated (34). On the other hand, it is shown that high risk behaviors of adhd patients during driving would persist despite controlling other factors . In other words, high risk behaviors during driving are an independent factor associated with adhd (25, 30). Different mechanisms are suggested including attention deficit, inability to control impulsivity, early fatigue, anger and aggressiveness (25, 3537). According to the mentioned studies, it may be concluded that there is a significant association between adult adhd and car accidents . It should be mentioned that in all studies, the patients with adhd and controls were either under investigation for driving - models or their driving situation while we compared the frequency of adhd in subjects with and without car accidents . The current study is the first report from tabriz- iran on the association of adhd to traffic injuries . The lack of difference between two groups regarding frequency of adhd may be due to the fact that the two groups were matched for job and educational level in this study . It was previously reported that adult adhd is one of the main causes of educational and job problems (30). Hence, matching of two groups for these variables may be a kind of selection bias . Also driving skill level unfortunately, we could not evaluate this condition in our sample . According to available reports, the low driving skill level and lack of sufficient information about driving rules (especially in young person) is one of the main causes for car accidents in our country (38, 39). In other words, high frequency of car accidents in our society is mainly due to lack of sufficient training of young drivers and lack of sufficient respect to the driving rules . Hence, it is very difficult to determine the role of adhd in this field . High frequency of motorcycle accidents in this study (64.3%) is a congruent factor because of higher rule destruction in this group . Difference in driving duration is another major factor and the mean daily driving hours were significantly higher in patients with car accidents . On the other hand, it has previously been shown that the mortality or major injuries and severity of injury in adhd patients experiencing car accidents is more than healthy subjects (26, 27). Despite the mentioned restrictions, accordingly, it seems that most car accidents involving driver injury are not related to adhd . This study showed that the odds of adult adhd was not significantly higher in drivers with traffic injuries compared with the matched controls . Therefore, larger scale studies stratified for different types of traffic accidents are recommended to further investigate such an association most of the traffic injuries cause death but these case were not included in our study.
Crh is a 41-amino - acid peptide hormone synthesized in the paraventricular nucleus of the hypothalamus . Its function in the central nervous system is to stimulate the hypothalamic - pituitary - adrenal (hpa) axis as part of the stress response . Crh is also produced in peripheral tissues including the placenta of humans and hominine primates [13]. Placental crh secretion results in an exponential rise of crh concentration in maternal plasma during the third trimester of gestation . The rate of the increase is related to gestational length, since the rise of crh level is accelerated in pregnancies ending with preterm birth, while the increase is retarded in pregnancies that continue after term . Because of this relationship, it is believed that the regulation of placental crh production is linked to the mechanism that determines the length of pregnancy and triggers labour and delivery . The mechanism regulating the exponential increase in placental crh expression remains unclear although positive feedback by glucocorticoids and increasing numbers of syncytial cell nuclei are suggested explanations [5, 6]. Spontaneous and agonist - induced syncytium formation by cytotrophoblasts is associated with crh expression with camp being a strong stimulant of both syncytial differentiation and crh gene activity . Molecular studies using crh promoter - reporter constructs indicated that transcription factor complexes bound to a consensus cyclic - amp response element (cre) at 224 bp upstream of the major transcription initiation site mediated the camp - stimulation, although a nonconsensus second promoter site was also implicated in the cyclic nucleotide response [811]. It has been suggested that these molecular interactions are involved in the gestational age dependent control of crh gene activity . Epigenetic chromatin modifications define cell - specific gene expression potential and alter gene expression patterns during cell differentiation and development . Methylation of cytosines at cpg dinucleotides in dna is a well - characterized epigenetic chromatin modification generally associated with closed chromatin structure and gene repression . Furthermore, methylation of cpg sites within particular transcription factor binding sequences may modify transcription factor binding affinity and alter regulatory changes in gene expression [1417]. The human crh proximal promoter contains 9 cpg dinucleotides with one located within the methylation sensitive cre sequence . In addition, the promoter is within 1000 bp distance from an intragenic cpg island, which corresponds to a cpg island shore region, the methylation of which is related to tissue specific gene expression . Therefore, in the present investigation we have explored the possibility that methylation of the promoter contributes to the control of crh expression in trophoblast cells . We used the bewo choriocarcinoma cell line in the experiments, which is a well - characterized trophoblast model exhibiting dynamic dna methylation as well as ability to syncytialise and increase crh expression when stimulated with camp [1921]. The bewo human choriocarcinoma - derived cell line was obtained from the american type culture collection (manassas, va, usa) (atcc #ccl-98). The culture medium was dmem / f12 (with hepes and l - glutamine, without phenol red) supplemented with 10% (v / v) fetal bovine serum (fbs) and 1x antibiotic - antimycotic (gibco / life technologies, mulgrave, vic, australia). Cells were cultured at 37c in a humidified atmosphere of 5% co2 in air . At approximately 80% confluence cells were detached with trypsin / edta solution (gibco), washed, counted, and transferred into six - well plates at a density of 0.8 10 cells / well . Cultures reaching 50% confluence were incubated with fresh medium with or without 8-br - camp (8-bromoadenosine-3,5-cyclic monophosphate, 2.5 10 mol l) and/or 5-aza - dc (5-aza-2-deoxycytidine, 5 10mol l) (sigma - aldrich, sydney, nsw, australia) followed by harvesting for rna and dna isolation . Drug concentrations were optimised in previous studies and showed no toxic effects in bewo cells at the concentrations and exposure times employed [19, 22, 23]. Rna was extracted from cells using the rneasy mini kit (qiagen, chadstone centre, vic, australia) according to the manufacturer's protocol . Rna was eluted from the rneasy spin columns with 30 l of rnase - free water and quantified using a nanodrop 1000 spectrophotometer (thermo fisher scientific australia, scoresby, vic, australia). Contaminating dna was removed by dnase treatment using the turbo dna - free kit (ambion / life technologies) following the routine protocol . The total reaction volume was 20 l including 2 l of 10x dnase buffer, 1 l of dnase, and up to 2 g of rna . Rna integrity in all samples was assessed by agarose gel electrophoresis . Prior to reverse transcription, the rna samples were spiked with 5 10 copies of alien rna transcript (supplied with the alien qrt - pcr inhibitor alert kit, stratagene / integrated sciences, chatswood, nsw, australia) per microgram rna . The alien rna transcript served as a reference rna of equal abundance in all samples and pcr runs . Rna was reverse transcribed using the superscript iii first - strand synthesis system for rt - pcr (invitrogen) with random hexamer primers . Real - time pcr was performed using an applied biosystems 7500 real - time pcr system with reagents supplied by the manufacturer (applied biosystems / life technologies). The amplification reaction contained template cdna from 20 ng of reverse - transcribed rna, 6 10 moles l forward and 3 10 moles l reverse primer, sybr green master mix, and milliq water to a total volume of 25 l . The crh cdna primers were designed and optimised by sehringer et al . And are listed in table 1 . Primer sequences for amplifying alien cdna are proprietary and were used according to the manufacturer's instructions (stratagen / integrated sciences). The temperature sequence was 50c for 2 min, 95c for 10 min, 40 cycles of 95c for 15 s, and 60c for 1 min, followed by melting curve analysis . No - template control and no - reverse transcriptase controls for all samples were included to detect residual genomic dna . Expression levels of the crh mrna were determined relative to alien rna according to the ct method . Cells grown in six - well plates were collected in 750 l pbs (phosphate - buffered saline; 137 10mol l nacl, 2.7 10 mol l kcl, 8 10mol l na2hpo4, and 2 10 mol l kh2po4, ph 7.4) using cell scrapers and centrifuged for 5 min at 300 g . Cell pellets were resuspended in 200 l of pbs and processed for dna isolation as per the manufacturer's instructions . Genomic dna was eluted from the mini spin columns with 200 l milliq water, quantified using the nanodrop 1000 spectrophotometer, and stored at 4c . Up to 300 ng of genomic dna was bisulfite - converted and purified using the methylseqr bisulfite conversion kit (applied biosystems). The crh proximal promoter regions were pcr - amplified using the toptaq master mix kit (qiagen) and two sets of nested primers designed with the methyl primer express software v.1.0 (applied biosystems). Pcr reactions contained purified bisulfite - converted dna template, 25 l of 2x toptaq master mix, 4 10 mol l of each primer, and milliq water up to 50 l final volume . The conditions for the first pcr amplification included an initial step at 94c for 3 min, followed by 30 cycles of 94c for 30 s, 54c for 30 s, and 72c for 1 min and a final extension step at 72c for 10 min . One microliter of a 20-fold diluted aliquot of the first pcr reaction was used as template for the second pcr amplification using the nested primer set . Pcr conditions were 94c 30 min, 30 cycles of 94c for 30 s, 50c for 30 s, and 65c for 1 min and an extension step at 65c for 10 min . Following amplification, 20 l of the pcr reaction mixture was separated by agarose gel electrophoresis and the amplification product was visualised with ethidium bromide . The gel slice containing the amplified dna fragment was excised, extracted, and purified using the wizard sv gel and pcr clean - up system (promega, auburn, vic, australia). The dna was collected in 50 l milliq water by the centrifugation of the sv minicolumn . The bisulfite - converted and pcr - amplified dna was ligated into the pgem - t easy vector using reagents provided by the manufacturer (promega). The 10 l reaction mixtures contained ligation buffer, 3 weiss units of t4 dna ligase, 50 ng of pgem - t easy vector, and pcr product at 3: 1 insert: vector molar ratio . A positive control using the control dna provided and a negative control (no pcr product) were also included . The ligation mixture was used subsequently to transform jm109 competent cells (promega) according to the manufacturer's protocol . Fifty l of transformed cell suspension was spread onto duplicate lb / ampicillin / iptg / x - gal plates and incubated at 37c overnight . The white streak colonies were picked the next day and cultured in 5 ml luria broth at 37c overnight . Plasmids were isolated from the minicultures using the genelute plasmid miniprep kit (sigma - aldrich, castle hill, nsw, australia). The presence of inserts was verified by digesting an aliquot from each plasmid preparation with ecor i (promega) followed by agarose gel electrophoresis . Plasmids containing the expected size inserts (300 bp) were sequenced from both directions by the australian genome research facility (agrf, brisbane, qld, australia). The sequencing primers were designed by promega and produced by invitrogen / life technologies (forward: 5-tatttaggtgacactatag-3, reverse: 5-tatttaggtgacactatag-3). Quality control was automatically performed and any sequence with an unacceptably low conversion rate or high number of sequencing errors was excluded . Ten randomly selected clones, representing individual gene copies, were processed for methylation frequency analyses from each dna sample using the chi - square test and fisher's exact test as appropriate . Significant one - sided tests are reported in cases when the two - sided tests showed borderline significance . The stata (college station, tx, usa) software package was used for the statistical calculations . As shown in figure 1, abundance was not significantly different between 0 h and 24 h and between 24 h and 28 h of incubation . Between 48 h and 72 h, a 2.9-fold increase (p = 0.029) was observed, which was coincident with the reported spontaneous syncytialisation of bewo cells . In the presence of 8-br - camp (2.5 10 mol l), a powerful stimulant of syncytial differentiation, significant increases of crh mrna level were detected between 0 h and 24 h (p <0.0001), 24 h and 48 h (p = 0.03), and 48 h and 72 h (p = 0.049, one - sided t - test) with a maximum at 72 h, which was 23.2-fold higher than the 0 h level (figure 1). In cultures treated with the dna methyltransferase inhibitor 5-aza - dc (5 10mol l) a slight, but significant, increase of crh mrna abundance was observed between 0 h and 24 h (1.43-fold, p = 0.028, one - sided t - test), but there was no further change between 24 h and 48 h and between 48 h and 72 h. combined treatment with 8-br - camp and 5-aza - dc resulted in robust increases of crh mrna levels between 0 h and 24 h (p <0.0001), 24 h and 48 h (p = 0.0026), and 48 h and 72 h (p = 0.039) reaching a 86.4-fold rise at 72 h compared to 0 h (figure 1). 8-br - camp increased crh mrna abundance relative to vehicle at all time points (24 h, p = 0.0042; 48 h, p = 0.0066; 72 h, p = 0.0004; figure 1), which confirmed previous findings of the stimulatory effects of 8-br - camp on crh gene expression in bewo cells . 5-aza - dc treatment had no effect at 24 h and 48 h and reduced crh mrna abundance relative to vehicle at 72 h, effectively blocking the increase seen between 48 h and 72 h in vehicle - treated cells (p = 0.0021, figure 1). Combined treatment with the cyclic nucleotide and the dna methyltransferase inhibitor upregulated crh mrna expression at all time points beyond the level reached in response to 8-br - camp alone (24 h, p <0.0001; 48 h, p = 0.0002; 72 h, p = 0.0029; figure 1). The significant effects of 5-aza - dc on crh mrna expression suggested that dna methylation was involved in the control of crh gene activity . To explore this further, we have determined the effects of 8-br - camp and 5-aza - dc on the methylation of the 9 cpg sites present in the crh proximal promoter . Bisulfite sequencing revealed partial methylation (38% of the 9 cpg sites combined) at 0 h, before treatments commenced (figure 2), which increased spontaneously to 57% (p = 0.001) by 72 h of culture . Treatment with 8-br - camp (250 m) resulted in a similar increase of methylation (to 61%), not significantly different from the vehicle - treated control . Treatment with 5-aza - dc for 72 h reduced promoter methylation to 23%, which was significantly less than the control (p = 0.001). Combined treatment with 5-aza - dc and 8-br - camp increased the level of methylation compared to 5-aza - dc alone (to 33%, p = 0.011) but did not reach the methylation level observed in cells treated with 8-br - camp only (p = 0.001, figure 2). Clonal bisulfite sequencing determines cytosine methylation with single base resolution in individual alleles (gene copies). The technique enabled us to determine the particular cpg sites that undergo methylation changes under the treatment conditions that influence methylation levels overall, as presented in figure 2 . The scheme in figure 3 shows the positions of the 9 methylatable cpg dinucleotides in the human crh proximal promoter . The two major transcription initiation sites and the two sequence regions implicated in the camp - response are also indicated with cpg2 residing within the cre [811, 29]. The heatmap in figure 4 illustrates the methylation levels of the 9 cpgs under the treatment conditions employed . Methylation levels were significantly different among the cpg sites ranging from 10% to 70% at 0 h and from 13% to 80% at 72 h of culture (p <0.001, figure 4) indicating site - specific differential methylation . The methylation level of cpgs 1, 2, 3, and 7, but not of cpgs 4, 5, 6, 8, and 9 increased significantly during the 72 h culture period demonstrating that methylation was dynamic at these sites . Figure 5 shows the methylation of each cpg in the cloned copies of the crh proximal promoter . The scattered distribution of methylated and unmethylated cpg sites suggests that the partial methylation observed was allele independent both at 0 h and at 72 h of culture . Cells treated with 8-br - camp and 5-aza - dc had similar scattered distribution of methylated cpgs in individual alleles (not shown). In cells treated with 8-br - camp for 72 h, the cpg sites remained differentially methylated (from 23% to 73%, p = 0.004), and the methylation levels of individual cpgs were not significantly different from the corresponding sites in the vehicle - treated control (figure 4). Treatment with the dna methyltransferase inhibitor 5-aza - dc decreased methylation at all cpg sites compared to vehicle, except for cpg 4, where methylation was relatively low . Furthermore, 5-aza - dc abolished the differences between the methylation levels of the individual cpgs . The methyltransferase inhibitor eliminated the methylation differences among individual cpgs in the presence of 8-br - camp as well (figure 4). Cotreatment with 8-br - camp prevented, however, the demethylating action of 5-aza - dc at cpgs 1, and 8, but not at cpgs 2, 3, 5, 7, and 9 (camp versus camp + aza in figure 4). Finally, no individual cpg site exhibited a statistically significant methylation difference in 8-br - camp + 5-aza - dc - treated cells compared to treatment with the methyltransferase inhibitor alone (aza versus camp + aza in figure 4) despite the small, but significant, overall increase in methylation (aza 72 h versus camp + aza 72 h, in figure 2). The aim of this study was to explore the involvement of promoter methylation in crh gene regulation in human trophoblast cells . Placental crh expression is predictive of gestational length and is influenced by pregnancy disorders [4, 30]. Dna methylation is a developmentally regulated epigenetic modification influenced by environmental inputs [31, 32], which can potentially control crh gene expression during pregnancy and in response to pathogenic factors . In our experiments we used the choriocarcinoma - derived bewo cell line, which is a well - characterized trophoblast model exhibiting increased crh gene expression during spontaneous and camp - induced syncytial differentiation similar to normal trophoblasts . Our dna sequencing data show that the crh proximal promoter sequence is identical in bewo cells and in normal trophoblasts including all reported transcription factor response elements and the methylation sensitive cpg sites (figure 3). We have shown by clonal bisulfite sequencing that the cpgs within the crh proximal promoter are partially methylated with significant differences among the individual sites . The size of the analysed sequence (261 bp) and the methylation level correspond to an intermediate methylation region, which is a genomic feature implicated in tissue specific epigenetic gene regulation . This is similar to normal trophoblasts, which also exhibit allele independent partial and differential methylation in the crh promoter region . Dna methylation increases in bewo cells during culture and the dna methyltransferase inhibitor 5-aza - dc changes cell phenotype and gene expression levels [21, 22, 35]. Our results show that the crh promoter follows this general trend, which is different from primary trophoblasts, where crh promoter methylation does not change in culture and remains unaltered by 5-aza - dc and 8-br - camp under the conditions where bewo cells show methylation changes . For this reason, bewo cells are uniquely suited to explore the relationship between crh promoter methylation level and gene activity . Methylation of cpg island promoters is associated generally with the repression of gene activity [12, 14, 21]. The crh promoter is located in a cpg island shore region relative to a crh intragenic cpg island (chr8:67,089,250 - 67,089,962 in the grch37/hg19 assembly, ucsc genome browser). Our results showed, however, that neither the spontaneous nor the 8-br - camp - evoked increase of crh gene activity was associated with the demethylation of the crh promoter (figures 1 and 2). Treatment with 5-aza - dc decreased crh promoter methylation and abolished the cpg site - specific methylation differences as expected (figures 2 and 4), but it also blocked the spontaneous increase of crh gene expression in culture (figure 1). 8-br - camp - stimulated crh expression strongly in the presence of 5-aza - dc (figure 1), but the cyclic nucleotide actually increased methylation in 5-aza - dc - treated cells (aza 72 h versus camp + aza 72 h in figure 2). Thus, crh expression was directly, and not reciprocally, related to promoter methylation under these conditions . This relationship may be unexpected in view of the well - documented global association between gene repression and promoter methylation, but genome wide trends do not necessarily predict the behavior of individual genes . In fact, the methylation level of the cpg - poor class of promoters was found to be uncorrelated with gene activity . The crh promoter falls into the cpg - poor class according to established criteria, with partial methylation in both the hypothalamus and the trophoblast [34, 37, 38] (figure 2). Methylation reduces crh gene expression in the hypothalamus as expected; in trophoblastic bewo cells, however, promoter methylation appears to have the opposite effect as detailed before . There is evidence to suggest that this cell - specific regulation may result from the methylation - dependent change of the functional properties of the camp - response element (cre) in the crh promoter (figure 3). The cre is critical in regulating the activity of transfected crh promoter - reporter constructs in trophoblast cells [911]. It contains a cpg dinucleotide that, when methylated, reduces the affinity of cre to its cognate transcription factor, creb, and renders it unresponsive to camp - stimulation [15, 39]. At the same time, the methylated cre has increased affinity to bind the transcription factor c / ebp - alpha, which often activates tissue specific genes during differentiation . The cpg within the cre was 30% methylated under basal (0 h incubation) conditions (cpg2 in figure 4). Culturing for 72 h increased cpg2 methylation to 60% concomitantly with enhanced gene expression, while treatment with 5-aza - dc reduced cpg2 methylation to 23% and diminished crh gene activity (figures 1 and 4). Considering that c / ebp - alpha is expressed in bewo cells, it is reasonable to conjecture that methylation - evoked changes in the transcription factor binding specificity of the cre may have contributed to the enhanced crh expression observed in association with increased promoter methylation . The cpg2 in the cre was partially methylated under all experimental conditions, which suggests that the unmethylated portion could have mediated stimulation by 8-br - camp using the canonical, creb - dependent pathway . Moreover, the crh proximal promoter contains a second, noncanonical camp - response element (figure 3), which contributes to the regulation of the gene specifically in the trophoblast [9, 11]. Methylation of the cre may thus function to influence the relative contribution of the two camp - response elements, their associated transcription factors, and the coupled signaling pathways to the overall activity of the crh gene under basal and camp - stimulated conditions . Cotreatment with 5-aza - dc strongly augmented the stimulation of crh mrna expression by 8-br - camp, although the dna methyltransferase inhibitor alone had no stimulatory effect under the same culture conditions (figure 1). Cotreatment with 5-aza - dc, however, decreased cpg methylation in the cre (cpg2, figure 4) to 43.3% from 70% measured after 8-br - camp treatment (p = 0.034, fisher's exact test, one - sided). The increased proportion of unmethylated cres in the cell population could explain, at least partially, the augmented response to 8-br - camp - stimulation . Moreover, 5-aza - dc decreases dna methylation globally increasing or repressing the activity of numerous genes [4143]. This suggests the possibility that 5-aza - dc may potentially influence crh expression indirectly through intervening gene products generating synergy between 8-br - camp and 5-aza - dc . Although gene activation occurs in 5-aza - dc - treated bewo cells [22, 23, 35], it remains to be established whether transcription factors or other gene products controlled by dna methylation regulate crh expression in trophoblasts . We have utilized the dynamic changes of dna methylation in the bewo cell line to explore the relationship between this epigenetic chromatin modification and crh gene expression in a trophoblastic cell type . Clonal bisulfite sequencing revealed the cpg site - specific and allele independent partial methylation of the crh proximal promoter and classified it as an intermediately methylated region of the genome . The data suggest that promoter methylation determines the contribution of the cre, its various associated transcription factors, and a trophoblast specific alternative camp - response element to crh gene regulation . Furthermore, our results are consistent with the possibility that dna methylation controls crh expression indirectly, but any intervening factor that may regulate crh expression by a dna methylation - dependent mechanism remains to be determined.
The three compounds lincomycin (a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961), clindamycin, and pirlimycin (two semi - synthetic derivatives of lincomycin) comprise a group of clinically important antibiotics known as lincosamides . The structure of lincomycin (fig . 1a) can be divided into two parts, a pyrrolidine derivative and a six - atom sugar ring (methylthiolincosamide), which are linked via an amide bond in the central part of the molecule . 1b) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin, and pirlimycin (fig . 1c) is obtained by trimming the propyl group of clindamycin to get an ethyl group . These compounds are soluble in water and chemically stable both in the dry state and in solution.fig . 1chemical structures of lincosamides: lincomycin (natural antibiotic, a) and its semi - synthetic derivatives clindamycin (b) and pirlimycin (c) chemical structures of lincosamides: lincomycin (natural antibiotic, a) and its semi - synthetic derivatives clindamycin (b) and pirlimycin (c) lincosamides block bacterial protein synthesis, which takes place on the ribosomes . The bacterial 70s ribosome can be separated into two subunits: large (50s) and small (30s), named after their sedimentation coefficients . The small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain, while the large subunit contains over 30 proteins and two rna chains (23s rna and 5s rna). Lincosamides bind to the 23s rna (interacting with the a- and p - trna binding sites) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction, i.e., peptide bond formation . The main spectrum of action of lincosamides includes bacteria associated with skin infections, and lincosamides are first - choice antibiotics used in veterinary dermatology . However, although they do have adverse effects, such as nausea, rash, or abdominal pain, lincomycin and clindamycin are also effectively used in human medicine . Lincosamides are effective against gram - positive bacteria, such as staphylococcus, streptococcus, most anaerobic bacteria (e.g., bacteroides fragilis), and some protozoa . In the latter case gram - negative bacteria are, in general, resistant to lincosamides, with one important exception: capnocytophaga canimorsus . Lincosamides exhibit excellent pharmacokinetic properties; they are well absorbed orally and can penetrate well into infected skin . Also, the minimum inhibitory concentration (mic90, the minimum concentration needed to inhibit growth overnight in 90% of organisms) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline . Unfortunately, extensive use of these antibiotics has led to increased resistance in many strains of bacteria, which have been developing various mechanisms to counter these drugs . One known mechanism is structural modification of the drug s target (ribosome) through the methylation of 23s rna (e.g., base no . 2058) and/or mutations (e.g., of bases g2057, a2058, a2059, c2452, and c2611). Other mechanisms of resistance are active efflux of the drug across the cell surface, or its enzymatic deactivation . Bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds . The conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [9, 10] as well as h and c nmr spectroscopy and molecular dynamics . However, verdier et al . Ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy (trnoesy), and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome . They found that the free conformers of clindamycin and linb (lincosamide nucleotidyltransferase) enzyme - bound clindamycin are similar . Rajeswaran et al . Solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . On the other hand, there are four crystallographically resolved structures of clindamycin bound to a target: three in complex with the ribosome [1315], and one with the linb molecule, the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation . Their crystal structures as well as the structure of the native ribosome are available through the protein data bank (pdb). These crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides . Two out of the three conformers of clindamycin, when bound to the ribosome, show significant differences between antibiotic conformations: in two structures, the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer . Theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond (ihb). Although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data, these calculations provide information about the shape of the potential energy surface (pes) without the need for any initial assumptions . Characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . To the best of our knowledge, no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported . The aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations . These ab initio calculations consist of the following steps: the choosing the model structures, geometry optimization, natural bond orbital analysis (nbo), atoms in molecules analysis (aim), and spectroscopic nmr parameter calculations, which are currently among the most popular methods used for conformational analysis . The methods applied in the calculations are described in the first section of the paper . We then characterize the conformations and molecular properties, focusing on the intramolecular hydrogen bonds . Four structures of clindamycin are available in the protein data bank (as of may 2011). Three of these structures are in complex with the bacterial ribosome [1315], while one is in complex with linb . The two ribosome - complexed structures show similar clindamycin conformers, and in one the pyrrolidynyl propyl group is rotated by 180. in this work, we used two significantly different conformers of clindamycin, which were taken from ribosome clindamycin complexes of deinococcus radiodurans (pdb code 1jzx) and haloarcula marismortui (pdb code 1yjn). Therefore, based on the known x - ray structures of clindamycin (fig . 2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome . Black conformer a, gray conformer b (only heavy atoms are shown for clarity) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome . Black conformer a, gray conformer b (only heavy atoms are shown for clarity) the second step involved optimizing the geometries of all of the antibiotic models constructed, using density functional theory (dft) at the b3lyp level [18, 19] with the 6 - 31 g basis set and a redundant coordinate algorithm ., we optimized the investigated molecules without any constraints . In the second case, the two dihedral angles d(c15c16n2c19) and d(c16n2c19c20), which are significantly different in both experimentally known conformers (a and b; see fig . 2), were kept constant in the optimization procedure to differentiate between the two conformers . The values of the dihedral angles d(c15c16n2c19) and d(c16n2c19c20) for conformer a were set to 47 and 180, while they were set to 119 and 178, respectively, for conformer b, in accordance with known experimental results . The corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points . No imaginary frequencies were observed, which means that the structures of the antibiotics are true minima . These structures, which we called exp - opt, were used during aim, nbo, and nmr calculations (see below). In a third step, in order to explore the conformational landscape of the molecules, we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner (i.e., all other geometrical parameters were optimized at each point) for both conformers . To describe the environment of the antibiotic, either the continuum solvation model or an atomic representation of the solvent can be used . Therefore, as a fourth step, in order to study the solvent effect, optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism (ief - pcm). In this model, the solvent is described by a dielectric constant, which was set to 80 in our work . The second model that was used to describe the environment of the antibiotic, and mimics the surroundings of the antibiotic in the ribosomal rna, was the model with point ions placed around the antibiotic [22, 23]. The positions of these point charges were obtained from the x - ray structures of the ribosome the coordinates of residues within 10 of each clindamycin atom were considered, and partial charges were assigned based on the g43b1 gromos96 force field . In this way, we studied the effect of the charged ribosome environment on the conformations of the lincosamides . Next, in order to gain a deeper insight into the nature of the conformational changes, nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin . The bond critical points (bcps) were characterized in terms of electron density and their laplacian values . Nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . In the last part of our work, we calculated the nmr chemical shifts and spin spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level . Shielding constants were calculated using the b3lyp / aug - pcs-1 method and the giao routine [2630]. The chemical shifts of the i - th nuclei were calculated as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta (i) = \sigma_i(x) - \sigma_i(c), $$\end{document} where i(c) and i(x) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule, respectively . Spin coupling constants were calculated at the b3lyp / aug - pcj-0 [31, 32] level . The nonstandard basis sets were taken from the emsl basis set library [33, 34]. The nbo calculations were performed with the nbo 5.0 program, while aim calculations were performed using aim2000 . Data were analyzed using gabedit, and the visualizations were carried out with vmd and xdrawchem . The energies, the zero - point vibrational energies (zpe), and the gibbs free energy (g) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase, taking solvent effects into account, are depicted in table 1 . The selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin, lincomycin, and pirlimycin in their bound modes are given in table 2 . 3.table 1total energies (e0, in au), zero - point energies (zpe, in kcal / mol), and relative gibbs free energies at 298.15 k (g, in kcal / mol). Top: values for the fully optimized a and b conformers of clindamycin, lincomycin, and pirlimycin in vacuum . Bottom: values for the a and b conformers of clindamycin, lincomycin, and pirlimycin (optimized using a redundant coordinate algorithm in vacuum) in the pcm model of solvent and in the point ions . Two dihedrals were kept constant, d(c15c16n2c19) = 47.0 and d(c16n2c19c20) = 180.0 for conformer a, and d(c15c16n2c19) = 119.0 and d(c16n2c19c20) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0 e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters (in and degrees) for the exp - opt structures of both conformers (a and b) of clindamycin, lincomycin, and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes . Top: clindamycin, middle: lincomycin, bottom: pirlimycin total energies (e0, in au), zero - point energies (zpe, in kcal / mol), and relative gibbs free energies at 298.15 k (g, in kcal / mol). Top: values for the fully optimized a and b conformers of clindamycin, lincomycin, and pirlimycin in vacuum . Bottom: values for the a and b conformers of clindamycin, lincomycin, and pirlimycin (optimized using a redundant coordinate algorithm in vacuum) in the pcm model of solvent and in the point ions . Two dihedrals were kept constant, d(c15c16n2c19) = 47.0 and d(c16n2c19c20) = 180.0 for conformer a, and d(c15c16n2c19) = 119.0 and d(c16n2c19c20) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters (in and degrees) for the exp - opt structures of both conformers (a and b) of clindamycin, lincomycin, and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes . Top: clindamycin, middle: lincomycin, bottom: pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin . Our results show that the most important is the energy profit from the formation of the ihb . The presence of hydrogen - bond donors (o h, n h, c h) and hydrogen - bond acceptors (c = o, cl) allows for a range of hydrogen - bond combinations and a number of stable forms [40, 41]. Because of the internal hydrogen bonds, one conformer is stabilized to a greater extent than the others . First and foremost, at the b3lyp/6 - 31 g level, the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol . The energetic picture significantly changes for lincomycin and pirlimycin . In the case of lincomycin, no single conformer is favored at 298.15 k, while the a conformer of pirlimycin is more stable than b according to the three models used: vacuum, pcm, and point ions . The clindamycin b conformer in vacuum displays both the longest o6h37 bond (0.980) and the shortest ihb distance, r(c19o9 h39c15) ihb distance in the less stable a conformer is 2.370, and r(c15h39) = 0.970 is the most important component of the conformer s stability . The differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . For the most stable conformer, b, an eight - atom ring is formed, whereas a six - atom ring is found in the a conformer . N single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase . The values of the starting dihedrals were different in the a and b conformers, as shown in fig . 2 . Full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out . The graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . Both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method . Table 1 confirms that solvation has a relatively small effect on the energy difference between conformers . The b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models, respectively.fig . 4changes in the energy (in au) of the c15c16n2c19 dihedral angle in both clindamycin conformers, a (top) and b (bottom) in vacuum; calculations were performed with the b3lyp/6 - 31 g method changes in the energy (in au) of the c15c16n2c19 dihedral angle in both clindamycin conformers, a (top) and b (bottom) in vacuum; calculations were performed with the b3lyp/6 - 31 g method in general, the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths, angles, and the electron density distribution over the whole structure . Natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . In this section analysis of the mlliken charges for the heavy atoms (data not shown) suggests relationships between the charges and geometrical parameters of the two conformers . The ring with ihb in the b conformer of clindamycin consists of one nitrogen (n2) atom with a charge of 0.53 au, two oxygen atoms (o9 and o6) with charges of 0.54 au and 0.59 au, and carbon atoms with charges ranging from 0.60 au to 0.02 au . In the a conformer of clindamycin, the negative charges of both oxygens are slightly decreased, while both carbon atoms become more positive (0.61 au, 0.07 au). Such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin . The second - order perturbation energy (e) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer (ct) between two moieties of the molecule are presented in table 3 . The selected orbital interactions (with a stabilizing effect of over 8 kcal / mol) are presented in fig . 5 for both clindamycin conformers.table 3selected second - order perturbation energy (e, in kcal / mol) between donor and acceptor orbitals and charge transfer (qi.j, in au) in the a (top) and b (bottom) conformers of clindamycin . Calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor (i)nboacceptor (j)e (kcal / mol)qi.j (au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig . 5representations of hyperconjugative intramolecular interactions, based on the nbo analysis of both clindamycin exp - opt conformers: red dashed line for a (left), blue dashed line for b (right); b3lyp/6 - 31 g in vacuum selected second - order perturbation energy (e, in kcal / mol) between donor and acceptor orbitals and charge transfer (qi.j, in au) in the a (top) and b (bottom) conformers of clindamycin . Calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions, based on the nbo analysis of both clindamycin exp - opt conformers: red dashed line for a (left), blue dashed line for b (right); b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model, the donor acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . In the case of the central h45n2c19o9 group, the ct from the lone pair orbital on n2 is mainly directed to the antibonding c19o9 orbital (0.118 in the a conformer and 0.119 au in the b conformer). Other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital (0.103 for the a conformer and 0.106 au for the b conformer), as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital (0.124 for the a conformer and 0.112 au for the b conformer). Additionally, for the a conformer, we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital (0.068 au). The lone pair orbital on o6 interacts with the antibonding c15c14 one (0.070 au), and the lone pair orbital on o5 with the antibonding orbital c13c12 (0.069 au). On the other hand, the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital (0.024 au) for the a conformer is lower than that for the h37o6 orbital of the b conformer (0.074 au). To summarize, nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability . Therefore, the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar (methylthiolincosamide), which are linked via an amide bond, is the dominant factor in the greater stability of the b conformer . The absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . In many cases, the atoms in molecules (aim) method is a practical tool for understanding the properties of hydrogen bonds . It identifies a unique line of communication between two nuclei, and provides a point describing the nature of this interaction . Topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a (3, 1) critical point (bcp), which is a key topological descriptor of internuclear interactions, while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond . It should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions . However, typical intermolecular as well as intramolecular h - bonds can be categorized properly, as has been proven in the literature [44, 45]. The popelier criteria [44, 46] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au, and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au . A negative laplacian reveals excess potential energy at the bcp, meaning that the electronic charge is concentrated into a bond . A positive bcp reflects an excess of kinetic energy in a bond, indicating a local depletion of the electron density along a bond path . In other words, generally, the laplacian of is positive when is locally reduced, and negative if it is locally concentrated . According to criteria elaborated by the aim theory, we found two types of intramolecular h - bonds in clindamycin: typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer, and unconventional h - bonds of type oh x (x = o, cl) in both conformers (fig . 6). The numerical values for the electron density (bcp) and figure 7 shows plots of bcp (top) and bcp (bottom) versus the length of the hydrogen bond ro...h . The general shape of the bcp curve is that of an exponential decay, as expected (see fig . 7) that, in accordance with chemical intuition, o...h is increased in an ihb.fig . Exp - opt conformers of clindamycin (top: a, bottom: b) based on critical points obtained from the aim analysis . The brown, red, blue, yellow, purple, and silver beads represent c, o, n, s, cl, and h atoms, respectively . The light violet and light green beads represent the (3, + 1) and (3, 1) critical points, respectively . The h - bonds [paths connecting the (3, 1) critical points] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a (top) and b (bottom) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9 7the electron density cp (top) and the laplacian cp (bottom) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both exp - opt conformers of clindamycin (top: a, bottom: b) based on critical points obtained from the aim analysis . The brown, red, blue, yellow, purple, and silver beads represent c, o, n, s, cl, and h atoms, respectively . The light violet and light green beads represent the (3, + 1) and (3, 1) critical points, respectively . The h - bonds [paths connecting the (3, 1) critical points] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a (top) and b (bottom) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp (top) and the laplacian cp (bottom) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer (bcp8) and 0.014 au for the a conformer (bcp6), which is in line with the most stable b structure . Bcp, the second measure of the bond properties according to aim, is barely below zero, and remains ca . 0.02 in the b conformer and 0.013 au in the a conformer, which could indicate weak hydrogen - bonding regions . However, for the ihbs found in this work, the laplacian at the bond critical point tends to be negative (although small), and smaller than that for an intermolecular hydrogen bond, suggesting that the threshold for this descriptor should be revised . This analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule . The experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm . The calculated values for the c chemical shifts are in fairly good agreement with the experimental data . As chemical shifts are sensitive to subtle changes in the electronic structure, which depends in a rather complex manner on the molecular structure, we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . As usual, the central part of the molecule is the most interesting part to consider for this purpose . The chemical shifts of the carbon atoms are predicted to be located in their usual ranges: (c = o) near to 185 ppm, (c h) close to 70 ppm . (c = o) exhibits a sensitivity to ihb: the highest value (188.6 ppm) occurs for the b conformer of clindamycin, which is stabilized by the c19o9 intramolecular hydrogen bond more than the a conformer (185.1 ppm) is stabilized by the c19o9 these chemical shifts have also been shown to depend strongly on the local properties of the electron density . The small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus (0.547 in the a conformer and 0.530 in the b conformer). Finally, it is clear that (o) can be classified into two groups . In the first group, the oxygen acts as the roton acceptor for the oh group, and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . The chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer (considering its absolute value), and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum . We used h (tms) = 31.50, c (tms) = 182.20, n (ch3no2) = 167.89, o (ch3no2) = 389.95 (+ 605), s (cs2) = 1131.94, and cl (nacl) = 151.02 as referencesatomchemical shifts (ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum . We used h (tms) = 31.50, c (tms) = 182.20, n (ch3no2) = 167.89, o (ch3no2) = 389.95 (+ 605), s (cs2) = 1131.94, and cl (nacl) = 151.02 as references the calculated oxygen chemical shifts correlate with the charges, but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra . Some coupling constants vary with changes in conformation; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer . However, more interesting is the jh37o9 sscc transmitted through the c = o h o ihb, which is equal to 5.3 hz in the b conformer . Spin constants j (in hz) for both exp - opt conformers of clindamycin, i.e., a (top) and b (bottom), optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin spin constants j (in hz) for both exp - opt conformers of clindamycin, i.e., a (top) and b (bottom), optimized at the b3lyp/6 - 31 g level in vacuum the energies, the zero - point vibrational energies (zpe), and the gibbs free energy (g) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase, taking solvent effects into account, are depicted in table 1 . The selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin, lincomycin, and pirlimycin in their bound modes are given in table 2 . 3.table 1total energies (e0, in au), zero - point energies (zpe, in kcal / mol), and relative gibbs free energies at 298.15 k (g, in kcal / mol). Top: values for the fully optimized a and b conformers of clindamycin, lincomycin, and pirlimycin in vacuum . Bottom: values for the a and b conformers of clindamycin, lincomycin, and pirlimycin (optimized using a redundant coordinate algorithm in vacuum) in the pcm model of solvent and in the point ions . Two dihedrals were kept constant, d(c15c16n2c19) = 47.0 and d(c16n2c19c20) = 180.0 for conformer a, and d(c15c16n2c19) = 119.0 and d(c16n2c19c20) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0 e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters (in and degrees) for the exp - opt structures of both conformers (a and b) of clindamycin, lincomycin, and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes . Top: clindamycin, middle: lincomycin, bottom: pirlimycin total energies (e0, in au), zero - point energies (zpe, in kcal / mol), and relative gibbs free energies at 298.15 k (g, in kcal / mol). Top: values for the fully optimized a and b conformers of clindamycin, lincomycin, and pirlimycin in vacuum . Bottom: values for the a and b conformers of clindamycin, lincomycin, and pirlimycin (optimized using a redundant coordinate algorithm in vacuum) in the pcm model of solvent and in the point ions . Two dihedrals were kept constant, d(c15c16n2c19) = 47.0 and d(c16n2c19c20) = 180.0 for conformer a, and d(c15c16n2c19) = 119.0 and d(c16n2c19c20) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters (in and degrees) for the exp - opt structures of both conformers (a and b) of clindamycin, lincomycin, and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes . Top: clindamycin, middle: lincomycin, bottom: pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin . Our results show that the most important is the energy profit from the formation of the ihb . The presence of hydrogen - bond donors (o h, n h, c h) and hydrogen - bond acceptors (c = o, cl) allows for a range of hydrogen - bond combinations and a number of stable forms [40, 41]. Because of the internal hydrogen bonds, one conformer is stabilized to a greater extent than the others . First and foremost, at the b3lyp/6 - 31 g level, the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol . The energetic picture significantly changes for lincomycin and pirlimycin . In the case of lincomycin, no single conformer is favored at 298.15 k, while the a conformer of pirlimycin is more stable than b according to the three models used: vacuum, pcm, and point ions . The clindamycin b conformer in vacuum displays both the longest o6h37 bond (0.980) and the shortest ihb distance, r(c19o9 h39c15) ihb distance in the less stable a conformer is 2.370, and r(c15h39) = 0.970 is the most important component of the conformer s stability . The differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . For the most stable conformer, b, an eight - atom ring is formed, whereas a six - atom ring is found in the a conformer . N single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase . The values of the starting dihedrals were different in the a and b conformers, as shown in fig . 2 . Full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out . The graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . Both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method . Table 1 confirms that solvation has a relatively small effect on the energy difference between conformers . The b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models, respectively.fig . 4changes in the energy (in au) of the c15c16n2c19 dihedral angle in both clindamycin conformers, a (top) and b (bottom) in vacuum; calculations were performed with the b3lyp/6 - 31 g method changes in the energy (in au) of the c15c16n2c19 dihedral angle in both clindamycin conformers, a (top) and b (bottom) in vacuum; calculations were performed with the b3lyp/6 - 31 g method in general, the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths, angles, and the electron density distribution over the whole structure . Natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . In this section analysis of the mlliken charges for the heavy atoms (data not shown) suggests relationships between the charges and geometrical parameters of the two conformers . The ring with ihb in the b conformer of clindamycin consists of one nitrogen (n2) atom with a charge of 0.53 au, two oxygen atoms (o9 and o6) with charges of 0.54 au and 0.59 au, and carbon atoms with charges ranging from 0.60 au to 0.02 au . In the a conformer of clindamycin, the negative charges of both oxygens are slightly decreased, while both carbon atoms become more positive (0.61 au, 0.07 au). Such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin . The second - order perturbation energy (e) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer (ct) between two moieties of the molecule are presented in table 3 . The selected orbital interactions (with a stabilizing effect of over 8 kcal / mol) are presented in fig . 5 for both clindamycin conformers.table 3selected second - order perturbation energy (e, in kcal / mol) between donor and acceptor orbitals and charge transfer (qi.j, in au) in the a (top) and b (bottom) conformers of clindamycin . Calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor (i)nboacceptor (j)e (kcal / mol)qi.j (au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig . 5representations of hyperconjugative intramolecular interactions, based on the nbo analysis of both clindamycin exp - opt conformers: red dashed line for a (left), blue dashed line for b (right); b3lyp/6 - 31 g in vacuum selected second - order perturbation energy (e, in kcal / mol) between donor and acceptor orbitals and charge transfer (qi.j, in au) in the a (top) and b (bottom) conformers of clindamycin . Calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions, based on the nbo analysis of both clindamycin exp - opt conformers: red dashed line for a (left), blue dashed line for b (right); b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model, the donor acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . In the case of the central h45n2c19o9 group, the ct from the lone pair orbital on n2 is mainly directed to the antibonding c19o9 orbital (0.118 in the a conformer and 0.119 au in the b conformer). Other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital (0.103 for the a conformer and 0.106 au for the b conformer), as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital (0.124 for the a conformer and 0.112 au for the b conformer). Additionally, for the a conformer, we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital (0.068 au). The lone pair orbital on o6 interacts with the antibonding c15c14 one (0.070 au), and the lone pair orbital on o5 with the antibonding orbital c13c12 (0.069 au). On the other hand, the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital (0.024 au) for the a conformer is lower than that for the h37o6 orbital of the b conformer (0.074 au). To summarize, nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability . Therefore, the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar (methylthiolincosamide), which are linked via an amide bond, is the dominant factor in the greater stability of the b conformer . The absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . In many cases, the atoms in molecules (aim) method is a practical tool for understanding the properties of hydrogen bonds . It identifies a unique line of communication between two nuclei, and provides a point describing the nature of this interaction . Topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a (3, 1) critical point (bcp), which is a key topological descriptor of internuclear interactions, while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond . It should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions . However, typical intermolecular as well as intramolecular h - bonds can be categorized properly, as has been proven in the literature [44, 45]. The popelier criteria [44, 46] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au, and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au . A negative laplacian reveals excess potential energy at the bcp, meaning that the electronic charge is concentrated into a bond . A positive bcp reflects an excess of kinetic energy in a bond, indicating a local depletion of the electron density along a bond path . In other words, generally, the laplacian of is positive when is locally reduced, and negative if it is locally concentrated . According to criteria elaborated by the aim theory, we found two types of intramolecular h - bonds in clindamycin: typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer, and unconventional h - bonds of type oh x (x = o, cl) in both conformers (fig . The numerical values for the electron density (bcp) and laplacian (bcp) are presented in table 4 . Figure 7 shows plots of bcp (top) and bcp (bottom) versus the length of the hydrogen bond ro...h . The general shape of the bcp curve is that of an exponential decay, as expected (see fig . 7) that, in accordance with chemical intuition, o...h is increased in an ihb.fig . Exp - opt conformers of clindamycin (top: a, bottom: b) based on critical points obtained from the aim analysis . The brown, red, blue, yellow, purple, and silver beads represent c, o, n, s, cl, and h atoms, respectively . The light violet and light green beads represent the (3, + 1) and (3, 1) critical points, respectively . The h - bonds [paths connecting the (3, 1) critical points] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a (top) and b (bottom) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9 h39c152.3700.01460.0131cp7c17cl28 7the electron density cp (top) and the laplacian cp (bottom) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both exp - opt conformers of clindamycin (top: a, bottom: b) based on critical points obtained from the aim analysis . The brown, red, blue, yellow, purple, and silver beads represent c, o, n, s, cl, and h atoms, respectively . The light violet and light green beads represent the (3, + 1) and (3, 1) critical points, respectively . The h - bonds [paths connecting the (3, 1) critical points] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a (top) and b (bottom) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp (top) and the laplacian cp (bottom) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer (bcp8) and 0.014 au for the a conformer (bcp6), which is in line with the most stable b structure . Bcp, the second measure of the bond properties according to aim, is barely below zero, and remains ca . 0.02 in the b conformer and 0.013 au in the a conformer, which could indicate weak hydrogen - bonding regions . However, for the ihbs found in this work, the laplacian at the bond critical point tends to be negative (although small), and smaller than that for an intermolecular hydrogen bond, suggesting that the threshold for this descriptor should be revised . This analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule . The experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm . The calculated values for the c chemical shifts are in fairly good agreement with the experimental data . As chemical shifts are sensitive to subtle changes in the electronic structure, which depends in a rather complex manner on the molecular structure, we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . As usual, the central part of the molecule is the most interesting part to consider for this purpose . The chemical shifts of the carbon atoms are predicted to be located in their usual ranges: (c = o) near to 185 ppm, (c h) close to 70 ppm . (c = o) exhibits a sensitivity to ihb: the highest value (188.6 ppm) occurs for the b conformer of clindamycin, which is stabilized by the c19o9 intramolecular hydrogen bond more than the a conformer (185.1 ppm) is stabilized by the c19o9 these chemical shifts have also been shown to depend strongly on the local properties of the electron density . The small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus (0.547 in the a conformer and 0.530 in the b conformer). Finally, it is clear that (o) can be classified into two groups . In the first group, the oxygen acts as the roton acceptor for the oh group, and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . The chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer (considering its absolute value), and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum . We used h (tms) = 31.50, c (tms) = 182.20, n (ch3no2) = 167.89, o (ch3no2) = 389.95 (+ 605), s (cs2) = 1131.94, and cl (nacl) = 151.02 as referencesatomchemical shifts (ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum . We used h (tms) = 31.50, c (tms) = 182.20, n (ch3no2) = 167.89, o (ch3no2) = 389.95 (+ 605), s (cs2) = 1131.94, and cl (nacl) = 151.02 as references the calculated oxygen chemical shifts correlate with the charges, but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra . Some coupling constants vary with changes in conformation; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer . However, more interesting is the jh37o9 sscc transmitted through the c = o h o ihb, which is equal to 5.3 hz in the b conformer . This value is large enough to be measured experimentally.fig . Spin constants j (in hz) for both exp - opt conformers of clindamycin, i.e., a (top) and b (bottom), optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin spin constants j (in hz) for both exp - opt conformers of clindamycin, i.e., a (top) and b (bottom), optimized at the b3lyp/6 - 31 g level in vacuum we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin, lincomycin, and pirlimycin at the b3lyp/6 - 31 g level . Internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . Using nbo analysis, and with the aid of the aim theory, we focused on the sensitivities of electronic structure parameters such as nbo atomic charges, bond critical points, nmr chemical shifts, and spin spin coupling constants to the conformation of clindamycin . The two most stable conformers of clindamycin exhibit c = oh o intramolecular hydrogen bonds . According to nbo and aim analyses, the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar (methylthiolincosamide) is the main influence on conformer stability in vacuum and in water.
The rising prevalence of alzheimer s disease and related dementias (henceforth referred to as dementia) is emerging as a leading global health system challenge . Effective early diagnosis and management models are required to mitigate its impact on patients, caregivers, and health - care systems . Enhancing primary care capacity however, dementia care predominantly resides with geriatric specialists, who are in short supply in canada and elsewhere, delaying access to care . To enhance the care of persons with dementia, many of these have been established as primary care - based memory clinics (pcmcs). Initial evaluations suggest that pcmcs can provide timelier assessment, lead to a high degree of satisfaction among referring physicians, patients, and caregivers, and streamline access to specialists . In order to retain the fidelity of such programs and consistency with initial training and practice guidelines, and practice drift and ensure ongoing high quality of care, quality assurance frameworks are needed . Quality indicators (qis), based on best practices, define achievable benchmarks and a quality assurance framework facilitates practice improvement through targeted, educational quality improvement mechanisms . To our knowledge, there is no quality assurance framework specific to dementia care in primary care - based settings . This paper describes the results of a consensus approach to identify qis and quality improvement mechanisms in an ontario - wide network of interprofessional pcmcs, and identify potential barriers and facilitators to the implementation of a quality assurance framework . A delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms . The delphi technique is an iterative consensus process, wherein surveys are used to solicit opinions from groups, and responses summarized and redistributed in a subsequent round for consideration . We identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods (table 1). Respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale, with 1 representing the least important and 9 the most important . Links to the web - based survey were electronically distributed to all pcmc clinicians (n = 283) and ontario specialists through the ontario medical association sections of geriatric medicine (n = 123) and neurologists (n = 134), and the canadian association of geriatric psychiatrists (n = 305). After each round, qis and improvement measures in the lower two tertiles of agreement (i.e., with mean ratings less than 7) were excluded, and those remaining were reviewed by the authors guided by respondent comments . Qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness . Data from the preceding round, including number of respondents, rating means, and standard deviations, were included in the subsequent round . Student s t - test was used to identify significant differences between pcmc clinicians and specialists . Spss version 23.0 (ibm corp .) Was used, with two - sided p values of <.05 as the threshold for statistical significance . Two authors (gh, vb) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round . A delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms . The delphi technique is an iterative consensus process, wherein surveys are used to solicit opinions from groups, and responses summarized and redistributed in a subsequent round for consideration . We identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods (table 1). Respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale, with 1 representing the least important and 9 the most important . Links to the web - based survey were electronically distributed to all pcmc clinicians (n = 283) and ontario specialists through the ontario medical association sections of geriatric medicine (n = 123) and neurologists (n = 134), and the canadian association of geriatric psychiatrists (n = 305). After each round, qis and improvement measures in the lower two tertiles of agreement (i.e., with mean ratings less than 7) were excluded, and those remaining were reviewed by the authors guided by respondent comments . Qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness . Data from the preceding round, including number of respondents, rating means, and standard deviations, were included in the subsequent round . Student s t - test was used to identify significant differences between pcmc clinicians and specialists . Spss version 23.0 (ibm corp .) Was used, with two - sided p values of <.05 as the threshold for statistical significance . Two authors (gh, vb) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round . Two survey rounds were conducted between april and june 2014 . In the first round, 842 surveys were distributed with a response rate of 21.3% . The majority of respondents were physicians, and nurses and other health professionals equally represented the remainder of respondents (table 2). Respondents had an average of 14.6 10.1 years of clinical practice with older adults . Among pcmc respondents, 31% were from urban centres, 42% rural settings, and 25% from mixed urban / rural populations . In contrast, 73% of specialists worked in urban settings, and 24% served mixed populations . Only 11 neurologists responded to the first survey, and their specialty was not included in round 2 . In round 2, respondents had an average of 17.42 10.08 years of clinical practice with older adults and practice settings were similar to round 1 . A third delphi round was not conducted due to the substantial drop in response rate between rounds 1 and 2 . Table 3 presents the results of the consensus process and table 4 presents the final list of quality indicators . Quality improvement mechanisms characterized by specialist integration, including case discussions, shared care, observerships, and mentorships, ranked highly (table 5). Other preferred quality improvement mechanisms included standardized electronic charting forms, self - directed learning activities, and interactive programs . Survey respondents recommended that between 1030% of patients seen in a pcmc also be reviewed by a specialist . Descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs: 1) its perceived importance, 2) collaboration and role clarity, and 3) the implementation process . Almost all respondents mentioned the need to maintain high - quality care through ongoing, targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams . Most recognized a formal quality assurance framework and individual qis as essential . Great indicators and very necessary [pcmc clinician, round 1]. Our challenge is to maintain evidence based salience, which ultimately facilitates improved quality of life for persons with dementia and their family [pcmc clinician, delphi round 2]. All are relevant quality indicators for a pcmc [specialist, round 2]. However, a few respondents were unsure why a quality assurance framework was needed at all . Others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . Is the intent of the questions to assess what we are doing now or [what] we think should be the standard? [pcmc clinician, round 1]. This is the canadian consensus guideline that every doc should know [specialist, round 1; emphasis added by authors]. A second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs, and identified a lack of clearly delineated responsibilities among referring clinicians, pcmcs, and specialists . Respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . Information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment, lifestyle modification and support resources [pcmc clinician, round 2]. This lack of clarity was considered most problematic with regard to patient follow - up . Patients with dementia with great plans should not need constant surveillance and follow up but...this is not my experience...they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [specialist, round 1]. Similar concerns were raised regarding the management of comorbidities . [my] assumption is that management of comorbidities is the primary care physician s domain . Recommendations are made for lifestyle changes, however [congestive heart failure] and diabetes management are only commented on, suggestions are made to the referring physician [pcmc clinician, round 2]. It was not clear which health care provider should conduct a physical examination, leading to gaps in assessment . Referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [pcmc clinician, round 1]. My biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [specialist, round 1]. Respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs . One of the main obstacles was the perceived burden of documentation required to implement qis . [it will take a lot of work to do the] searching and documenting as a group of patients needs a process to set up how to search, then doing the search [pcmc clinician, round 2]. Integrating qis into existing electronic medical records was touted as a solution, though potentially a resource - intensive one . Making up stamps to collect this information is doable but takes time for someone to make the [standardized template] and then to test it a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores . I think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist, done on both patients referred to the specialist by the pcmc, and unselected patients seen in the clinic that would not have been referred to the specialist [specialist, round 1]. Access to other health care professionals, within pcmcs and in the community, was identified as important for quality care, though access was not perceived as uniform . Having a pharmacist on our team is a great asset .... the local [pharmacists] volunteer their time to assist us at our once monthly day long clinics [pcmc clinician, round 2]. The [social work] and [occupational therapy] members of the team do an excellent job the [alzheimer s society] representative has been excellent as well when given an opportunity [pcmc clinician, round 2]. An important finding is the relative lack of importance ascribed to end - of - life planning . While respondents agreed on its importance, many expressed that such planning discussions were not appropriate for patients with less advanced disease . Palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced . Family physician team will do this in following up patient [pcmc clinician, round 1]. Lastly, many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement . They also identified specialists as agents to promote care quality, particularly in the context of a shared care approach . This [case review] could be done at the same time, as the specialist mentoring the clinic is in a clinic day with the team [pcmc clinician, round 1]. Barriers to the implementation of this framework included role confusion among stakeholders and limited resources . Descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs: 1) its perceived importance, 2) collaboration and role clarity, and 3) the implementation process . Almost all respondents mentioned the need to maintain high - quality care through ongoing, targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams . Most recognized a formal quality assurance framework and individual qis as essential . Great indicators and very necessary [pcmc clinician, round 1]. Our challenge is to maintain evidence based salience, which ultimately facilitates improved quality of life for persons with dementia and their family [pcmc clinician, delphi round 2]. All are relevant quality indicators for a pcmc [specialist, round 2]. However, a few respondents were unsure why a quality assurance framework was needed at all . Others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . Is the intent of the questions to assess what we are doing now or [what] we think should be the standard? [pcmc clinician, round 1]. This is the canadian consensus guideline that every doc should know [specialist, round 1; emphasis added by authors]. A second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs, and identified a lack of clearly delineated responsibilities among referring clinicians, pcmcs, and specialists . Respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . Information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment, lifestyle modification and support resources [pcmc clinician, round 2]. This lack of clarity was considered most problematic with regard to patient follow - up . Patients with dementia with great plans should not need constant surveillance and follow up but...this is not my experience...they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [specialist, round 1]. Similar concerns were raised regarding the management of comorbidities . [my] assumption is that management of comorbidities is the primary care physician s domain . Recommendations are made for lifestyle changes, however [congestive heart failure] and diabetes management are only commented on, suggestions are made to the referring physician [pcmc clinician, round 2]. It was not clear which health care provider should conduct a physical examination, leading to gaps in assessment . Referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [pcmc clinician, round 1]. My biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [specialist, round 1]. Respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs . One of the main obstacles was the perceived burden of documentation required to implement qis . [it will take a lot of work to do the] searching and documenting as a group of patients needs a process to set up how to search, then doing the search [pcmc clinician, round 2]. Integrating qis into existing electronic medical records was touted as a solution, though potentially a resource - intensive one . Making up stamps to collect this information is doable but takes time for someone to make the [standardized template] and then to test it [pcmc clinician, round 2]. A second issue identified in relation to the implementation of the quality assurance framework i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist, done on both patients referred to the specialist by the pcmc, and unselected patients seen in the clinic that would not have been referred to the specialist access to other health care professionals, within pcmcs and in the community, was identified as important for quality care, though access was not perceived as uniform . Having a pharmacist on our team is a great asset .... the local [pharmacists] volunteer their time to assist us at our once monthly day long clinics [pcmc clinician, round 2]. The [social work] and [occupational therapy] members of the team do an excellent job the [alzheimer s society] representative has been excellent as well when given an opportunity [pcmc clinician, round 2]. An important finding is the relative lack of importance ascribed to end - of - life planning . While respondents agreed on its importance, many expressed that such planning discussions were not appropriate for patients with less advanced disease . Palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced . Family physician team will do this in following up patient [pcmc clinician, round 1]. Lastly, many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement . They also identified specialists as agents to promote care quality, particularly in the context of a shared care approach . This [case review] could be done at the same time, as the specialist mentoring the clinic is in a clinic day with the team [pcmc clinician, round 1]. Barriers to the implementation of this framework included role confusion among stakeholders and limited resources . Almost all respondents mentioned the need to maintain high - quality care through ongoing, targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams . Most recognized a formal quality assurance framework and individual qis as essential . Great indicators and very necessary [pcmc clinician, round 1]. Our challenge is to maintain evidence based salience, which ultimately facilitates improved quality of life for persons with dementia and their family [pcmc clinician, delphi round 2]. All are relevant quality indicators for a pcmc [specialist, round 2]. However, a few respondents were unsure why a quality assurance framework was needed at all . Others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work . Is the intent of the questions to assess what we are doing now or [what] we think should be the standard? [pcmc clinician, round 1]. This is the canadian consensus guideline that every doc should know [specialist, round 1; emphasis added by authors]. A second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs, and identified a lack of clearly delineated responsibilities among referring clinicians, pcmcs, and specialists . Respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement . Information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment, lifestyle modification and support resources [pcmc clinician, round 2]. This lack of clarity was considered most problematic with regard to patient follow - up . Patients with dementia with great plans should not need constant surveillance and follow up but...this is not my experience...they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [specialist, round 1]. Similar concerns were raised regarding the management of comorbidities . [my] assumption is that management of comorbidities is the primary care physician s domain . Recommendations are made for lifestyle changes, however [congestive heart failure] and diabetes management are only commented on, suggestions are made to the referring physician [pcmc clinician, round 2]. It was not clear which health care provider should conduct a physical examination, leading to gaps in assessment . Referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [pcmc clinician, round 1]. My biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [specialist, round 1]. Respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs . One of the main obstacles was the perceived burden of documentation required to implement qis . [it will take a lot of work to do the] searching and documenting as a group of patients needs a process to set up how to search, then doing the search [pcmc clinician, round 2]. Integrating qis into existing electronic medical records was touted as a solution, though potentially a resource - intensive one . Making up stamps to collect this information is doable but takes time for someone to make the [standardized template] and then to test it a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores . I think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist, done on both patients referred to the specialist by the pcmc, and unselected patients seen in the clinic that would not have been referred to the specialist [specialist, round 1]. Access to other health care professionals, within pcmcs and in the community, was identified as important for quality care, though access was not perceived as uniform . Having a pharmacist on our team is a great asset .... the local [pharmacists] volunteer their time to assist us at our once monthly day long clinics [pcmc clinician, round 2]. The [social work] and [occupational therapy] members of the team do an excellent job the [alzheimer s society] representative has been excellent as well when given an opportunity [pcmc clinician, round 2]. An important finding is the relative lack of importance ascribed to end - of - life planning . While respondents agreed on its importance, many expressed that such planning discussions were not appropriate for patients with less advanced disease . Palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced . Family physician team will do this in following up patient [pcmc clinician, round 1]. Lastly, many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement . They also identified specialists as agents to promote care quality, particularly in the context of a shared care approach . This [case review] could be done at the same time, as the specialist mentoring the clinic is in a clinic day with the team [pcmc clinician, round 1]. Barriers to the implementation of this framework included role confusion among stakeholders and limited resources . This study used a consultative process among primary and specialty providers to identify a core group of qis and preferred quality improvement mechanisms for dementia care . Quality assurance can be an effective method to ensure fidelity to best practices and maintain a high standard of care quality . Selected qis address care processes, assessment and reassessment, medication review and management, investigations, non - pharmacological management, pharmacological management, and managing concomitant conditions . Selected quality improvement mechanisms emphasize a desire for multimodal education and closer collaboration with specialists . Assessing care quality and publically reporting the findings are increasingly commonplace . However, quality assurance still mainly focuses on specific care sectors or episodes, rather than on overall care for conditions that require coordination and collaboration across multiple sectors . In that context critical considerations include educating clinicians about the role of quality assurance, improving role clarity among providers involved in dementia care, expanding access to allied health professionals, and creating standardized electronic medical record templates to simplify qi documentation . Quality assurance must not create additional burden on clinicians often working with limited resources and time, a burden that could also impact the quality of the clinician - patient interaction . Respondents proposed that 1030% of patients seen in pcmcs be reviewed with a specialist, preferably in a shared care approach . Closer integration of urban specialists with rural pcmcs represents a tremendous opportunity to extend quality dementia care, particularly to rural patients ., it will be necessary to identify barriers (e.g., allocation of funding) and facilitators (e.g., resources for coordination, electronic medical records, telemedicine). Another important finding was the relatively low rankings of qis related to end - of - life planning with dementia patients . In addition to potential discomfort among clinicians to address such issues, this finding may also stem from the understandable desire to maintain hope and engagement early in the course of the illness, though undue delays may leave patients and caregivers less able to meaningfully participate in such discussions . The most important identified barrier to the implementation of the quality assurance framework is the need for greater clarity on responsibilities of referring family physicians, pcmc clinicians, and specialists, particularly with respect to follow - up, physical examination, and care of complex comorbidities . Quality assurance often targets relatively simple conditions, such as hypertension, or restricts its scope to specific aspects, processes or locations of care . In contrast, dementia, like all major chronic conditions, follows a course of progressive decline punctuated by increasingly frequent health complications (related to dementia itself, as well as to exacerbations of concurrent comorbidities), multiple care transitions, progressive caregiver stress and health service utilization, and ultimately death . Optimal dementia care thus requires a systems approach of integration and coordination . Addressing greater role clarity among all dementia stakeholders, a task that with proper resources could be coordinated from within pcmcs, requires immediate attention, in order to ensure a stable clinical infrastructure that is able to safely and effectively address the needs of these patients, wherever they may be and whenever they arise . Until such integration is achieved first, only two delphi rounds were conducted because the response rate fell markedly after the first . However, the response rate remained above what is considered appropriate for delphi surveys, and ratings of individual qis, except those related to physical examination, remained stable between rounds . A second limitation is that the survey was solely distributed within the previously described network of pcmcs, though this model is widely implemented across ontario . As the qis were selected by both primary and specialist providers, they are likely applicable to other dementia care settings . Third, participation of neurologists in the first round was low and their response was not solicited in the second round . The relatively lower participation of neurologists in this work, compared to geriatricians and geriatric psychiatrists, is notable and requires further investigation . Fourth, allied health providers were under - represented and, given their importance in dementia care, additional work is required to further understand and develop their role in an integrated system of dementia care . Fifth, patient and caregivers were not surveyed regarding what they consider to be important qis . Finally, candidate qis and quality improvement mechanisms were not identified through a systematic literature review, but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care, which would thus have remained highly ranked . First, only two delphi rounds were conducted because the response rate fell markedly after the first . However, the response rate remained above what is considered appropriate for delphi surveys, and ratings of individual qis, except those related to physical examination, remained stable between rounds . A second limitation is that the survey was solely distributed within the previously described network of pcmcs, though this model is widely implemented across ontario . As the qis were selected by both primary and specialist providers, they are likely applicable to other dementia care settings . Third, participation of neurologists in the first round was low and their response was not solicited in the second round . The relatively lower participation of neurologists in this work, compared to geriatricians and geriatric psychiatrists, is notable and requires further investigation . Fourth, allied health providers were under - represented and, given their importance in dementia care, additional work is required to further understand and develop their role in an integrated system of dementia care . Fifth, patient and caregivers were not surveyed regarding what they consider to be important qis . Finally, candidate qis and quality improvement mechanisms were not identified through a systematic literature review, but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care, which would thus have remained highly ranked . While this study has identified qis and quality improvement mechanisms to assess care quality for dementia, findings underscore the importance of system integration for the provision of quality dementia care, with specifically defined and mutually understood roles among stakeholders and, where necessary, the reallocation of existing resources to support this approach to care . An approach whereby clear roles are negotiated among dementia stakeholders can provide sufficient flexibility to meet regional needs (especially in rural areas where access to specialists is more limited), foster more effective collaboration and accountability, and thus facilitate the delivery and measurement of care quality for dementia . Within that context, proper field - testing, validation, and evaluation of selected qis and quality improvement mechanisms this work has significant implications on the organization of care for aging patients with complex conditions . Primary and specialist providers share the responsibility of providing and supporting integrated dementia quality care . As such, the care of persons with cognitive impairment would be enhanced by the development of a practical and realistically feasible quality assurance framework, under whose umbrella both pcmc and specialist services are integrated, and which ensures high fidelity to intended design and best practices, and thus maintains a high level of care quality.
Symptomatic supravesical obstructive uropathy in a patient with dual functioning kidneys is classically characterized by bilateral hydroureters / hydronephrosis and an empty urinary bladder . This obstruction may be secondary to metastatic abdomino - pelvic and retroperitoneal malignancies, ureteric calculi and retroperitoneal fibrosis [25]. The evidence for obstruction may be partially or totally absent on ultrasound or computerized tomography, in one or both kidneys [68]. Our recent experience with a uraemic 56-year - old caucasian showing only unilateral moderate right - sided hydronephrosis is presented . A 56-year - old caucasian male patient with a past medical history that included hypertension and coronary artery disease and a recent serum creatinine of 88.4 mol / l, was diagnosed 10 days earlier with urinary tract infection (uti). Two days before presentation to us, he developed worsening anorexia, nausea with vomiting and oliguria . His father had prostate cancer and died at age 75 years from colon cancer; a sister had breast cancer and a maternal uncle had throat cancer . Physical examination revealed a blood pressure of 165/89 mmhg, pulse rate of 56/min and respiratory rate of 16/min . He was not dehydrated nor orthostatic . Except for trace ankle oedema and bilateral costo - vertebral angle punch tenderness, mol / l, co2 14 mmol / l and potassium 5.5 mmol / l . Urinalysis showed 510 wbc per high - power field (phf) and 25 rbc phf . Alanine aminotransferase (alt), aspartate aminotransferase (ast) and total creatine kinase (ck) levels were normal . A post - void urinary bladder scan had demonstrated only 60 ml of residual urine . A renal ultrasound showed unilateral moderate right - sided hydronephrosis with suspected mass effect on the inferior urinary bladder . The left kidney appeared normal, measuring 16.2 cm 8.1 cm, with preserved cortical thickness . The next day, with increasing vomiting, serum creatinine 894.6 mol / l, phosphorus 2.6 mmol / l, potassium 6.2 mmol / l and oliguria (table 1), he started daily haemodialysis . A non - contrast computerized tomography examination, on day 2, again showed unilateral right - sided hydronephrosis, bilateral nephric stranding and urinary bladder wall thickening suspicious for transitional cell cancer (figure 1). Cystoscopy, on day 3, revealed a sessile urinary bladder tumour, which was resected . The right ureter was successfully cannulated and a right ureteric stent was placed with prompt urine drainage . The next day, with a strong push from nephrology, the patient consented to a percutaneous left nephrostomy procedure despite the normal appearing left kidney . A percutaneous left nephrostogram revealed a previously unrecognized mild hydronephrosis / hydroureter with obstruction at the ureterovesical junction . The pathology report revealed high - grade urothelial carcinoma, grade 3 of 3, with invasion of the muscularis propria . A patient with dual functioning kidneys presenting with uraemic symptoms and suspected to have obstructive uropathy must be presumed to necessarily have bilateral renal obstruction [610]. This is without prejudice to the findings on conventional renal imaging with ultrasound or computerized tomography [3,610]. There are false negative tests with these imaging modalities, the so - called syndrome of non - dilated obstructive uropathy or non - dilated obstructive nephropathy [610]. Clinical conditions associated with the absence of hydronephrosis on ultrasound and computerized tomography despite obstructed kidney(s) include acute early obstruction, the presence of retroperitoneal fibrosis or infiltrative metastatic abdomino - pelvic cancers, dehydration or septic shock and severe oliguria [210]. Our patient was not dehydrated and was not hypotensive but was severely oliguric (table 1). We note that we did not rule out the presence of retroperitoneal fibrosis in our patient . The classic picture of bilateral hydronephrosis with hydroureters and an empty urinary bladder, in symptomatic uraemia following supravesical obstruction, in patients with dual functioning kidneys, is well acknowledged . However, the presentation of new - onset symptomatic uraemia concurrent with only unilateral hydronephrosis / hydroureter on conventional imaging (ultrasound, computerized tomography) should raise the plausibility of non - apparent obstruction of the contra lateral kidney . In such instances, the more sensitive albeit invasive percutaneous nephrostogram of the apparently normal appearing kidney is therapeutic and will lead to greater renal salvage [68]. We would like to remind practicing providers that symptomatic uraemia presenting in the setting of suspected obstructive uropathy must be assumed to imply bilateral renal obstruction, regardless of the results / interpretations of any form of conventional renal imaging . Therefore, necessarily, every attempt to decompress both kidneys must be the rule . This approach would result in early and improved renal salvage . Left undiagnosed and therefore untreated, this potentially reversible cause of renal failure can lead to irreversible renal failure if bilateral,, or to significant residual loss of renal function if missed on one side only . We note that we were not able to carry out any split renal functional testing after recovery as the patient's primary attention at this point was to find out treatment options for his cancer.
In 1985, smith pioneered phage display technology, an in vitro methodology and system for presenting, selecting and evolving proteins and peptides displayed on the surface of phage virion . Since then, phage display has developed rapidly and become an increasingly popular tool for both basic research such as the exploration of protein - protein interaction networks and sites [24], and applied research such as the development of new diagnostics, therapeutics, and vaccines [510]. Usually, the protein used to screen the phage display library is termed as target and the genuine partner binding to the target is called template . Peptide mimicking the binding site on the template and binding to the target is defined as mimotope, which was first introduced by geysen et al . . One type of the most frequently used targets is monoclonal antibody . In this situation, the template is the corresponding antigen inducing the antibody, and the mimotope is a mimic of the genuine epitope . . Goes a mimotope is defined as a molecule able to bind to the antigen combining site of an antibody molecule, not necessarily identical with the epitope inducing the antibody, but an acceptable mimic of the essential features of the epitope . Mimotopes and the corresponding epitope are considered to have similar physicochemical properties and spatial organization . The mimicry between mimotopes and genuine epitope makes mimotopes reasonable solutions to epitope mapping, network inferring, and new diagnostics, therapeutics, and vaccines developing . Powered by phage display technology, mimotopes can be acquired in a relatively cheap, efficient and convenient way, that is, screening phage - displayed random peptides libraries with a given target . However, not all phages selected out are target - specific, because the target itself is only one component of the screening system . From time to time, phages reacting with contaminants in the target sample or other components of the screening system such as the solid phase (e.g., plastic plates) and the capturing molecule (e.g., streptavidin, secondary antibody) rather than binding to the actual target are recovered with those target - specific binders (displaying mimotopes) during the rounds of panning . Peptides displayed on these phages are called target - unrelated peptides (tup), a term coined recently by menendez and scott in a review . The results from phage display technology might be a mixture of target - unrelated peptides and mimotopes, and it can be difficult to discriminate tup from mimotopes since the binding assays used to confirm the affinity of peptides for the target often employ the same components as the initial panning experiment . Therefore, target - unrelated peptides might be taken into study as mimotopes if the researchers are not careful enough . Obviously, target - unrelated peptides are not appropriate candidates for the development of new diagnostics, therapeutics, and vaccines . For mimotope - based epitope mapping, if tup is included in the mapping, the input data is improper and the result might be misleading . There are now quite a few programs for mimotope based epitope mapping, none of them, however, has a procedure to scan, report and exclude target - unrelated peptides [1523]. In this study, we describe a web server named sarotup, which is an acronym for scanner and reporter of target - unrelated peptides . Sarotup was coded with perl as a cgi program and can be freely accessed and used to scan peptides acquired from phage display technology . It is capable of finding, reporting, and precluding possible target - unrelated peptides, which is very helpful for the development of mimotope - based diagnostics, therapeutics, and vaccines . The power and efficiency of sarotup was also demonstrated by preliminary tests in the present study . Recently, menendez and scott reviewed a collection of target - unrelated peptides recovered in the screening of phage - displayed random peptide libraries with antibodies . They divided their collection into several categories according to the component of the screening system to which target - unrelated peptides bind . Very recently, brammer et al . Reported a completely new type of target - unrelated peptides . In the review of menendez and scott, target - unrelated selection is due to the binding to contaminants or components other than target; however, in the report of brammer et al ., target - unrelated selection is due to a coincident point mutation in the phage library [12, 13]. We compiled a set of 23 tup motifs from the above two references [12, 13], including 12 motifs specific for the capturing agents, 5 motifs specific for the constant region of antibody, 3 motifs specific for the screening solid phase, 2 motifs specific for the contaminants in the target sample, and 1 motif for a mutation in phage library (table 1). The sarotup was implemented as a free online service, powered by apache and perl . Three pages are designed and integrated into a tabbed web interface with cascading style sheets codes . The core program of sarotup was sar.pl, a cgi script coded with perl . In this script, the 23 tup motifs were converted to regular expressions, which were then used to match each input peptide sequence . We constructed two - test data sets from [12, 13, 1523, 25, 26]. The first data set contains 8 cases; 6 of them are sourced from test cases used in extant programs for mimotope - based epitope mapping [1523]; the left 2 are cases studies published recently [25, 26]. As shown in table 2, the target of each case in the first data set is monoclonal antibody and the structure of corresponding antigen - antibody complex has been resolved, which is used to derive its structural epitope as the golden standard for evaluation . For each case if target - unrelated peptides were found, a new panel of peptides excluding tup was produced . The old and the new panel of peptides were then used to predict epitope using mapitope or pepsurf [15, 21, 22]. Finally, the results were compared to show if sarotup could improve the performance of mimotope - based epitope mapping . The second data set is composed of 100 peptides in raw sequence format . The first group has 77 sequences compiled from the first data set without any known tup motifs; the second group has 23 sequences sourced from [12, 13] with various tup motifs . The mixture of the two groups of sequences made the second data set, which was then used as the sample input and can be used to evaluate the efficiency of sarotup . As a free online service, the web interface of sarotup has successfully been implemented as a tabbed web page . The left tab is the default page, providing a brief introduction to this web service . The users can either paste a set of peptide sequences in the text box or upload a sequence file to the sarotup server for scanning . As shown in figure 1, a panel of peptides in raw sequence format taken from the b12 test case was pasted in the text box . Besides the raw sequences however, only the standard iupac one - letter amino acid codes are accepted at present . The lower section of the form has a series of options (figure 2). It includes three drop lists for the screening target, screened library, and screening solid phase, respectively . It also has two groups of check boxes for the capturing reagents and contaminants in the target sample or screening system . By default however, the users can customize their scan according to their experiment at this section . After the users submit their request, the scanning results of sarotup will be displayed on the middle tabbed page . If any target - unrelated peptides are found, they will be reported in a table . At the same time, a new panel of peptides excluding target - unrelated peptides is produced and can be downloaded from the hyperlink created by the sarotup server (figure 3). The file of the new panel of peptides will be stored on the server for a month and then automatically deleted . We have tested sarotup on the internet explorer (version 6.0), mozilla firefox (version 3.5.2), and google chrome (version 3.0). Although sarotup looks a little bit different among different browsers, it works normally on all browsers tested . As shown in table 2, the first test data set has 11 panels of peptides acquired from phage display libraries screened with 8 targets . In the 11 panels of peptides sarotup scanned, there were target - unrelated peptides in 3 panels from cetuximab, 80r, and b12 test case, respectively (table 3). This result suggested it was not rare that target - unrelated peptides sneaked into biopanning results and then were taken as mimotopes in study . In all, 7 target - unrelated peptides were found; 4 of them were due to binding to plastic; the left 3 were due to binding to the fc fragment (table 3). For the above 3 cases, the genuine epitopes recognized by cetuximab, 80r, and b12 monoclonal antibodies are compiled according to the ced records and pdbsum entries . Mapitope or pepsurf [15, 21, 22] were used to perform mimotope - based epitope prediction with or without sarotup procedure . For mapitope and pepsurf algorithm,; the stop codon modification was set to none; and all other options were in default . The cluster with best score was taken as the predicted epitope . In the cetuximab case, pepsurf was used because there are only four or three peptides in the panel, statistically too few for mapitope . In the case of 80r and b12, mapitope was used because many peptides in the two cases exceeding the length limit of pepsurf, that is, 14 amino acids . If a predicted residue is identical with a residue in the true epitope, it is underlined (table 4). As shown in table 4, the number of true positives improved from zero to four in the cetuximab case with sarotup procedure . When it came to the b12 case sarotup did not improve the number of true positives in the 80r case when the parameters are same to the cetuximab and b12 cases . However, when the distance parameter was adjusted from default (i.e., 9) to 10, sarotup did increase the number of true positive residues from eight to eleven . These results indicate: (1) epitope prediction based on mimotope will be interfered if target - unrelated peptides are taken as mimotopes; (2) sarotup can improve the performance of mimotope based epitope mapping through cleaning the input data . The second data set has 100 peptides, varying from 6 to 22 residues long . Suppose that matching each pattern to each peptide manually costs 10 seconds, then it would take a researcher more than 6 hours (23,000 seconds) to look through the second data set for target - unrelated peptides, even if he is as prompt during the whole period . Besides, a table of target - unrelated peptides and a new panel of peptides excluding tup was produced at the same time by sarotup . It is true that some target - unrelated peptides can be identified through control and binding competition experiments . However, using sarotup first will certainly save a lot of labor, money, and time for researchers in this area . Although the target of all tests described previously were monoclonal antibodies, sarotup can be customized and used in scanning the results from phage display technology using other targets such as enzymes and receptors . For the same reason, we can also expect that sarotup will extend its use to other similar in vitro evolution techniques, such as ribosome display [2931], yeast display, and bacterial display [3335]. Furthermore, sarotup will not only benefit the mimotope - based epitope mapping, but also the development of new diagnostics, therapeutics, and vaccines . Target - unrelated peptides are not appropriate candidates for mimotope based diagnostics, therapeutics, and vaccines, since they are mimics to components or contaminants of the screening system rather than target . Therefore, it is reasonable to find and exclude possible target - unrelated peptides from the candidate list of new diagnostics, therapeutics, and vaccines . Screened a phage - displayed random peptides library with the cetuximab and got four different peptides, that is, qfdlstrrlk, qynlssralk, vwqrwqksyv, and mwdrfsrwyk . As described previously, we scanned the four mimotopes with sarotup and the result suggested that the peptide vwqrwqksyv might be a tup . Indeed, the dot blot analysis of riemer et al . Showed that qynlssralk bound the cetuximab with high affinity but vwqrwqksyv was less reactive with the cetuximab . Trying to develop a mimotope vaccine, riemer et al . After immunization mice with these constructs, they found that either the cetuximab or the antibodies induced by the qynlssralk vaccine construct inhibited the growth of a431 cancer cells significantly . The inhibition of the antibodies induced by the vwqrwqksyv vaccine construct however, was not statistically significant when compared with the inhibition caused by the isotype control antibody . Sarotup must be used with caution since it is a tool only based on pattern matching at present . As these tups are not embedded in sarotup at present, it is possible that a true tup cannot be detected by sarotup . To reduce this kind of false negatives besides the motif - based search, the database - based search can find out the known tup without known motifs . It is also possible that a sarotup predicted target unrelated peptide is actually target - specific . To decrease this kind of false positives, the users should customize the scan according to their experiment at the section of advance options . For example, the user should select antibody without fc fragment as the target if fab was used in biopanning; this will prevent sarotup from reporting peptides bearing the fc - binding motifs as tup . As described above, sarotup in future will also provide an exact match tool based on database search . In this way, a match might mean that different research groups have isolated the same peptide with a variety of targets . Thus, the false positive rate of sarotup can be decreased further when its new feature become available . At last, we must point out that the controlled experiment is still the gold standard to distinguish tups from the specific mimotopes . Sarotup, a web application for scanning, reporting and excluding target - unrelated peptides has been coded with perl . It is also useful in the development of diagnostics, therapeutics, and vaccines . To our knowledge
Reversible focal lesions in the splenium of the corpus callosum (scc) have been found in patients with various conditions including seizures, antiepileptic drug toxicity or withdrawal, viral encephalitis, hypoglycemia, wernicke's encephalopathy, marchiafava - bignami disease, sympathomimetic - induced kaleidoscopic visual illusion syndrome, hemolytic uremic syndrome, altitude brain injury, and acute axonal injury . Analysis of the current literature revealed that the most frequent cause of reversible focal lesions of the scc is influenza virus - associated encephalitis / encephalopathy (iaee). The abnormality has been reported in association with various viruses (e.g., epstein - barr virus, rotavirus, influenza virus) (1 - 3). The brain lesions described in patients with iaee include restricted diffusion involving the cerebral cortex and subcortical white matter in various locations, symmetric lesions in the brainstem, basal ganglia, thalamus, and cerebellar white matter with or without brain edema, and mild brain atrophy . Transient restricted diffusion of the scc in patients with iaee has also been described (4). Here, we report an adult who presented with non - specific dizziness and lesions of the scc on magnetic resonance imaging (mri) who was diagnosed with hemorrhagic fever with renal failure syndrome (hfrs) with serologically proven hantaan virus infection . A previously healthy 53-yr - old man presented with a 6 - 7-day history of fever up to 40 and dizziness . The initial diffusion - weighted images revealed high signal intensity in the corpus callosum with a low signal on the apparent diffusion coefficient (adc) map (fig . 1). The next day, he developed thrombocytopenia (4010/l) and was transferred to our emergency room for further evaluation . He stated that he had been tying rice straw into sheaves several days earlier, but not using any drugs, like metronidazole, antiepileptic drug or alcohol, and had not been exposed to toxins or experienced weight loss . On neurologic examination, he was slightly confused, and scored 24/30 on the mini mental status exam (mmse). The cranial nerve, motor, and sensory examinations, deep tendon reflexes, and cerebellar function tests were normal . His blood tests showed marked thrombocytopenia (2110/l) and azotemia (bun / cr 23 mg / dl/1.5 mg / dl). He was started on cefotaxime and clindamycin for a suspected infection and transfused with fresh frozen plasma, but his fever (> 38.0) was sustained . Two days after admission, he was alert and his fever had subsided, but the oliguria, azotemia (bun / cr 49 mg / dl/3.0 mg / dl), and thrombocytopenia (610/l) had worsened (figs . 2, 3). Four days later, the thrombocytopenia had recovered, although the azotemia (bun / cr 82 mg / dl/7.1 mg / dl) had not improved . He was diagnosed with hfrs based on the detection of hantaan virus antibody in his serum . Six days later, he was alert, and his electroencephalograph (eeg) was normal . His platelet count had stabilized (31110/l), and a lumbar puncture was performed . The cerebrospinal fluid (csf) analysis revealed no cells, a nor mal protein level of 26.0 mg / l, a normal glucose level of 80 mg / dl, and no organisms on gram staining . The mri findings had resolved completely in follow - up studies conducted on day 14 (fig . Hemorrhagic fever with renal failure syndrome occurs mainly in europe and asia and is characterized by a fever and renal failure associated with hemorrhagic manifestations . Hfrs is caused by airborne contact with secretions from rodent hosts infected with viruses belonging to the genus hantavirus in the family bunyaviridae . The clinical features consist of a triad of fever, hemorrhage, and renal insufficiency (5). Other common symptoms during the initial phase of the illness include, myalgia, abdominal and back pain, and diarrhea . In most cases, mild neurological symptoms such as headache, vertigo, and nausea the pathogenesis is largely unknown, but findings from several studies have suggested that immune mechanisms play an important role . After the infection, marked cytokine production, kallikrein - kinin activation, complement pathway activation, and increased levels of circulating immune complexes occur . Damage to the vascular endothelium, capillary dilatation, and leakage are clinically significant features of the disease . Myelin sheaths in the scc have a relatively high water component, and the scc is more susceptible to cytotoxic edema than are other brain areas (2). Reversible brain lesions have been attributed to the transient development of intramyelinic edema due to the separation of myelin layers, which is a possible mechanism for the transiently decreased diffusion of the scc lesion (2, 11). The influx of inflammatory cells and molecules, possibly combined with related cytotoxic edema, might have decreased the adc (2, 12, 14). The most common causes of reversible focal lesions of the scc are viral encephalitis, antiepileptic drug toxicity / withdrawal, and hypoglycemic encephalopathy . They are not specific to iaee and have been reported secondary to various infectious agents, including rotavirus, measles, herpesvirus, salmonella organisms, mumps, varicella - zoster virus, adenovirus, o157 escherichia coli - associated hemolytic - uremic syndrome, legionnaires' disease, and unknown pathogens (4, 9, 14). The similar clinical and imaging features suggest a common mechanism induced by different pathological events leading to the same results (1). The most likely causes of these transient lesions of the scc have been explained as a rapidly resolving intramyelinic infiltrate or the influx of inflammatory cells and macromolecules, combined with related cytotoxic edema, which is very similar to the pathogenesis of hfrs (2, 7, 9, 10, 13). This argues for a non - specific cause related to vasogenic edema, probably secondary to the inflammatory response (2, 7) our patient was diagnosed with hfrs, and his mri showed scc lesions . We report a reversible scc lesion associated with hfrs . To our knowledge, this is the first case of virus - associated encephalitis / encephalopathy in which the pathogen was a hantaan virus . Although it is not certain whether the neurologic symptoms in this patient might have been caused by fever, or scc lesions, or both, we suggest that hfrs patients with neurologic symptoms like dizziness and mental slowing should be considered to have structural brain lesions and to require brain imaging studies.
As is the most common cause of left ventricular outflow obstruction in children and adults . It is most common type of valvular heart disease in europe and north america, occurring in 27% of the population over 65 years of age . Medical therapy alone is not effective for the long - term management of aortic valve disease, thus valve replacement remains the standard of care in patients with an acceptable risk profile . We present a case of critical as with significant symptom manifested as chest pain that was managed successfully without surgical avr or tavr . We will also briefly review incidence, etiology, grading of as and current guidelines for avr . A 91-year - old gentleman with history of hypertension, dyslipidemia and as presented to our office on 7/2015 with complaints of new onset sub sternal burning pain of 6 weeks duration . Prior to 6 weeks patient could walk 2 blocks on level ground without shortness of breath or chest pain . Blood pressure was 140/90 mm hg and pulse was regular at 60 beats per minute . Cardiovascular exam revealed a grade 4/6 ejection systolic murmur best heard in the right second intercostal region radiating bilaterally to the neck . Ecg revealed left ventricular hypertrophy (lvh) with non specific st - t wave changes in inferior leads (figure 1). Echocardiography (echo) done in may of 2014 had revealed critical as with aortic valve area of 0.6 - 0.7 centimeter square and ejection fraction of 65 percent . The aortic valve (av) was heavily calcified . Aortic valve peak velocity (av vmax) 4.66 m / s (figure 2). The patient was directly referred for a cardiac catheterization to evaluate his coronary anatomy prior to avr . Hemodynamic measurement revealed left ventricle (lv) pressure 240/0 with left ventricular end diastolic pressure (lvedp) of 10 . Pulmonary artery wedge pressure (pawp), mean of 24, right atrium (ra) mean of 8, right ventricle (rv) 60/10 with an right ventricular end diastolic pressure (rvedp) of 14, pulmonary artery (pa) 60/17 . The aortic valve area was calculated at 0.6 centimeter square, which was unchanged from echo done may 1, 2014 . The left ventriculogram in the right anterior oblique (rao) view revealed normal lv systolic motion and ejection fraction of 55 - 60 percent . Left main coronary artery revealed a common ostium - giving rise to the left anterior descending (lad) and the circumflex . Right coronary artery (rca) revealed a 99% ostial narrowing (figure 3). Lad revealed a 60% narrowing in its proximal segment and a 90% ostial diagonal narrowing (figure 4). After a detailed discussion involving the patient, his son, interventional cardiologist and the cardio thoracic surgeon, patient requested that only pci of the rca be considered, as the chest pain was of recent duration with no change in aortic valve finding . Thus, a successful pci was done in the proximal rca with a bare metal stent (figure 6). Patient did extremely well after pci of the rca and the patient has remained asymptomatic to date . Thus, this new onset chest pain in patient with critical as was due to concomitant coronary artery lesion and was amenable to stenting and thus spared this elderly patient from the aortic valve replacement which would have been a high risk surgery for him . As is the most common valvular heart condition in the developed world, affecting 3% of people between ages 75 and 85 and 4% of people over age 85 . Congenitally unicuspid, bicuspid, tricuspid, or even quadricuspid valves may be the cause of as . In adults who develop symptoms from congenital as the main causes of acquired as include degenerative calcification and, less commonly, rheumatic heart disease . Other, infrequent causes of as include obstructive vegetations, homozygous type ii hypercholesterolemia, paget disease, fabry disease, ochronosis, and irradiation . Based upon a variety of hemodynamic and natural history data, clinicians generally grade the severity of stenosis as mild, moderate, severe, or critical . Grading of as are as follows: i) mild: valve area exceeds 1.5 cm; transvalvular velocity 2.0 to 2.9 m / s; mean gradient <20 mmhg; ii) moderate: valve area of 1.0 to 1.5 cm; transvalvular velocity 3.0 to 3.9 m / s; mean gradient 20 to 39 mmhg; iii) severe: valve area is less than 1.0 cm; transvalvular velocity 4 m / s; mean gradient 40 mmhg . The term critical stenosis was defined based upon theoretical considerations showing that the aortic valve area must be reduced to one - fourth of its natural size before significant changes in circulation occur . As a result, since the triangular orifice area of the normal (adult) aortic valve is approximately 3.0 cm, an area exceeding 0.75 cm would not be defined as critical . Avr and tavr remain the only treatment proven to reduce the rates of mortality and morbidity in this condition . Under current guidelines, the onset of symptoms of exertional angina, syncope and dyspnea in a patient who has severe as is a class i indication for surgery . The annual rate of sudden death in patients with this condition is estimated at 1% to 3% but the surgical mortality rate in avr has been as high as 6% . With improvements in surgical techniques and prostheses, mortality rates have been reduced to 2.42% making a case for earlier intervention . Tavr has become widely available, but further investigation into its use in this patient cohort is warranted . While assessing the cases of asymptomatic as we have both traditional as well as novel markers at our disposal now . Left ventricular ejection fraction (lvef) <50 percent, peak aortic jet velocity> 4.0 m / s, valve area <1 cm and mean pressure gradient> 40 mm hg are the traditional markers to denote severe as in asymptomatic patients . While, indexed left atrial size> 12.2 cm / m, lvh with wall thickness> 15 mm, global left ventricular longitudinal strain <15.9, bnp (b - natriuretic peptide) level> 130 pg / ml and increase in mean pressure gradient of> 20 mm hg during exercise testing are the novel markers of asymptomatic severe as . Bnp level does not appear to be significantly associated with the degree of as severity but does reflect heart failure status . The american college of cardiology and american heart association (acc / aha) have issued the following recommendations for avr, based on the severity of stenosis and on whether the patient has symptoms: i) severe stenosis, with symptoms: class i recommendation (surgery should be done). Without surgery, these patients have a very poor prognosis, with an overall mortality rate of 75% at 3 years; ii) severe stenosis, no symptoms, in patients undergoing cardiac surgery for another indication (example coronary artery bypass grafting, ascending aortic surgery, or surgery on other valves): class i recommendation for concomitant aortic valve replacement; iii) moderate stenosis, no symptoms, in patients undergoing cardiac surgery for another indication: class iia recommendation (i.e., aortic valve replacement is reasonable); iv) very severe stenosis (aortic peak velocity> 5.0 m / s or mean pressure gradient 60 mm hg), no symptoms, and low risk of death during surgery: class iia recommendation; v) severe stenosis, no symptoms, and an increase in transaortic velocity of 0.3 m / s or more per year on serial testing or in patients considered to be at high risk for rapid disease progression, such as elderly patients with severe calcification: class iib recommendation (surgery can be considered). On revisiting the above case description we realize that the patient did have critical as but was asymptomatic . His chest pain was only due to concomitant coronary artery disease but was not due to as per se . Age and comorbidity of the patient posed a high risk for coronary artery bypass graft (cabg) with avr . Besides, the patient s symptoms were of new onset and subsequent cardiac catheterization revealing critical rca stenosis and the patient s preference for treating the cause of his recent symptoms, encouraged us to think otherwise . The decision to perform pci alone with the belief that this chest pain and cad would be amenable to the minimal risk procedure paid dividends . In addition, patient received a bare metal stent with the option of undergoing surgical avr if symptoms were not relieved . Awareness amongst physicians about the fact that all critical aortic stenosis with chest pain may not require aortic valve replacement is important . This can lead to less invasive treatment tailored to the need of the patient especially in those with advanced age, significant comorbidities and an extremely high risk for cabg with avr and can also result in decreased cost of care . Pharmacological nuclear stress testing may be another modality that can be used to differentiate etiology of the symptoms.
The various blood cell lineages in mammals arise from a multipotent haematopoietic stem cell via particular differentiation pathways . One of these pathways, erythropoiesis, leads to the production of red blood cells (rbcs), which transport oxygen and carbon dioxide around the body by means of the intracellular metalloprotein haemoglobin (hb). Hb is a tetramer, consisting of two -globin and two -globin subunits . In mammals, these globin chains are encoded by two gene loci: the -globin locus and the -globin locus . In humans, the -globin locus consists of the embryonic - and adult -globin genes, and the -globin locus comprises the embryonic -, foetal - and -, and adult - and -globin genes [1, 2]. The globin genes expressed from these loci differ from embryonic to adult erythropoiesis in order to meet varying oxygen demands and facilitate placental transfer of oxygen from mother to embryo . There are a number of severe diseases caused by the disruption of adult globin genes, including thalassaemias and certain types of anaemia . According to the world health organisation, approximately 5% of the world's population carry genes involved in hb disorders, and as such, they present an enormous health burden . Thalassaemia is caused by a reduction or abolition of the expression of one or more globin genes, resulting in an imbalance of - and -globin chains in red blood cells and consequent anaemia [1, 4]. Sickle cell anaemia is another prevalent haemoglobinopathy and is caused by a mutation in the adult -globin gene which generates a single glutamic acid to valine amino acid substitution . This mutation leads to the polymerisation of globins in venous circulation [5, 6], which can trigger a rigid and sickled cell phenotype [7, 8] and results in a number of acute conditions such as vaso - occlusion, splenic sequestration, and haemolytic anaemia . There are currently a number of treatments available for patients suffering from thalassaemia and sickle cell anaemia . The most common is packed red blood cell transfusion, but this is associated with a number of problems, such as sufficiency of supply, bacterial and viral infection, biochemical and biomechanical changes during storage (red blood cell storage lesions), and the risk of immune rejection from the patient [10, 11]. Furthermore, blood transfusions are ongoing throughout a patient's life and often lead to a potentially fatal buildup of iron and associated reduction in organ activity . Residual production of foetal -globin persists naturally throughout life, and levels vary between individuals [12, 13]. This persistent expression allows two -globin chains to combine with two adult -globin chains to form what is known as foetal hb (hbf). As only the adult -globin gene is mutated in sickle cell anaemia, affected infants are protected from severe symptoms until they reach several months of age, due to the large amount of hbf still in circulation at birth . Furthermore, patients who have inherited alleles associated with increased levels of hbf, known as hereditary persistence of foetal hb (hpfh), are protected into adulthood . Similarly, a more asymptomatic disease phenotype has also been shown in patients with -thalassaemia who exhibit higher levels of hbf . Together, these observations indicate that increased foetal -globin is able to compensate in part for the loss of adult -globin function and thus ameliorates the symptoms of certain adult haemoglobinopathies . Accordingly, a number of drug treatments for -thalassaemia and sickle cell disease, for instance, 5-azacitidine, hydroxyurea, and butyrate, all act by nonspecifically reactivating foetal -globin gene expression by various mechanisms . There is evidence that long - term administration of these drugs has chronic side effects, consistent with their lack of specificity [8, 17]. As the existing methods of treatment for these haemoglobinopathies remain inadequate, alternative forms of therapy are currently being sought, and stem cell therapies should be considered . This paper will discuss progress in utilising novel cellular reprogramming techniques to treat rbc diseases . Stem cells, both embryonic and adult, have the ability to differentiate into various cell types, making them a potentially attractive treatment option . Embryonic stem cells (escs) and adult stem cells (ascs) each have their own strengths and disadvantages in these strategies . Escs are more easily grown in culture and are pluripotent, meaning that they are able to differentiate into any cell of the body . The practicality of widespread esc use for therapeutic purposes, however, has been questioned due to issues of supply and ethical and legal considerations . Moreover ascs, on the other hand, overcome some of these problems as they can be harvested from each individual patient . They are not abundant and are difficult to obtain, often being harboured in internal organs such as the gut and bone marrow . They have proven difficult to culture in vitro, and furthermore, they are widely believed to be multipotent rather than pluripotent and are thus only able to differentiate into certain cell types . Cellular reprogramming potentially overcomes these issues and may offer new treatment methods for a range of diseases, including those of rbcs . Takahashi and yamanaka were the first to show that it is possible to take differentiated somatic cells and transform them into cells with pluripotent potential . They began by identifying a pool of 24 transcription factors which are important in maintaining stem cell traits and used retroviral transduction to express these factors in murine embryonic and adult fibroblasts . They found that these cells then displayed characteristics and properties comparable to those of pluripotent escs . They were able to further refine the required transcription factors by setting up numerous combinations to determine which were essential to this process and identified four factors, oct4, sox2, klf4, and c - myc, needed to transform a somatic cell to an induced pluripotent stem cell (ipsc). Takahashi et al . Then applied these four factors to human cells and transformed neonatal and adult human fibroblasts into human ipscs (hipscs). This was a valuable subsequent step as it showed that this process can be reproduced with human cells and could thus potentially be utilised for stem cell - based therapies of human disease . Several other transcription factor combinations have since been shown to be sufficient to reprogram somatic cells [2224] and one particular combination, involving oct4, sox2, nanog, and lin28, is particularly efficient and is now widely utilised . For example, a number of the transcription factors used to generate these cells, including c - myc, klf4, and lin28, are oncogenes, and their misexpression can lead to cancer [2628]. In order for ipscs to be differentiated into a cell type of choice, the retrovirally transmitted genes need to be switched off or removed to reduce the possibility of their inducing tumours [22, 29]. Another potential issue is that the transplantation of any cells that have not been fully differentiated from the ipsc state could lead to the formation of cancerous teratomas . Any cells remaining which are still in a pluripotent state could multiply and, without the appropriate growth controls, would have the potential to result in tumours in transplant patients . To potentially avoid the issue of tumour formation and uncontrolled proliferation of ipscs, an alternative methodology known as transdifferentiation is also being considered as a potential treatment strategy . Transdifferentiation is achieved by introducing various exogenous factors into a differentiated cell, such as a fibroblast, to directly convert it into another type of differentiated cell, thereby bypassing the pluripotent state (figure 1(b)). As early as 1990, choi et al . Were able to convert various cells types, including dermal fibroblasts and chondroblasts, into mononucleated, striated myoblasts that were indistinguishable from normal myoblasts in vivo this was achieved through the expression of the myogenic regulatory factor myod, a transcription factor known to be involved in the determination of muscle cells . Shortly after this, another group showed that it was possible to turn myeloid 416b cells into mast cells through the forced expression of gata-2 and gata-3, two transcription factors which play important roles in haematopoiesis . Investigated the effects of ectopically expressing the gene eyeless (ey), an orthologue of the mammalian pax6 gene, in drosophila and found that eye structures formed in places such as the wings and legs . Since takahashi and yamanaka's pioneering studies in cellular reprogramming, the field of transdifferentiation has advanced considerably . Zhou et al . Investigated the effects of expressing ngn3, pdx1, and mafa, transcription factors involved in -cell differentiation, on exocrine cells of the adult pancreas . They found that the coexpression of these factors was able to convert the exocrine cells into -cells . The induced -cells were identical to endogenous -cells in morphology and showed similar expression of genes associated with -cell function . These cells can also rescue the phenotype of hyperglycaemia, as they are able to secrete insulin and remodel surrounding vasculature . In other work, utilised the neural - specific transcription factors, ascl1, brn2, and myt1l, to rapidly convert both murine embryonic fibroblasts and adult fibroblasts directly into functional neurons . These induced neuronal cells express neuron - specific proteins, generate action potentials, and form functional synapses . A further advance has shown that it is also possible to transdifferentiate fibroblasts into functional neural progenitor cells by transient induction of oct4, sox2, klf4, and c - myc in cells cultured in a defined neural reprogramming medium . This process bypassed the generation of ipscs and gave rise to multipotent progenitors with the capacity to expand and differentiate into a number of neural lineages . All of these experiments indicate that it is possible to direct a differentiated cell to another cell fate through the application of extragenic factors . Advances in cellular reprogramming have raised the attractive possibility that this technology could be utilised to generate a limitless source of immune - matched, pathogen - free erythrocytes for transfusion . They found that hipscs are capable of generating haematopoietic cells with phenotypic and morphological characteristics similar to those derived from hescs; however, these hipsc - derived cells exhibited a dramatically reduced capacity (by greater than 1000-fold) to generate erythroid cells . A subsequent study by lapillone et al ., however, showed that it was indeed possible to produce significant numbers of mature erythroid cells from hipscs in vitro . This group employed the methods outlined by thomson's group using oct4, sox2, nanog, and lin28 to convert fibroblasts to hipscs . They then cultured these cells in medium containing the cytokines scf, tpo, flt3 ligand, rhu bmp4, rhu vegf - a165, il-3, il-6, and erythropoietin (epo). They analysed the expression profiles of these cells over 20 days of culture and found that pluripotent stem cell markers decreased whilst the erythroid markers cd36, cd235a, and cd71 increased . Cells at day 20 were found to have a high erythroid potential and were plated in sequential cocktails of cytokines comprising scf, il-3, and/or epo . Erythroid maturation was achieved after another 25 days and was confirmed by morphological examination and by flow cytometric analysis of erythroid markers (cd235a, cd71, and cd36). Furthermore, these erythroid cells were able to enucleate, albeit with reduced capacity compared to hesc - derived erythroid cells, and were found to express functional hb (predominantly hbf). These hipsc - derived erythroid cells were compared to those differentiated from hescs, and no significant differences were detected in terms of erythroid commitment, expression of erythroid markers, and type and functionality of hb . Revealed that while functional rbcs could be generated from hipscs, when compared to hescs, hipscs were shown to have reduced (approximately 8-fold) amplification potential in producing mature erythroid cells . A recent study has set out to determine whether this reduced efficiency is an intrinsic property of hipscs, or whether it can be increased by altered culture conditions . This study showed not only that it is possible to generate rbcs from hipscs with similar efficiency to hescs, but also that these cells can be differentiated from hipscs generated using episomal vectors, obviating the need for potentially deleterious retroviral transfection . This group cultured hipscs derived from human fibroblasts with the op9 bone marrow stroma line to induce hematopoietic differentiation and followed this with selective expansion of erythroid cells in serum - free media with cytokines supporting rbc differentiation . The erythroid cultures established in this study consisted of a pure population of cd235a+ cd45 leukocyte - free rbcs which had robust expansion capabilities, as well as a long lifespan of up to 90 days . These hipsc - derived cells can enucleate and were shown to express foetal - and embryonic -globin demonstrating successful reprogramming of the -globin locus . The results from this study show that it is possible to produce significant numbers of erythroid cells from fibroblast - derived hipscs, and that thus cellular reprogramming could contribute to the treatment of haemoglobinopathies . Furthermore, since these rbcs are generated from transgene - free hipscs, they circumvent problems associated with genomic integration and undesired reactivation of reprogramming factors . Another recent study has shown that it is possible to use transdifferentiation to produce haematopoietic cell lineages, including mature erythrocytes . In this work, szabo et al . Transduced human dermal fibroblasts with oct4 alone and showed that these cells then express the panhaematopoietic marker cd45 but lack the pluripotency marker tra-1 - 60 . They then cultured these cells in a haematopoietic cytokine cocktail containing scf, g - csf, flt3lg, il-3, il-6, and bmp-4 supplemented with epo and observed an emergence of cells expressing adult -globin protein and the red blood cell marker glycophorin - a . In addition, the authors noted that these erythroid cells express adult rather than embryonic globins, unlike cells derived from human pluripotent stem cells [39, 40], perhaps suggesting that they have indeed been transdifferentiated from adult fibroblasts, bypassing the hipsc stage . As the techniques used in this study avoid the pluripotent state as well as have a high yield, expansion capacity, and clinical feasibility, this strategy could provide a reasonable basis for autologous cell replacement therapies . These studies demonstrate that it is possible to differentiate hipscs into mature erythrocytes in vitro for transfusions . In order to utilise these methods to treat rbc diseases successfully, fibroblasts could potentially be taken from either a healthy immunomatched individual, or a universal donor, and be converted to erythrocytes via the hipsc state . A study has already developed hipscs from an individual with a bombay phenotype of the abo blood group system, where the abh antigen is not expressed on rbcs, and blood can be donated to anyone . This group has further demonstrated that it is possible to differentiate haematopoietic lineages from these cells although they have not explicitly shown mature erythrocyte differentiation . This possible treatment method circumvents many of the problems associated with current transfusions, such as questionable quality, lack of supply, and immune - rejection . However, issues still remain, such as cost and the risk of iron overloading . In order to cure, rather than treat, rbc diseases, healthy progenitor cells must be transplanted into patients and subsequently repopulate the haematopoietic system . A promising study has already shown that it is possible to use reprogrammed cells to treat sickle cell anaemia in mice . Hanna et al . Took cells from the tail tips of a humanized sickle cell anaemic mouse model, in which the mouse -globin genes have been replaced with human -globin, and the mouse -globin genes have been replaced with human and (sickle) globin genes, and through adaptation of the protocol developed by takahashi and yamanaka, who reprogrammed these cells into ipscs . Once this was accomplished, they were able to correct the mutant -globin gene by replacing it with a copy of the wildtype human -globin gene through homologous recombination . Following this, the ipscs were differentiated into haematopoietic progenitors through the ectopic expression of hoxb4, and by growing in haematopoietic cytokines on an op9 bone marrow stroma cell line . This group then carried out extensive testing to determine whether the treated mice displayed any further signs of the disease . Firstly, functional correction was evaluated by electrophoresis for the human -globin proteins and, and they found a significant increase in - and a decrease in -globin . Blood counts were also performed 12 weeks after transplant, which showed that treated mice had increased rbcs, hb, and packed cell volume as well as reduced reticulocytes, a common indicator of sickle cell disease and severity . Lastly, they examined symptomatic indicators of sickle cell anaemia such as urine concentration, body weight, and breathing rate and found that all three of these parameters were ameliorated in the treated mice . This study thus provides an important proof of principle that cellular reprogramming can be employed to correct erythroid disorders, albeit in this case, in conjunction with gene therapy . Normal erythropoiesis is dependent upon the correct expression of globin genes . Where globin genes are incorrectly expressed, or are mutated, anaemia or thalassemia results . Current therapy for these disorders involves packed red blood cell transfusions, which are limited by supply, risk of infection, expense, and patient rejection . Drug - based therapies involve the nonspecific reactivation of foetal globins and have long - term side effects . In seeking alternative strategies, recent advances have shown that cellular reprogramming can now generate large quantities of red blood cells in culture, potentially for use in transfusions . Furthermore, these strategies have been successfully combined with gene therapy to treat a sickle cell anaemia mouse model, suggesting that cellular reprogramming will provide a realistic future alternative to conventional treatment of haemoglobinopathies.
He had a left ventricular ejection fraction of 42% and regional wall motion abnormalities compatible with multi - vessel territories . Coronary angiography findings revealed total occlusion at the proximal left anterior descending artery (lad), 50 - 75% stenosis at the mid to distal left circumflex artery, and 90% stenosis at the mid right coronary artery (rca). Cardiac surgery was performed through a median sternotomy, and the heart was displaced using a deep pericardial retraction suture, gauze swab, and tissue stabilizer . After confirming the lack of atheroma burden in the ascending aorta by epiaortic ultrasound examination, the left radial artery was connected to the ascending aorta . After completing the lad grafting using the left internal thoracic artery, the patient was in the trendelenburg position, and norepinephrine was infused at a dose of 0.15 g / kg / min . After stabilizer application, there was a temporary deterioration in the patient's hemodynamic status, which was stabilized without the aid of incremental doses of norepinephrine dosage . After opening the anastomotic site at the om branch, the surgeon informed us the patient was bleeding from the arteriotomy site despite placement of a proximal snare . The co2 blower was more aggressively used to aid grafting at a flow rate of 3 - 5 l / min . Bleeding was replaced by colloid solution, and all hemodynamic variables were stable: systemic blood pressure 121/58 mmhg, heart rate 59 beats / min, pulmonary arterial pressure 24/11 mmhg, cardiac index 2.6 l / min / m and mixed venous oxygen saturation 82% . Approximately 5 minutes after the commencement of grafting, the surgeon decided to insert an intracoronary shunt for better visualization . Suddenly, the patient's systemic blood pressure decreased to 64/56 mmhg despite increasing the norepinephrine dose from 0.05 g / kg / min to 0.30 g / kg / min . The pulmonary arterial pressure increased to 34/20 mmhg, and the mixed venous oxygen saturation decreased to 66% . Vasopressin 0.6 iu was quickly administered intravenously, and the transesophageal echocardiography (tee) revealed a massive gas floating at the orifice of the rca (fig . Although the patient remained in normal sinus rhythm, complete heart block occurred within minutes, and ventricular pacing was commenced . Over the next several minutes, ventricular fibrillation developed; despite aggressive treatment, including defibrillation, epinephrine, and direct manual cardiac massage, adequate circulation could not be restored, and cardiac massage was continued . Upon preparing for emergent conversion to cardiopulmonary bypass (cpb), epinephrine 300 g was injected directly to the aortic root by the surgeon using a 26-gauge needle . Subsequently, manual cardiac massage was continued for several cycles, and spontaneous recovery of unspecified cardiac rhythm was observed . Then the patient's systemic mean arterial pressure gradually rose from 38 to 103 mmhg, and recovery of normal sinus rhythm and contractile performance followed immediately . The remaining grafts were performed successfully without conversion to cpb, and no sign of bleeding was observed at the puncture site in the aorta . The patient had an unremarkable postoperative course without neurologic deficits and was discharged 10 days after the surgery . Despite using a proximal coronary snare, this method of hemostasis can be incomplete in the presence of severe atheroma or calcification, causing bleeding through the arteriotomy site . Cardiac displacement invariably causes hemodynamic deterioration, and when the diastolic systemic blood pressure decreases below the pressure generated by the co2 gas blower, gas may migrate in a retrograde fashion to the aortic root . Even in the presence of a proximal snare, the proximal coronary lumen may not be completely obstructed due to the presence of calcified atheroma, as evidenced by continued bleeding from the arteriotomy site after application of the snare . As the rca ostium is in the highest position of the sinus of valsalva, gaseous materials would be prone to migrate into the rca, causing embolic infarction . A minor gas embolism may pass unnoticed or be overcome with supportive measures to aid emboli clearance . However, in the case of a massive embolism with the possible introduction of room air due to venturi effect, hemodynamic collapse can occur and may require aggressive treatment, such as cardiopulmonary resuscitation (cpr) with aspiration of the air by direct puncture of the aortic root . It may even require emergent conversion to cpb, which is known to be associated with increased morbidity and mortality . Therefore, when bleeding from the arteriotomy site persists despite the use of a proximal snare or intracoronary shunt, anesthesiologists should be aware of the possibility of a gas embolism and advise the surgical team to stop or limit the use of the co2 blower . When a gas embolism results in hemodynamic deterioration, supportive measures should be immediately undertaken to increase the coronary perfusion pressure and facilitate emboli clearance . Gas embolism should also be confirmed with tee, and a pacemaker should be at hand given that complete heart block due to atrioventricular nodal ischemia may occur in the case of an embolism involving the rca . Finally, in the case of failure of supportive measures such as drug administration to the venous side, direct aortic injection of the drugs can be attemped before initiating cpb . In coronary artery bypass graft surgery, direct cardiac massage may not be aggressively and effectively performed due to the fear of disrupting the previously grafted conduits, limiting the drug delivery from the venous side to the systemic circulation . Prior to the 1960s, intracardiac injection of medications was used blindly or directly, but this approach was abandoned due to the devastating complication of lacerating the epicardial coronary arteries and cardiac tamponade . However, direct injection to the aortic root following a sternotomy appears to be feasible and may be life - saving, as severe right ventricular dysfunction may limit systemic drug delivery injected through the venous side despite the performance of direct cardiac massage . In the current case, intra - aortic injection of epinephrine may have resulted in a more effective increase in the systemic vascular resistance, thereby increasing the coronary perfusion pressure and thus facilitating emboli clearance . In conclusion, in the rare case of a gas embolism occurring during opcab occurs, the potential for catastrophic results necessitates immediate and appropriate managements including supportive measures, needle aspiration of gas, and cpr with direct cardiac massage . In refractory cases, while considering emergent conversion to cpb, direct injection of drugs to the aortic root can be attempted before initiating cpb . This may be a simple, relatively safe, and life - saving approach for the management of a gas embolism during opcab because a sternotomy has already been conducted.
Multiple sclerosis (ms) is a chronic progressive inflammatory and neurodegenerative disease of human central nervous system (cns) which usually affects young adults, with a reported median age of onset of approximately 30 years.1 in approximately 85% of patients with ms, the disease is initially recognized by a relapsing - remitting course which may eventually lead to a progressive deterioration of neurological status along with significant loss of neurological function.2 neuropathologically, ms presents with various patterns, however, inflammatory demyelination and oligodendrocyte loss and neuronal and axonal degeneration3 constitute salient features of microscopic examination of ms lesions . Currently, the cause and cure for ms remain elusive and in many cases ms ends with significant neurological disabilities . While massive activation of the inflammatory arm of the immune system against putative cns antigen(s) such as myelin basis protein plays a major role in the pathogenesis of the demyelinating process of ms, the neurodegenerative process prevails in the long run and causes significant cognitive and functional deterioration in affected patients . Electrophysiologic examination of demyelinated lesions indicates conduction block as the predominant feature of these lesions . The pathophysiologic basis of such conduction block in ms rests on segmental demyelination with loss of whole internodes of myelin . In normal individuals, blocking the voltage - gated potassium channels does not have any significant impact on conduction of the action potential since these channels are enclosed by layers of myelin sheath . However, loss of myelin during pathogenesis of ms, exposes the potassium channels and interferes with production and conduction of the action potential . In fact, the impulse conduction fails specifically at the site of demyelination, while the normal segments of the axons on both sides continue their impulse conduction normally . Certain features which determine the impulse conduction block in demyelinated axon consist of the size of demyelinated area, extent of myelin loss along the internode, and the time elapsed since demyelination happened . The duration of demyelination is an important factor since myelin loss sets a number of adaptive axonal responses in motion . For example, development of a revised group of ion channels along the demyelinated membrane may result in restoration of impulse conduction . The size of the demyelinated lesion is another significant factor since axons repair centripetally inside the lesion and extensive lengths of demyelination are repaired slowly which in turn reflects the fact that the chance of functional recovery correlated inversely with the lesion size . It appears that myelin loss involving the paranodal area is stronger factor in blocking conduction than a comparable loss of distributed along the internode . Apart from the segmental demyelination which is associated with conduction block, histopathologic examination of ms tissue has shown that some node are inappropriately wide in ms lesions due to paranodal demyelination or retraction of the paranodes.4 another salient feature of demyelinated axons is abnormal reorganization of the ion channels . The speedy conduction velocity and uninterrupted transmission of electric signals in mammalian myelinated axons rest on the appropriate spatial distribution ofion - selective channels . Na channels are gathered at nodes of ranvier in concentrations which are ~25 times that of internodal regions.5 these clusters are essential for efflux of ion currents at nodes and allow fast saltatory conduction . On the contrary, other studies have demonstrated that k currents appear only after loosening of the myelin sheath from the axonal membrane.6 k channels are located only in paranodal and internodal areas and normally they do not contribute to generation of the action potential.7 scientific studies which have focused on action potential propagation in normal and demyelinated axons have revealed a reorganization of axolemmal ion channels, which in turn leads to conduction impairments . Loss or decrease of sodium channels from the nodes of ranvier8 causes the nodes to be inexcitable and leads to conduction block . Sherratt et al9 demonstrated that in demyelinated regions sodium channel concentrations are reduced, while voltage - sensitive potassium channels (which are not active on normal myelinated axons) become detectable . This exposure of k channels at juxtaparanodal region leads to efflux through fast k channels and causes axonal conduction block by preventing sufficient depolarization and initiation of the action potential at the node of ranvier.10 demyelination also exposes slow k channels, further interrupting normal hyperpolarization and blunting normal repetitive impulse release from the presynaptic area terminals.11 in 1981, bostock et al12 demonstrated that potassium channel blockers dendrotoxin and 4-aminopyridine improved conduction impairments in experimentally demyelinated axons and laid the scientific rationale for clinical studies of potassium channel blockers in ms patients . Fampridine (or 4-aminopyridine [4-ap]) is a broad - spectrum blocker of voltage - sensitive potassium channels, which has been demonstrated to improve conduction and propagation of the action potential along the demyelinated axons . In addition, fampridine increases the duration of the pre - synaptic action potential and amplitude which in turn leads to increased transmitter release.13 apart from being a voltage - activated potassium channel blocker, 4-aminopyridine potentiates synaptic and neuromuscular transmission by affecting the beta subunit of voltage - activated calcium channels.14 this novel mode of action of 4-aminopyridine which occurs independent from its blocking effect on the potassium channels, provides another rationale for its positive effect on the neuromuscular function in patients with spinal cord injury, myasthenia gravis and ms . Hawthorne, ny, usa) is one of the three isomeric amines of pyridine with chemical formula h2nc5h4n, which acts a selective potassium - channel blocker . Dalfampridine, which is a sustained - release oral form of fampridine, is rapidly and completely absorbed from the gastrointestinal tract, however, its absolute bioavailability has not been determined . Compared to an aqueous oral solution, the extended release tablet shows a relative bioavailability of 96%, with a delayed absorption pattern which provides a less rapid rise to a lower maximum plasma concentration . In healthy volunteers who took a single 10 mg dose of dalfampridine, peak serum concentrations were reached 3 to 4 hours following oral administration and it was almost completely and rapidly eliminated by urinary excretion . The elimination half - life of dalfampridine is 6.4 hours in healthy individuals . As a lipid - soluble agent, dalfampridine passes through the blood brain barrier and blocks potassium channels in both the central and peripheral nervous systems.15 daldampridine binds reversibly to the cytoplasmic side of potassium channels, blocking the ion conductance pathway which in turn prolongs the action potentials in unmyelinated nerve fibers and enhances neurotransmitter release at synaptic endings.16 dalfampridine has been studied extensively in symptomatic treatment of ms patients with walking impairment . Impairment of ambulation is a major neurological deficit in ms patients and significantly interferes with their quality of life . It is hypothesized that dalfampridine improves clinical symptoms of ms by restoring conduction in demyelinated axons through voltage - dependent potassium channel blockade.15 in addition, dalfampridine has been used for treatment of lambert - eaton myasthenic syndrome,17 where it prolongs neurotransmitter release at the neuromuscular junction . Studies involving animal models have demonstrated that can reverse tetrodotoxin toxicity.18 pharmacokinetic assessmentsof both immediate - release and sustained release formulations of fampridine have been done in normal individuals as well as those patients with ms and spinal cord damage . Uges et al19 performed a pharmacokinetic study involving 9 healthy individuals (7 men and 2 women) to assess immediate - release fampridine in intravenous, entric - coated, and uncoated oral formulations . The bioavailability of the enteric coated tablets was calculated from the area under the serum concentration curve 95% 29% (mean sd), which was not much different from that calculated from urinary excretion 98% 8% . There was no evidence that metabolism of fampridine involved glucoronidation, sulfonation, or n - acetylation . Investigators reported that 89.6% 7.5% of the medication was excreted in the urine unchanged after 24 hours of the intravenous formulation and 86.1% 2.7% with the entric coated formulation . One dose - ranging pharmacokinetic study included 12 ms patients who received increasing doses of sustained - release fampridine from 7.5 to 25 mg every 12 hours.20 the investigators reported a mean time to peak concentration of 5 hours and mean serum half - life of 5.2 hours . Vollmer et al21 studied the pharmacokinetics of dalfampridine in clinical trials of ms patients and found that multiple dosing of this medication was associated with its accumulation . In addition, the investigators noted that the steady - state pharmacokinetic profile of fampridine sustained - release 20 mg bid administered for 2 weeks appeared to support the administration of twice - daily dosing in this population . Dalfampridine (ampyra) is available as 10 mg extended release tablets with relative bioavailability of 96%, peak of plasma concentration of 34 hours and half life of 5.26.5 hours . The major goals of treating ms patients consist of prevention of exacerbations, limiting disability progression, and improving cognitive decline . The available treatments for ms fall under two categories: disease modifying agents such as beta - interferons and glatiramer acetate and medications for symptomatic treatment such as baclofen for treatment of spasticity and dalfampridine which is used to restore the impulse conduction along the demyelinated axons and improve muscle strength and walking ability . In january 2010, dalfampridine was approved by the fda for symptomatic treatment of ms patients with specific indication for improving walking . In the past 4-aminopyridine has been used as an experimental agent which presumably enhances nerve conduction of demyelinated axons through its effects on potassium channels for treatment of fatigue in ms . Sheean et al22 reported that use of fampridine in ms patients was associated with improvements in fatigue as well motor and visual symptoms . In addition, dalfampridine has been assessed and studied in other cohorts of ms patients in the context of various clinical trials to determine its efficacy in improving ms patients ambulation . In this manuscript only some of the more significant clinical trials have been cited and discussed . Using quantitative measures of motor function, schwid et al23 performed one of the earlier clinical trials of sustained - release fampridine - sr in ms patients . This was a randomized, double - blind, placebo - controlled, crossover clinical trial included 10 patients with clinically definite ms, limb weakness and stable motor deficits (edss 6.07.5). Study subjects were randomized to be treated with sustain - release fampridine 17.5 mg orally twice daily for a period of one week or placebo for a period of one week . Study outcomes included time to walk 8 meters, time to climb four stairs, maximum voluntary isometric contraction measured quiantitatively, manual muscle testing, grip strength, edss, and the patient s global impression . Of these outcomes, only timed gait progressed on sustain - release fampridine compared with placebo in 9 of the study participants . Seven participants preferred the medication over placebo based on the findings from the patient s global impression . Goodman et al24 reported the results of a four - center randomized, double - blind placebo - controlled study of controlled release aminopyridine in 31 ms patients . The study participants were treated with increasing doses of 20 to 80 mg of aminopyridine daily in divided doses . A total of 25 participants received the active medication and 11 participants were treated with placebo . Outcome measures of this clinical trial consisted of timed ambulation, manual testing of leg strength, paced auditory serial addition task, 9-hole peg test, and a fatigue diary . The results of this clinical trial indicated a statistically significant and dose - related enhancement of timed ambulation in ap - treated patients compared to those who received placebo . A significant enhancement of leg strength was also observed in approximately 11% of ap - treated participants compared with a 4% worsening of placebo - treated participants, p = 0.01 . The reported side effects consisted of dizziness, insomnia, paresthesia, nausea, headache, tremor, pain and anxiety . On daily doses of 60 and 70 mg a phase 2, multi - center, randomized, double - blind, placebo - controlled, parallel - group, dose - comparison (10, 15, or 20 mg twice daily orally) clinical trial of sustained - release fampridine in ms patients demonstrated significantly more consistent responders as determined by improvement in ambulation as compared to the placebo - group: 36.7% versus 8.5%.25 fampridine was generally well tolerated and serious and severe adverse effects were more common in those patients who received the highest dose . A multi - center randomized, double - blinded, placebo - controlled phase 2 dose - raning clinical trial was conducted by goodman et al which assessed the concept that whether the dose of fampridine - sr could be safely elevated to 80 mg / daily in patients with ms.26 a cohort of 36 ms patients were randomized in a 2 to 1 ratio to be treated with an increasing dose of fampridine - sr for a period of 8 weeks . The dosage of fampridine - sr was escalated from 10 to 40 mg twice daily by increments of 5 mg twice daily on a weekly schedule . A group of evaluations were done once a week which included ms functional composite (msfc), fatigue questionnaires, and lower extremity manual muscle testing . The objectives of this clinical trial included assessment of the safety of higher doses of fampridine - sr as well as the efficacy and dose - response to this drug by ms patients . Five study participants were terminated from fampridine - sr due to adverse events at doses larger than 25 mg and these events consisted of seizures in two individuals at doses of 30 and 35 mg twice daily . No subjects in the placebo arm of the study dropped out due to the adverse events . In the group which was treated with fampridine - sr compared to the group which was treated with placebo showed improvement in the lower extremity muscle strength (prospective analysis) and walking speed (post - hoc analysis). The investigators concluded that future clinical trials should restrict the dose of fampridine - sr to 20 mg twice daily and walking speed and lower extremity muscle strength tests are potential efficacy measurements . Another phase 3 multicenter clinical trial of dafampridine which was controlled and double - blind included 301 ms patients of different types (27% with relapsing - remitting ms and 73% with progressive ms). During the 14 weeks duration of this trial, participants were assigned in a ratio of 3 to 1 to be treated with dalfampridine 10 mg (n = 229) or placebo (n = 72) orally twice daily for a period of 14 weeks.27 the primary aim of this study included steady progress on timed 25-foot walk to define responses with proportion of timed walk responders in each arm . The investigators reported a higher proportion of timed walk responders in the fampridine - treated arm (78/224 or 35%) compared to those who were treated with placebo (6/72 or 8%). Based on the results of this study, treatment of ms patients with fampridine was associated with gait improvement with a decrease in patient s gait impairment . A more recent executed phase 3 multi - center double - blind clinical trial of extended - release dalfampridine in patients with definite ms of any clinical course assessed the efficacy, safety, and pharmacodynamics of extended release oral dalfampridine in these patients.28 the length of this study was 9 weeks and the participants were randomized to be treated with dalfampridine (10 mg twice daily; n = 120) or placebo (n = 119) for a period of 9 weeks . The primary aim of this study consisted of constant progress on the timed 25-foot walk, with percentage of time walk responders (twrs) in each treatment arm . The last on - treatment assessment collected information from 8 to 12 hours postdose, to assess and determine the persistence of the impact of the medication . The results of this clinical trial indicated that the quantity of twrs was higher in the dalfampridine - treated group (51/119 or 42.9%) compared to the placebo - treated group (11/118 or 9.3% . The average enhancement of walking speed among the dalfampridine - treated twrs during the 8-week efficacy assessment period was 24.7% compared to baseline; the mean enhancement during the finalon - treatment visit was 25.7%, reflecting continuation of the impact of medication over the interdosing phase . The authors concluded that this clinical trial generated class 1 evidence that treatment of patients with definite ms with extended - release dalfampridinetablets caused meaningful enhancement of walking ability in and the effect of the medication continued persisted between doses . Based on the animal studies, dalfampridine demonstrates properties of a powerful convulsant stemming from its broad - spectrum suppressing activity on the potassium channels in the peripheral and central nervous systems.11 based on a number of clinical trials of 4-ap side effects consist of dizziness, insomnia, paresthesia, nausea of mild to moderate intensity, tremor, pain, anxiety and seizures.2934 dalfampridine is neither a substrate for nor an inhibitor of the p - glycoprotein transporter, therefore its pharmacokinetics is not affected by the medications which suppress this transporter . It does not interact with the medications which are substrates for the p - glycoprotein transporter . Dalfampridine is contraindicated in patients with history of epilepsy or history of severe renal insufficiency.35 the reason that dalfampridine use is not recommended in epileptic patients is that epilepsy is characterized by abnormal neuronal depolarization with excessive inhibition on potassium channels, hence a further reduction of potassium channel activity by dalfampridine is not advised . Pathogenesis of ms involves loss of oligodendrocyte / myelin complex which turn results in re - organization of axolemmal ion channels which leads to conduction abnormalities including conduction delay or block . Pharmacological agents which block potassium channels have been investigated in the context of clinical trials with positive impact on impulse conduction in experimentally - induced demyelination as well as in patients with ms . Dalfampridine (which is marketed as ampyra), is an extended release form of fampridine (4-aminopyridine), which has been recently approved by the us fda for symptomatic treatment of ms patients . While this new oral blocker of voltage - gated potassium channels does not have any impact on the underlying pathology of ms, it has been demonstrated to improve fatigue and walking ability in these patients.
Several imaging modalities including pa, occlusal and panoramic radiography, cone beam computed tomography (cbct) and ct are used before and after implant treatment as well as in the maintenance sessions . After successful implant placement, periapical radiography is usually a suitable imaging modality for long - term follow - ups . Radiographically, a thin radiolucent margin around the implant indicates implant mobility and is an important sign of failed osseointegration [13]. The popularity of digital radiography in dentistry is mainly due to the adjustability of digital images by using image inversion, embossed tools, brightness, contrast and magnification enhancement filters [49]. By applying image inversion to digital images, inverted digital images are used for various purposes in dentistry such as the measurement of bone loss due to periodontal disease and localization of the mandibular canal and mental foramen [10,1117]. Several previous studies have compared the diagnostic accuracy of unprocessed digital images (psp and charge - coupled device or ccd), film - based radiographs and inverted images for detection of peri - implant bone defects and simulated periodontal lesions using image tool and adobe photoshop software programs [1214]. This study aimed to compare the diagnostic value of unprocessed (psp) and inverted digital images for detection of peri - implant defects using scanora software . Soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . In between imaging stages, the rib segments were stored at 1c temperature to minimize moisture loss . Osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another . A total of 30 implant placement sites including 10 control sites, 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created . The sicace screw implants (sicace, basel switzerland) (3.4mm/14.5 mm; cylindrical parallel walled) were placed in the marked areas . Osteotomy sites were prepared in two steps . In step 1, holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length (sic extension drill 3.10 mm, short). In step 2 of osteotomy, the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill (sic extension drill, basel, switzerland)(4.25 mm, short) to create 0.425 mm peri - implant space . The coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill (astra tech drill, stockholm, sweden) (4.85, 819 mm) to create 0.725 mm peri - implant space . To stabilize fixtures in bone, drilling was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap . All fixtures measuring 3.414.5 mm (the sicace screw implants 3.4mm/14.5 mm incl .) Were placed into the osteotomy sites at the level of the superior border of the rib (bone level) and cover screws were inserted (figure 1). To simulate soft tissue, an acrylic block (polymethyl methacrylate) with 1 cm thickness periapical radiographs were obtained from the bone segments at the same day the fixtures were placed . All radiographs were obtained using digora optime imaging system (soredex corporation, helsinki, finland), psp size 2 sensor (40.0mm30.0 mm) and minray dental x ray system (soredex, tuusula, finland) with exposure settings of 60 kv and 7ma for 0.16s . Digital intraoral sensor holder (kerr dental europe, bioggio, switzerland) the x ray tube (2) the simulated soft tissue (3) bone segment with implant fixtures placed . After exposure of sensors, images were stored in dicom files on a computer using a scanora lite software (palodex, tuusula, finland) with standard resolution . By applying image inversion, 60 digital images including 30 unprocessed and 30 inverted images were obtained . Four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale: definite defectprobable defectnot sureprobably no defectdefinitely no defect examiners viewed the images on an 18.5 samsung monitor (samsung syncmaster e1945nx, 1360 768) under adequate lighting . The examiners were only allowed to adjust the brightness and contrast of digital images (figure 3). Study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers . The sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared . Independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities . Soft tissue residues were removed and the rib was trimmed to prepare bone segments suitable for the placement of 30 implants . In between imaging stages, the rib segments were stored at 1c temperature to minimize moisture loss . Osteotomy sites were marked on the superior border of the rib with 15 mm distance from one another . A total of 30 implant placement sites including 10 control sites, 10 sites with 0.425 mm peri - implant space and 10 sites with 0.725 mm peri - implant space were created . The sicace screw implants (sicace, basel switzerland) (3.4mm/14.5 mm; cylindrical parallel walled) were placed in the marked areas . Osteotomy sites were prepared in two steps . In step 1, holes were drilled using sic drill system with 3.1 mm diameter and 14.5 mm length (sic extension drill 3.10 mm, short). In step 2 of osteotomy, the coronal 8 mm of 10 osteotomy sites was enlarged using a 4.25 mm drill (sic extension drill, basel, switzerland)(4.25 mm, short) to create 0.425 mm peri - implant space . The coronal 8 mm of another 10 sites was enlarged using a 4.85 mm diameter drill (astra tech drill, stockholm, sweden) (4.85, 819 mm) to create 0.725 mm peri - implant space . To stabilize fixtures in bone, drilling was performed at a diameter of 3.1 mm and height of 6 mm for the apical part of fixtures and the remaining coronal 8 mm was prepared with larger drills to form a gap . All fixtures measuring 3.414.5 mm (the sicace screw implants 3.4mm/14.5 mm incl .) Were placed into the osteotomy sites at the level of the superior border of the rib (bone level) and cover screws were inserted (figure 1). To simulate soft tissue, an acrylic block (polymethyl methacrylate) with 1 cm thickness periapical radiographs were obtained from the bone segments at the same day the fixtures were placed . All radiographs were obtained using digora optime imaging system (soredex corporation, helsinki, finland), psp size 2 sensor (40.0mm30.0 mm) and minray dental x ray system (soredex, tuusula, finland) with exposure settings of 60 kv and 7ma for 0.16s . Digital intraoral sensor holder (kerr dental europe, bioggio, switzerland) the x ray tube (2) the simulated soft tissue (3) bone segment with implant fixtures placed . After exposure of sensors, images were stored in dicom files on a computer using a scanora lite software (palodex, tuusula, finland) with standard resolution . By applying image inversion, 60 digital images including 30 unprocessed and 30 inverted images were obtained . Four oral and maxillofacial radiologists with at least two years of work experience evaluated all 60 images for presence or absence of peri - implant defects using a efive - point scale: definite defectprobable defectnot sureprobably no defectdefinitely no defect examiners viewed the images on an 18.5 samsung monitor (samsung syncmaster e1945nx, 1360 768) under adequate lighting . The examiners were only allowed to adjust the brightness and contrast of digital images (figure 3). In this study the basis of calculating the absolute sensitivity / specificity was the number of the definitely correct diagnoses and the complete sensitivity / specificity was determined based on the total number of definite and probable correct answers . The sensitivity and specificity for detection of small and large peri - implant defects were calculated and compared . Independent t - test was used to compare the diagnostic value of unprocessed and inverted digital imaging modalities . A total of 60 digital pa radiographs including 30 unprocessed and 30 inverted radiographs were obtained of 30 implants with the same diameter and length: 10 implants with no defects, 10 with small (0.425 mm) diameter peri - implant defects in the coronal 8 mm and 10 with large (0.725 mm) diameter peri - implant defects in the coronal 8 mm . Based on the results, unprocessed images had a higher diagnostic value than inverted images . According to the t - test, in the equality of the means, no significant difference was detected in the sensitivity of unprocessed and inverted images for detection of peri - implant defects (absolute positive predictive value) and for detection of a probable lesion (complete positive predictive value)(p>0.05). Considering the equality of variances, the t - test showed that unprocessed images had higher absolute negative predictive value (p=0.049) and complete negative predictive value (p=0.017) than inverted images (table 1). The mean standard deviation of absolute and complete negative and positive predictive values of unprocessed and inverted images the mean standard deviation of sensitivity and specificity of unprocessed and inverted images for detection of small and large defects significantly different from zero (p<0.05) considering the equality of variances, the t - test showed that unprocessed images had a higher absolute sensitivity for detection of small defects than inverted images (p=0.48). However, the mean complete sensitivity of the two modalities for detection of small defects despite the non - equality of variances (p=0.001) and equality of the means, was not significantly different (p=0.81) (table 2). Considering the equality of variances (p=0.055), the t - test failed to find a significant difference in the absolute sensitivity for detection of large defects between the unprocessed and inverted images (p=1.0). However, despite the equality of variances (p=0.003), the t - test found a statistically significant difference in complete sensitivity for detection of large defects and unprocessed images had a higher mean value in this respect (p=0.019) (table 2). In the non - equality of the means, the mean absolute specificity of unprocessed and inverted images was significantly different based on the t - test (p<0.05) and unprocessed images had a higher diagnostic value . However, in the equality of the means, the complete specificity of unprocessed and inverted images was not significantly different (p>0.05) (table 2). Periapical radiography is conventionally used as the standard follow up radiography after implant placement to assess the peri - implant tissue status . The digital imaging techniques and different enhancement filters have enabled more accurate diagnoses in dentistry . This study showed that unprocessed images had a higher diagnostic value than inverted images for detection of small and large peri - implant defects . This finding may be due to the unfamiliarity of the inverted images to the eyes of the observers . Our obtained results were in accord with those of kavadella et al, de molon et al, and jorgenson et al, despite the differences in the type of receptors (film and digital sensor), type of defects and method of creating the defects . However, our results were in contrast to those of scaf et al, in 2007 . Compared unprocessed and inverted digitized images for detection of bone loss due to periodontal disease and found no significant difference in the diagnostic value of unprocessed and inverted images . Scaf et al, in their study used pa radiographs available in the records of patients with periodontal disease . The difference between our results and those of scaf et al . May be attributed to the different methodology and the software programs used . Kavadella et al, in their in - vitro study compared film - based conventional radiography with unprocessed and inverted digital radiography with ccd sensors for detection of peri - implant lesions . Our results had some differences with those of kavadella et al . In our study, the sensitivity and specificity of unprocessed images were higher than those of inverted digital images . The complete sensitivity for detection of small defects was 0.90 for unprocessed and 0.62 for inverted images . The complete sensitivity for large defects was 1 for unprocessed and 0.85 for inverted images . . However, kavadella et al . Showed high specificity and low sensitivity values . Such differences may be due to the methodology of studies i.e. Method of defect formation, using fresh bone in our study and use of magnification enhancement (x2) by the observers in the study by kavadella et al . However, our results regarding the lower accuracy of inverted digital images compared to unprocessed images were similar to those of kavadella et al . Molon et al, in their study compared the diagnostic accuracy of unprocessed and converted (cmos sensor) images with film - based conventional radiographs for detection of bone loss due to simulated periodontal disease . The results showed that inverted digital images had lower accuracy than film - based radiographs . Despite the difference between the type of sensors used in our study and the study by molon et al, similar results were obtained indicating the higher diagnostic accuracy of unprocessed digital images and film - based radiographs than inverted images . Jorgenson et al, in their comparative study evaluated the diagnostic accuracy of conventional f - speed film radiographs, unprocessed digital radiographs and inverted digital images (psp, digora) in patients with vertical bone defects . The digital sensor used in our study was similar to that used by jorgenson et al, and our results were in accord with their findings . Based on the results, we found that unprocessed digital images had higher diagnostic accuracy than inverted images for detection of small and large peri - implant defects.
Upper gastrointestinal bleeding (ugib) is a major cause of mortality and morbidity accounting for about 56.5/100,000 hospitalization with a mortality rate of 8.2% . Aorto - esophageal fistula (aef), an abnormal anatomical communication between the aorta and esophagus is a rare cause of ugib . As the thoracic aneurysm grows in size, the increasing tension on its wall will erode the aorta and rupture into the surrounding esophagus leading to uncontrolled bleeding . As ulcer related or variceal bleeding constitutes for most of the ugib bleeding, even in elderly who are at risk of aef and the inability of the esophagogastroduodenoscopy (egd) to detect most cases, aef is often not diagnosed timely . All these factors make the timely diagnosis of aef challenging, and at times missed altogether leading to catastrophic consequences . We present a case of aef which was initially treated as variceal bleeding and later promptly diagnosed with computed tomography (ct). A 70-year - old male patient with hypertension with regular ethanol use, presented to a local hospital with complaints of hematemesis and melena from the past 1-day . He was hemodynamically stable on presentation with initial hemoglobin level of 7.2 g / dl . He underwent timely egd, which showed grade 1 mid and distal esophageal varices without any active bleeding . Subsequently, his condition deteriorated with the development of hypotension and transferred to our medical center for further management . Weiss tear without active bleeding along with the presence of large blood clots in the gastric fundus . As the source of bleeding was not clear despite two egd and clinical evidence of gi bleeding, he underwent emergent colonoscopy which once again failed to detect any source lower gi bleeding . However, later in the day, he developed massive hematemesis and hematochezia leading to persistent hemorrhagic shock . Emergent intubation was done for airway protection, massive transfusion protocol was initiated, and a ct angiogram was performed . It revealed type b aortic dissection with rupture and the active extravasation of blood into the esophagus, confirming the diagnosis of aef [figure 1]. He underwent emergency percutaneous thoracic endovascular aortic repair with aortic stent placement [figure 2]. His shock resolved hemoglobin stabilized and did not have any more episodes of bleeding thereafter . Repeat egd showed a 3-cm opening in the midesophagus with ragged and irregular borders, not seen earlier egds . His hospital course was complicated by the infection of the endograft which was replaced and fortified with an omental flap and being is presently being, followed by for reconstruction surgeries . (a) communication and extravasation between aortia and esophagus (blue arrow head) in the arterial phase . (b) accumulation of blood in lower esophagus in venous phase (yellow arrow head). (c) significant accumulation of blood in the stomach in the kidney delay images (red arrow head). (d) sagittal reconstruction images shows commination (blue arrow) and extravasation (yellow arrow) of blood between aorta and esophagus endovascular graft in the thoracic aorta aorto - esophageal fistula is an abnormal anatomical communication between the aorta and esophagus . In 1818, dubrueil described this condition for the 1 time in a french soldier who ingested a beef rib, leading to massive hematemesis . Almost a century later, chiari described the triad of midthoracic pain, herald hemorrhage, and fatal hematemesis seen in these patients . The exact incidence of this condition is not known, as the vast majority of these patients die before a definitive diagnosis can be made . In the past, infections such as tuberculosis and syphilis, leading to aortitis were the common cause of this condition . In this antibiotics era, thoracic aortic aneurysm constitutes for more than half of these cases . Other causes described in the literature are cancer, acid reflux disease, trauma, caustic ingestion, and esophageal biopsies . Aefs are classified as primary if they are caused by the spontaneous erosion of the aortic wall into the esophagus and as secondary if they occur as a complication following aortic or esophageal surgeries . Owing to the increasing number of interventions of the aorta, secondary aef is 10 times more common than primary . The herald bleeding described by chiari is only seen in half of these patients . Once the bleeding starts, rapid extravasation from aorta will cause a sudden drop in blood pressure ., the blood pressure will raise again dislodging the fresh and loose clot around the aef leading to terminal bleeding . Commonly used resuscitation fluids including pseudo random binary sequence lack adequate clotting factors add to the compromise of clot integrity . Unlike in other structures, the lax mediastinum around the aorta and esophagus prevent autotamponade, to prevent persistent extravasation of blood . These factors can contribute to torrential bleeding, the final hematemesis as described by chiari . The risk of aortic aneurysm increases with age, with an incidence of 12.5% in elderly americans . The sensitivity of egd to identify the source of bleeding in other causes of ugib is 90%, but for aef it is <10% . It may show pulsatile mass, bluish discoloration, or simply blood clotting in the esophagus in one - third of cases . A ct arteriogram is diagnostic in most cases, which show active extravasation of blood from the aorta to esophagus . Once diagnosed, aef should be treated with emergent aortic stenting to control the bleeding . Owing to contamination from the gi tract, one - quarter of stents become infected . Apart from broad - spectrum antibiotics and antifungals, once the infection is controlled, the complex aortic and esophageal reconstruction is done in multiples stages . Aef, once considered to be uniformly fatal, is now a treatable condition with timely intervention . Aorto - esophageal fistula is a rare, but life - threatening condition presenting as ugib . A high level of suspicion is needed, as routine tests performed for ugib typically only detect one - third of cases . Avoiding over - resuscitation can be helpful in preventing subsequent hemorrhages.
The discovery of the silicon pn junction is a landmark in the development of modern microelectronics . Silicon and other inorganic semiconductors are the predominant materials in electronic manufacturing today; however, the development of organic - based electronic components has been a focus of intensive research and development . Organic electronics have become an active research area in recent years because they are an alternative to the traditional semiconductor technology and challenges in design on a smaller scale from microscale to nanoscale . Moreover, organic materials have vast potential for integration in low - cost microelectronic devices . Organic - based semiconductors, for example polymeric gels, may have superior properties due to their flexibility against to regulate some physical properties of the materials even after the production, e.g., by changing the volume of the gel . Circuits and displays based on organic electronics may also be flexible, low - weight, and environment friendly . Due to great diversity and functionality of the polymers, they may also be produced for specific requirements, like biocompatible semiconductors . Last, some water - tolerant organic devices may be biocompatible, that is, they may be used in circuits that directly reside on or are implanted inside animal and human tissue and could perform various sensing, interfacing, and controlling functions for drug delivery, prosthetics, and neural - electronic integration . Insulating polymers have been made conductive so far by using various methods such as ion implantation [1 - 4], press contacting, and photochemical doping . The impressive properties of these materials that have been taken advantage are the ability to manipulate electronic properties by changing their molecular structure . Differently current rectifying in semiconductor diodes via diffusion of ionic charges across a pn junction sets up a built in potential; there is unidirectional current is observed across a membrane where a built in potential arises from diffusion of anions and cations in organic junctions . We considered that the principle of the electrical conductivity in polymeric gels can be changed by doping static (inactive) ions having counter ions as charge carriers and these gels can be used to form pn junctions that work similar to inorganic semiconductors . These semiconductor polymeric gels can be doped with both negative (n - type) and positive (p - type) counter ions as charge carriers . We have shown that the pn junction formed by combining p - type and n - type gels together in much closer contact rectifies the current . P - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide (aam) and n, n-methylene bisacrylamide (bis) as crosslink agent in the presence of pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt) in varying amount, 10, 10, 10 mol / l [10 - 12]. The ratios of aam to bis and aam to aps were kept fixed for all samples; aam / bis = 31 and aam / aps = 43 in bidistilled water . The samples were deoxygenated by bubbling nitrogen during 10 min, and the gelation was performed in the 1 1 cm square - shaped holders (4 cm of volume) at 60c in a heat bath . After gelation processes were completed, gels were washed with pure water during 15 days with changing their water every day . Thus, pollutions and unreacted chemicals washed out from the gel, and only pyranines, which were bound to the polymer strands chemically [10 - 12], were stayed in the gel . After the gels dried at 40c furnace, they were cut into thin slices with ~1 mm thickness for electrical measurements . N - type gels were synthesized, similar to p - type, as free radical cross - linking co - polymerization of n - isopropylacrylamide (nipa) and n, n-methylene bisacrylamide (bis) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride (maptac) in varying amount, 3 10, 3 10, 3 10 mol / l [13 - 15]. The ratios of nipa to bis and nipa to aibn were kept fixed for all samples; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide (dmso). These gels were prepared for electrical measurements in similar way with p - type gels . Aam, nipa, and doping concentrations of the samples were summarized in table 1 . Properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties, samples p1, p5, n1, and n5 were synthesized without doping agents for comparison with the other samples . The gels, prepared in the slice shapes of area a and thickness d, were swollen to a certain swelling ratio, and then placed between platinum electrodes, and sealed from the air against drying . The current densities per unit mass, j / m, of the gel were measured against the time, the applied voltage and the swelling ratio of the gels . P - type hydrogels were synthesized via free radical cross - linking co - polymerization of acrylamide (aam) and n, n-methylene bisacrylamide (bis) as crosslink agent in the presence of pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, trisodium salt) in varying amount, 10, 10, 10 mol / l [10 - 12]. The ratios of aam to bis and aam to aps were kept fixed for all samples; aam / bis = 31 and aam / aps = 43 in bidistilled water . The samples were deoxygenated by bubbling nitrogen during 10 min, and the gelation was performed in the 1 1 cm square - shaped holders (4 cm of volume) at 60c in a heat bath . After gelation processes were completed, gels were washed with pure water during 15 days with changing their water every day . Thus, pollutions and unreacted chemicals washed out from the gel, and only pyranines, which were bound to the polymer strands chemically [10 - 12], were stayed in the gel . After the gels dried at 40c furnace, they were cut into thin slices with ~1 mm thickness for electrical measurements . N - type gels were synthesized, similar to p - type, as free radical cross - linking co - polymerization of n - isopropylacrylamide (nipa) and n, n-methylene bisacrylamide (bis) as crosslink agent in the presence of methacrylamidopropyltrimethylammonium chloride (maptac) in varying amount, 3 10, 3 10, 3 10 mol / l [13 - 15]. The ratios of nipa to bis and nipa to aibn were kept fixed for all samples; nipa / bis = 50 and nipa / aibn = 71 in dimethylsulphoxide (dmso). These gels were prepared for electrical measurements in similar way with p - type gels . Aam, nipa, and doping concentrations of the samples were summarized in table 1 . Properties and codes of synthesizing semiconducting gels to be able to evaluate the net effect of the doping on the electrical properties, samples p1, p5, n1, and n5 were synthesized without doping agents for comparison with the other samples . The gels, prepared in the slice shapes of area a and thickness d, were swollen to a certain swelling ratio, and then placed between platinum electrodes, and sealed from the air against drying . The current densities per unit mass, j / m, of the gel were measured against the time, the applied voltage and the swelling ratio of the gels . The pyranine binds to the polymer chains, over its oh group, chemically during the polymerization as it is discussed in detail in references [10 - 12], and they form stable charged sites doped with positive counter ions . Thus, the polyacrylamide gel is doped with pyranine having ions as side groups and na as counter ions, so - called p - type semiconducting gel . Similarly, nipa gel was doped with maptac, having cl as counter ions, so - called n - type semiconducting gel . The maptac binds to the polymer chains chemically during the polymerization, thus they form stable charged sites doped with negative counter ions as represented in scheme 1together with the p - type gel . This doping process was also discussed in detail in references, . When these gels are in the collapsed state, no current is observed . When the gels start to swell, the current starts to flow through the gels . Schematic representation of the p - type (a) and n - type (b) gels doped with pyranine and maptac the currents decrease with time for a constant applied voltage as shown in fig . As the charge carriers (the ions in water phase of the gel) were gathered on the electrodes, the density of the charge carriers decrease in time, and thus the current . As seen, the initial values of the currents measured for the doped gels are considerably bigger than that of the neat gel (the gel that was not doped with ions) at a certain swelling ratio, v / v0 = 1.28 and under constant applied voltage, u = 5 v. furthermore, the initial amplitude of the currents increases with increasing doping concentration . This clearly shows that the net charge in the gel increases with increasing doping concentration . Aj / mgel vs time plots for the doped (p2, p3, p4) and neat (p1) gels under constant applied voltage, 5 v, and swelling ratio, v / v0 ~ 1,28 . Bj / mgel versus time plots upon short - cut of the circuit for samples p4, p3, p2, p1 . The vertical bar represents the standard error for each plot examples for the short - cut experiments -the source of the voltage was removed and the current was measured by means of an ammeter without changing its polarity- were also indicated in (fig . 1b) where all the current takes first a negative value, as it is expected, and then goes to zero with time . Figure 1 clearly indicates that the free charges leading the current are accumulated on the surfaces of the gel on which the voltage is applied (similar to charging a parallel plate capacitor) and the gels have ionic charge carriers . Figure 2a shows typical current - time plots of the slab gels for increasing pyranine content . The gels were swollen in pure water up to certain swelling degree (v / v0 = 1,3), under a fixed applied voltage, 1 v. as seen, the currents for all samples decrease with time; however, the initial values of them increase with increasing doping agent (pyranine content). Aj / mgel versus time plots of the samples p6, p7, and p8, b the ratios of j / mgel for the doped and neat gels (p8/p5) as function of the applied voltage for varying swelling ratio, v / v0 figure 2b shows the ratio of the normalized current densities (j / mgel) for doped (p8) to neat (p5) gels as function of the applied voltage for varying swelling ratio . The currents were measured 5 s later just after applying voltage . As seen, the doping effect on the current decreases when the swelling ratio is increased . This may be due to the vast increase in the free ions coming from the water which causes screening . This picture may indicate that doping effect (p - type behavior) can be fulfilled up to a certain swelling degree of the above - mentioned gel which semiconducting behavior will be lost . The instantaneous currents the currents that were measured 5 s later just after changing the voltage- as function of the volume of the gels for varying voltages were also measured, and the results were indicated in fig . As seen from this figure, distinct peaks appear as the solvent uptake, v / v0, increases . The peaks almost disappear for the gels doped with pyranine due to increasing homogeneity of the gels in the presence of the ions . The results coincide with the recent literature where three dominant peaks, each corresponding to a different swelling stage in the conductivity, were observed . Figure 3 indicates that the volume of the gel is one of the main parameters determining the current . Variation in j / mgel against v / v0 for (a) the neat gel, p1, and for (b) the doped gel, p2 these peaks were related to the distribution of dense polymer regions; they are defined as the blobs appearing in a microstructure of the given paam gel having at least some average sizes . This special behavior in these gels may result specific features related to the electrical features . The heterogeneity of the gel may decrease essentially in case when it is prepared with the charged groups . The internal electric field due to the charged sites could disturb the tendency between the polymer units (affinity of the aam momoners to the cross - linker molecules) because the charge excess has a tendency to form denser polymer regions especially in mesoscale regions . The formation of the rigid network in the charged gel makes it more homogenous in comparison with the neutral gel . This could cause the decreasing amplitude of the peaks, and overall decrease in the conductivity as it is confirmed by fig . 3 (see also fig . 2 of reference). Therefore, in the charged gel, the penetration of neutral water molecules through the system of large channels located in the vicinity of small blobs is facilitated . Figure 4 represents the normalized current density per unit mass as function of the voltage for the gels (sample p3) swollen with water (fig . Dmso is not a polar molecule; therefore, the ions like h or oh are absent in it . As seen from this figure, the current density decreases fairly for the gels swollen with dmso since no contribution to the current occurs via h and oh ions . The current occurs mainly due to doped ions (the counter ions of the doping agent). This observation clearly indicates that the possible negative effects of the water upon the pnjunctions that may be formed by means of our gels can be eliminated . J / mgel versus applied voltage, u, for the doped gel (p3) swollen in (a) pure water, b and in dmso all the above - mentioned measurements were repeated for n - type gels, and the similar results were reproduced . Here the results are not given to avoid the repetition . In fig . 5, representative examples of i - u(current voltage) character of polymeric diode are given for the neat and the doped gels . Figure 5a shows the current voltage character that was made by means of close connection of p-(sample p4) and n - type (sample n4) slab gels- putting one of the gel on the top of the other for each gel . The swelling ratio of each gel was kept the same, v / v0 = 1,2 . When the neat gels are used for both the p - and n - type sides, figure 5a clearly shows that the current of the interface between the cationic and anionic gels, which is passing through only one direction, originates directly from the doping effect of the gels . A i versus u plot for pn junction formed with p - and n - type gels (p4-n4), b i versus u plot for the junction formed with neat gels (p1-n1) in this study, we have shown that p- and n - type doped hydrogels can be synthesized and be used to built pn junction . The synthesis of these gels and making junctions are more simple, inexpensive, and scalable than traditional pn junctions . The current voltage characteristic of the polymeric pn junction can be changed by changing doping concentration, swelling ratio etc that will be discussed in our feature works . The percentage of the standard deviations changes around 20% as can be seen from fig . The reasons for this high standard deviations can be explained by the following arguments: (1) the electrical futures of these gels depend strictly on the swelling ratio of the gels as can be seen from fig . 3 and from the detailed discussion given in a recent work and (2) on the internal morphology (nonergodicity or random distribution of the heterogeneous regions in the gels). This strict volume dependence and the randomness of the internal distributions of the heterogeneous regions set a barrier for comparison of the samples from the point of the current amplitudes due to the difficulties to bring the gels to exactly the same swelling ratios and to repeat the synthesis with exactly the same internal morphology . Therefore, the stability and reversibility problems need detailed experiments and probably more controllable polymer synthesis . This article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
In june 2014, a 61-year - old female was admitted to keimyung university dongsan medical center because of a severe dry cough that had lasted for over 20 days . According to her medical history, she had undergone a hysterectomy in 2005, and the pathologic diagnosis was leiomyoma of the uterus . In 2006, chest computed tomography (ct) findings demonstrated multiple nodules in both lungs (fig . Microscopic examination revealed a proliferating spindle cell tumor showing relatively bland nuclei, no pleomorphism, and no mitoses . In addition, immunohistochemical staining analysis was positive for smooth muscle actin, desmin, vimentin, estrogen receptor (er), and progesterone receptor (pr), and negative for cd34, pan - ck, and s100 proteins . The result of the pathologic study was benign leiomyoma, and the nodules were diagnosed as pulmonary benign metastasizing leiomyoma (pbml)., the patient did not return to our outpatient clinic for follow - up . Upon the patient s return, a posteroanterior chest x - ray showed a very large mass on the right cardiac border (fig . Chest ct demonstrated a 101-mm mass on the right lower lobe that compressed the posterobasal segmental bronchus (fig . A ct - guided needle biopsy of the huge mass revealed it to be leiomyoma . After right thoracotomy, a very large mass, 100 mm in size, originating from the visceral pleura of the right lower lobe was observed (fig . The mass was in contact with the parietal and diaphragmatic pleura, but no invasion was noted . Near the mass, multiple fine, scattered, whitish nodules measuring less than 1 cm were found on the diaphragmatic, parietal, and visceral pleura . We performed an excision of the mass in the right lower lobe and excised fine nodules from the parietal and diaphragmatic pleura for biopsy . Intrapulmonary nodules found in the middle lobe and upper lobe were not excised, because they had not increased in size . The necrotic lesion showed severe cellular atypia, frequent mitoses (more than 15 per 10 high power fields [hpfs]), and continuity of the leiomyoma and leiomyosarcoma (fig . 2c). Immunohistochemical staining revealed that the tumor was positive for smooth muscle actin (sma), er, and pr, but negative for cd34, pan - ck, s100, and hmb45 (fig . The nodular lesions taken from the parietal and diaphragmatic pleura showed no malignant cells . The patient was discharged 8 days after the operation without complications . Large heterogeneous hypermetabolic masses were found from the left para - aortic area to the left pelvic cavity (fig . 3), and we diagnosed them as metastatic leiomyosarcoma . Additionally, increased fluorodeoxyglucose (fdg) uptake was observed in the peribronchial lymph nodes, the mediastinum, and the supraclavicular lymph nodes . Two months later, en - bloc resection of the retroperitoneal masses with left nephrectomy was performed, and the pathologic diagnosis was metastatic leiomyosarcoma . We decided to continue observing fdg uptake in the mediastinal and peribronchial lymph nodes rather than prescribing chemotherapy, because metastasis of leiomyosarcoma to the lymph nodes is rare . A chest ct scan done 1 year after surgery demonstrated no changes in the pulmonary nodules and no recurrence of the mass . Benign metastasizing leiomyoma (bml) results from metastasis of a uterine leiomyoma; thus, bml patients have a history of uterine leiomyoma . The condition may occur at any age, but occurs most often in women from 30 to 74 years old . Bml can occur in a number of different areas of the body, including but not limited to the lungs, para - aortic lymph nodes, abdominal lymph nodes, heart, breast, liver, and esophagus . The most common site of occurrence is in the lungs, in which case it is known as pbml . The pathogenesis of pbml is unknown, but the most common theory is hematogenous spreading of leiomyoma during uterine surgery . Immunohistochemical staining of affected tissue is positive for desmin, sma, pr, and er . Most cases of pbml do not require treatment, but pbml that metastasizes or increases in size does need to be treated, as it can cause respiratory failure, chest pain, or hemoptysis . Surgical excision is the treatment of choice, but this is only possible in cases of resectable pbml . In some cases of pbml that a repositive for er or pr, reducing hormone release via hysterectomy, bilateral adnexectomy, and/or hormone therapy may decrease the tumor s size . The incidence of the sarcomatous transformation of benign uterine leiomyomas is only 0.1%0.8%, and is most often seen in women in their 50s . Pbml can also undergo malignant transformation; excluding our case, just 1 case of the malignant transformation of pbml has been reported . Radiologically, it is difficult to differentiate between normal pbml and pbml that has become malignant . On fdg pet - ct, heterogeneous fdg uptake of the tumor may reflect necrosis within the leiomyosarcoma . In our case, pathologically, the current criteria used to differentiate a malignant smooth muscle tumor from a benign mass are the presence of necrosis, a high mitotic index (> 5 mitoses seen in 50 hpfs), nuclear atypia, cellularity, and an ill - defined tumor border . However, in primary pulmonary leiomyosarcoma (ppl), progress is slow, and metastasis of ppl occurs late in the disease process . If the condition is diagnosed early, wide excision with negative margins (r0) is the standard treatment . The 5-year survival rate in ppl patients who undergo complete resection is over 50%, and recurrence is rare . Lymph node resections are generally not necessary because ppl rarely shows lymph node involvement . For advanced lesions, depending on the size and grade of the tumor, radiation therapy, chemotherapy, or both may be indicated as an adjuvant therapy . However, the median survival of patients with an unresectable sarcoma or metastatic disease is about 12 months . Overall, early diagnosis and complete mass resection are vital in cases of the malignant transformation of pbml . However, preventive resection or diagnostic excisional biopsy is not recommended for all pbml patients, since the occurrence of a malignant change is extremely rare if there is a very large, newly developed mass that is increasing in size rapidly, or if a malignant mass is found on pet - ct, diagnostic mass resection should be considered.
Copd is a common, usually progressive disease characterized by chronic inflammation of the airways and persistent airflow limitation . Its prevalence is increasing worldwide, and it is expected to be the third largest global cause of mortality by the year 2030.1 in japan, a large epidemiological study (the nippon copd epidemiological study) reported that the prevalence of airflow limitation was 10.6%, and at least 8.6% of subjects were sought to have copd.2 several studies have shown that the prevalence of potentially undiagnosed airflow limitation in both western35 and asian6 countries is ~ 3%15% . Many patients with copd continue to be underdiagnosed and untreated.2,7 since copd is a preventable and treatable disease, the importance of early detection has been emphasized.8 the use of simple copd screening questionnaires to detect persistent airflow limitation may help in the early diagnosis of copd . These tools reliably detect airflow limitation in the general population and may facilitate the early, accurate diagnosis of copd in general practice settings.9,10 some copd diagnostic questionnaires have already been reported.1114 the copd population screener (copd - ps), which was developed by a clinician working group in the united states, is a five - item, self - administered questionnaire that was validated for screening individuals in the general population who are at high risk of copd . It is composed of three copd - related items (breathlessness, productive cough, and activity limitation) and one question, each regarding smoking history and age.15 in a previous population - based study, we verified the validity of the japanese version of the copd - ps questionnaire for the identification of individuals at increased risk of airflow limitation.16 this instrument is easy to score and would be suitable for large - scale screening of possible airflow obstruction (ao). Another copd screening tool, the international primary care airway group (ipag) questionnaire,11 is in use worldwide . It consists of eight items and takes more time to administer than the copd - ps questionnaire . The purpose of this study was to compare these two questionnaires in the general japanese population, as no previous research has done so, as well as to assess the ability of both instruments to discriminate between subjects with and without persistent ao . This study was based on data from the hisayama study, which is an ongoing population - based epidemiologic study designed to investigate the morbidity, mortality, and risk factors of cardiovascular and smoking - related diseases in the town of hisayama, japan . The town is located in a suburban area adjacent to fukuoka city, a large urban center on kyushu island in the southern part of japan . The population of the town is ~8,000 and has been stable for over 50 years . According to national census data, the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17 this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . In 2012, registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry . Of the 2,643 subjects who were enrolled between june 2012 and october 2012, 307 were excluded for the following reasons: 105 had physician - diagnosed asthma, 22 had a previous lung resection, 159 had poor studied data, and 21 had records with missing data . The final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements . The subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires, and then, in addition to their usual clinical tests, underwent spirometry using a chestgraph hi-105 spirometer (chest mi, tokyo, japan). Each subject performed at least three forced vital capacity (fvc) maneuvers according to the recommended method . The results were assessed by two pulmonary physicians, who visually inspected the flow volume curve and excluded subjects without at least two satisfactory tests . The highest forced expiratory volume in 1 second (fev1) and fvc values were used for analysis . The subjects who had pre - bronchodilator (bd) fev1/fvc <0.70 were eligible for post - bd testing, in which spirometry was performed 15 minutes after inhalation of salbutamol (glaxosmithkline, tokyo, japan) via a metered - dose inhaler with a spacer, according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc <0.70 . The subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria (mild, fev1 80% predicted; moderate, 50% fev1 <80% predicted; severe, 30% fev1 <50% predicted; very severe, fev1 <30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university (numbers 2137, 2482, and 24123) and of kagoshima university (numbers 156 and 279), and all subjects provided their written informed consent prior to participation in the study . The copd - ps is a brief, reliable, self - scored questionnaire to identify individuals likely to have copd . It consists of five items, three assessing copd - related symptoms on a 5-point scale, one on cigarette use (3-point scale), and one on the subject s age (four categories). These five items are scored 0, 1, or 2 with a summed total score ranging from 0 to 10 . In the japanese version of the copd - ps questionnaire, a cutoff point of 4 has been found to be useful for copd screening.16 the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items: one on the subject s age (four categories), one on body mass index (3-point scale), one on cigarette use (4-point scale), and five on symptoms / history (2- or 3-point scales). Each question is scored individually, with a summed total score ranging from 0 to 38 . A suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan . However, there is no cutoff score for persistent ao in the general japanese population, including never - smokers, and we therefore investigated this issue in this study . Baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . The kruskal spearman correlations were used to examine the strength of associations between informant and performance measures . For the subjects who used a bd, another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity; larger numbers provide more convincing evidence of a disease, smaller numbers indicate that the disease is less likely, and ratios close to one lack diagnostic value . Likelihood ratios (positive and negative) were calculated for both the copd - ps and the ipag questionnaires . Receiver operating characteristic (roc) curves and areas under the roc curves (aucs) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose; this approach assesses the ability of a model to reclassify case and control subjects, respectively, on the basis of the individual - estimated probability of an event . The ability of the model to reclassify is summarized by the net reclassification improvement (nri) and integrated discrimination improvement (idi). We defined four strata dividing the risk of persistent ao into four quartiles, namely, q1, q2, q3, and q4 . The nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another . In contrast, the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable . We computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao . All statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 . This study was based on data from the hisayama study, which is an ongoing population - based epidemiologic study designed to investigate the morbidity, mortality, and risk factors of cardiovascular and smoking - related diseases in the town of hisayama, japan . The town is located in a suburban area adjacent to fukuoka city, a large urban center on kyushu island in the southern part of japan . The population of the town is ~8,000 and has been stable for over 50 years . According to national census data, the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17 this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . In 2012, registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry . Of the 2,643 subjects who were enrolled between june 2012 and october 2012, 307 were excluded for the following reasons: 105 had physician - diagnosed asthma, 22 had a previous lung resection, 159 had poor studied data, and 21 had records with missing data . The final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements . The subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires, and then, in addition to their usual clinical tests, underwent spirometry using a chestgraph hi-105 spirometer (chest mi, tokyo, japan). Each subject performed at least three forced vital capacity (fvc) maneuvers according to the recommended method . The results were assessed by two pulmonary physicians, who visually inspected the flow volume curve and excluded subjects without at least two satisfactory tests . The highest forced expiratory volume in 1 second (fev1) and fvc values were used for analysis . The subjects who had pre - bronchodilator (bd) fev1/fvc <0.70 were eligible for post - bd testing, in which spirometry was performed 15 minutes after inhalation of salbutamol (glaxosmithkline, tokyo, japan) via a metered - dose inhaler with a spacer, according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc <0.70 . The subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria (mild, fev1 80% predicted; moderate, 50% fev1 <80% predicted; severe, 30% fev1 <50% predicted; very severe, fev1 <30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university (numbers 2137, 2482, and 24123) and of kagoshima university (numbers 156 and 279), and all subjects provided their written informed consent prior to participation in the study . The copd - ps is a brief, reliable, self - scored questionnaire to identify individuals likely to have copd . It consists of five items, three assessing copd - related symptoms on a 5-point scale, one on cigarette use (3-point scale), and one on the subject s age (four categories). These five items are scored 0, 1, or 2 with a summed total score ranging from 0 to 10 . In the japanese version of the copd - ps questionnaire, the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items: one on the subject s age (four categories), one on body mass index (3-point scale), one on cigarette use (4-point scale), and five on symptoms / history (2- or 3-point scales). Each question is scored individually, with a summed total score ranging from 0 to 38 . A suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan . However, there is no cutoff score for persistent ao in the general japanese population, including never - smokers, and we therefore investigated this issue in this study . Baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . The kruskal spearman correlations were used to examine the strength of associations between informant and performance measures . For the subjects who used a bd, another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity; larger numbers provide more convincing evidence of a disease, smaller numbers indicate that the disease is less likely, and ratios close to one lack diagnostic value . Likelihood ratios (positive and negative) were calculated for both the copd - ps and the ipag questionnaires . Receiver operating characteristic (roc) curves and areas under the roc curves (aucs) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose; this approach assesses the ability of a model to reclassify case and control subjects, respectively, on the basis of the individual - estimated probability of an event . The ability of the model to reclassify is summarized by the net reclassification improvement (nri) and integrated discrimination improvement (idi). We defined four strata dividing the risk of persistent ao into four quartiles, namely, q1, q2, q3, and q4 . The nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another . In contrast, the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable . We computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao . All statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 . The baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 . The majority of subjects (88.9%) showed an initial fev1/fvc 0.70 . Following post - bd spirometry, almost all the ao subjects (94.0%) were classified as having mild or moderate copd; only 6.0% of the ao subjects had severe or very severe copd . The ao subjects were older, were more likely to be men, had lower body mass index, had a higher number of pack - years smoked, and were more likely to be former or current smokers (table 1). The mean and for the ao and non - ao subjects, respectively, the mean scores were 3.9 and 2.4, while the median scores were 4 and 2 . The ipag and the copd - ps questionnaires were correlated with fev1/fvc (r=0.356 and r=0.301, respectively, p<0.001), and the two questionnaires correlated with each other (r=0.622, p<0.001; table 2). The previously identified cutoff point of 4 was used for the copd - ps questionnaire . The crude odds ratio (or) of the copd - ps questionnaire for ao was 5.52, and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% . In this population - based study that included never - smokers, a cutoff point of 20 on the ipag questionnaire would be adequate for screening for ao (table 3). The crude or of the ipag questionnaire for ao, using a cutoff point of 20, was 6.56 . If a cutoff point of 17 was used on the ipag questionnaire, the sensitivity was higher (86.0%) but both the specificity and auc were much lower, 46.2% and 0.66%, respectively . If a cutoff point of 20 was used instead of 17, the sensitivity was slightly lower (71.3%) but both the specificity and auc were higher, 75.2% and 0.72%, respectively . Roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao . The auc values obtained from the roc curve by discriminating ao from no ao were 0.747 (95% confidence interval [ci], 0.7070.788) for the copd - ps questionnaire and 0.775 (95% ci, 0.7350.816) for the ipag questionnaire (figure 1). There was no significant difference in the auc values with the two questionnaires (p=0.09). Between the copd - ps and ipag questionnaires, no statistically significant differences were founded in terms of sensitivity, specificity, or positive and negative predictive values . However, compared with the copd - ps questionnaire, the ipag questionnaire had superior likelihood ratios for both a positive (2.51 vs 2.59) and negative test (0.45 vs 0.40), with nonoverlapping cis (table 4). Reclassifications of subjects with and without ao are summarized in table 5 . For 672 (30.7%) subjects without ao, classification improved using the model with the copd - ps questionnaire, and for 549 (25.1%) subjects, it became worse, with the net gain in reclassification proportion of 0.056 . For subjects with ao, classification was improved in 21 subjects (14.0%) and less accurate in 28 subjects (18.7%), with the net gain in reclassification proportion of 0.047 . Thus, the categorical nri was 0.0096 (95% ci, 0.08680.106; p=0.845). The continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 (95% ci, 0.2730.058; p=0.203) and 0.014 (95% ci, 0.0330.006; p=0.182), respectively, and these were not statistically significant (table 5). The copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao . The baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 . The majority of subjects (88.9%) showed an initial fev1/fvc 0.70 . Following post - bd spirometry, almost all the ao subjects (94.0%) were classified as having mild or moderate copd; only 6.0% of the ao subjects had severe or very severe copd . The ao subjects were older, were more likely to be men, had lower body mass index, had a higher number of pack - years smoked, and were more likely to be former or current smokers (table 1). The mean and for the ao and non - ao subjects, respectively, the mean scores were 3.9 and 2.4, while the median scores were 4 and 2 . The ipag and the copd - ps questionnaires were correlated with fev1/fvc (r=0.356 and r=0.301, respectively, p<0.001), and the two questionnaires correlated with each other (r=0.622, p<0.001; table 2). The previously identified cutoff point of 4 was used for the copd - ps questionnaire . The crude odds ratio (or) of the copd - ps questionnaire for ao was 5.52, and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% . In this population - based study that included never - smokers, a cutoff point of 20 on the crude or of the ipag questionnaire for ao, using a cutoff point of 20, was 6.56 . If a cutoff point of 17 was used on the ipag questionnaire, the sensitivity was higher (86.0%) but both the specificity and auc were much lower, 46.2% and 0.66%, respectively . If a cutoff point of 20 was used instead of 17, the sensitivity was slightly lower (71.3%) but both the specificity and auc were higher, 75.2% and 0.72%, respectively . Roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao . The auc values obtained from the roc curve by discriminating ao from no ao were 0.747 (95% confidence interval [ci], 0.7070.788) for the copd - ps questionnaire and 0.775 (95% ci, 0.7350.816) for the ipag questionnaire (figure 1). There was no significant difference in the auc values with the two questionnaires (p=0.09). Between the copd - ps and ipag questionnaires, no statistically significant differences were founded in terms of sensitivity, specificity, or positive and negative predictive values . However, compared with the copd - ps questionnaire, the ipag questionnaire had superior likelihood ratios for both a positive (2.51 vs 2.59) and negative test (0.45 vs 0.40), with nonoverlapping cis (table 4). Reclassifications of subjects with and without ao are summarized in table 5 . For 672 (30.7%) subjects without ao, classification improved using the model with the copd - ps questionnaire, and for 549 (25.1%) subjects, it became worse, with the net gain in reclassification proportion of 0.056 . For subjects with ao, classification was improved in 21 subjects (14.0%) and less accurate in 28 subjects (18.7%), with the net gain in reclassification proportion of 0.047 . Thus, the categorical nri was 0.0096 (95% ci, 0.08680.106; p=0.845). The continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 (95% ci, 0.2730.058; p=0.203) and 0.014 (95% ci, 0.0330.006; p=0.182), respectively, and these were not statistically significant (table 5). The copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao . In the present population - based study, the japanese version of the copd - ps questionnaire was compared with the ipag questionnaire in a general japanese population at the age of 40 years or older . The ipag questionnaire had superior likelihood ratios of both positive and negative tests compared with those of the copd - ps questionnaire . However, in comparison with two questionnaires, no significant differences were founded in the auc values obtained from the roc curves discriminating between subjects with and without ao and in the sensitivity, specificity, or positive and negative predictive values . The two questionnaires were correlated with each other, fev1/fvc, and for both of them, the ors were significantly greater than 1.0 . These findings suggest that the copd - ps and the ipag questionnaires are useful screening tools for detecting persistent ao . In addition, the subjects with a 4 point on the copd - ps questionnaire and those with a 20 point on the ipag questionnaire are at increased risk for ao . Overall, the categorical and continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire were 0.0096 (95% ci, 0.08680.106; p=0.845), 0.107 (95% ci, 0.2730.058; p=0.203), and 0.014 (95% ci, 0.0330.006; p=0.182), respectively . Reclassifications showed the ipag questionnaire to be superior to the copd - ps questionnaire; however, there was no significant difference between the two questionnaires . A cutoff point of 17 on the ipag questionnaire is used in general health checkup settings and general practices in japan.24 in this study, we found that a cutoff point of 20 was superior . The reason for this discrepancy is not clear, although it may be due at least in part to the backgrounds of the study subjects . The present study was population based and included never - smokers . In contrast, in a previous study that enrolled subjects in general health checkup settings, a cutoff point of 17 resulted in a higher sensitivity of 86.0% and a smaller auc of 0.66 (lower than 0.70) than a cutoff point of 20.24 the present study was implemented in the town of hisayama, in which the age and occupational distributions of the population have been almost identical to those of japan as a whole, and thus, we recommend a cutoff point of 20 for the general japanese population . There were only marginal differences between the two questionnaires in terms of ability to discriminate between subjects with and without ao . Moreover, the copd - ps questionnaire consists of fewer items than the ipag questionnaire and requires less time to complete . It takes ~5 minutes to fill out copd - ps questionnaire, however, the completion of the ipag questionnaire takes ~510 minutes . In this regard, the japanese version of the copd - ps questionnaire should be an adequate measure for large - scale screening for possible ao.
The human cytomegalovirus (cmv) or human herpesvirus 5 is one of the major causes of congenital infections . Its clinical manifestations range from asymptomatic forms (90% of cases) to severe fetal damage and, in rare cases, death due to abortion . Furthermore, 10%15% of the children who are asymptomatic at birth may develop late sequelae, especially hearing defects, after a period of months or even years . Latency following a primary infection (first contact with the virus) may be punctuated by periodic reactivations that give rise to recurrent infections, and in utero transmission may occur during either primary or recurrent infections . Actually recurrent infections may be due to reinfection with a new strain or to reactivation, but it is likely that most recurrent infections are due to reinfection . The risk of congenital infection is much higher during primary infection [25], when the rate of transmission from mother to fetus is 30%40% [1, 6], as against 0.15%2.2% during reactivations or reinfections [1, 69] when, furthermore, most of the newborns are asymptomatic . Symptomatic cases are due more to reinfection than reactivation [2, 10]. It has been reported that the risk of fetal damage is greater if the primary infection occurs during the first trimester of pregnancy [1113]. The prevalence of congenital infection ranges from 0.2% to 2.5% in different populations [1420], in which the risk factors include particular races or ethnic groups, a low socioeconomic status, premature birth, and admission to an intensive care unit [6, 17]. Furthermore, the prevalence of congenital infection varies with the prevalence of the infection in the population . The seroprevalence of cmv among women of childbearing age ranges from 35% to 95% in different countries [12, 2124] and, as well as increasing with age, may also depend on sexual activity and occupation, particularly occupations involving close contacts with children in a community setting . In the case of parents, contact with the urine or saliva of their children is a major source of infection [2527]. The incidence of primary infection among pregnant women ranges from 0.5% and 4% [28, 29]; the rate of seroconversion during pregnancy ranges from 0.4% to 2% [12, 13, 30, 31] and depends on the seroprevalence of the infection in the population, being 3.7% among women belonging to populations with a low seroprevalence (55%) and 1.0%1.6% among those belonging to populations with a high seroprevalence (85%). The risk of acquiring infection during pregnancy is 0.71.38 100 pregnancies [23, 29] among seronegative women, and 0.20.8 100 pregnancies among women as a whole . As far as prevention is concerned, in addition to health education campaigns, the serological screening of pregnant women has been proposed . However, there is no consensus in the scientific community concerning the implementation of screening, and it is not recommended by any public health system because of its cost / benefit ratio, although many doctors in israel, belgium, and france do test their pregnant patients . Furthermore, although the current public health legislation in italy (law decree 245 of 10 september 1998) does not include free cmv antibody screening during pregnancy, it is prescribed by many general practitioners . The aim of this study was to assess the incidence and risk of acquiring cmv infection in pregnant women in an urban area in northern italy in the period 20052007 . During the three years 20052007, the microbiology unit of hospital of legnano, received samples for the detection of cmv antibodies from 2817 pregnant women (mean age 32 years, range 1546; 2522 (89.5%) were italian and 295 (10.5%) of foreign origin). Forty - eight women (1.7%) were 20 or less than 20 years of age, 928 (32.9%) women were aged between 21 and 30, 1750 (62.1%) aged between 31 and 40, and 91 (3.2%) between 41 and 50 . 2318 women (82.3%) underwent their first screening in the first trimester (group a), 316 (11.2%) in the second trimester (group b), and 183 (6.5%) in the third (group c). The requests were made by the general practitioners as part of the routine screening required during pregnancy . All of the samples were analyzed for the presence of anti - cmv igg and igm antibodies by means of an enzyme - linked immunosorbent assay (elisa) (eti - cytok - g - plus, eti - cytok - m reverse plus, diasorin, saluggia, italy). The cutoff value used to determine igg was 0.4 iu / ml, whereas the samples were considered igm - positive when their absorbence was equal to, or greater than the control cutoff value . The igm - positive samples were confirmed using an enzyme - linked fluorescent assay (elfa) (vidas cmv igm, biomrieux, lyon, france) and were considered positive when their index was 0.90, borderline when their index was between 0.70 and 0.90, and negative when their index was> 0.70 . As igm anti - cmv antibodies may test positive for more than 12 months and may be produced during reactivation or reinfection, the samples that were igm - positive at elisa were also tested for igg avidity (liaison cmv igg avidity, diasorin saluggia, italy), which was considered low if the index was <0.2, moderate if it was between 0.2 and 0.3, and high if it was 0.3 . Low igg avidity levels strongly suggest an infection contracted less than three months before, whereas a high avidity tends to exclude this .the elisa igm - positive samples were also tested for the presence of rheumatoid factor (arthri - slidex, biomrieux, lyon, france). In the case of positivity for igm, the patients' general practitioners were contacted and advised to evaluate the case and refer patients to a reference center . At the first screening, the elisas showed that 1925 women (68.3%; 95% ci: 66.6%70.0%) were positive for anti - cmv igg (positive or negative for igm) and 26 (0.9%; 95% ci: 0.55%1.25%) were positive for igm antibodies (25 in the first trimester, and one in the second trimester for whom no previous data were available as she did not undergo screening during the first trimester). Table 1 shows the results of igm and igg elisa by trimester of first screening (groups a elfa of the 26 elisa igm - positive samples showed that 17 (65.4%) were positive or borderline, and nine were negative (34.6%), including the sample that was igm - positive at elisa screening in the second trimester . None of the differences in the prevalence of igg and igm between contiguous age classes was statistically significant . Of the 892 women who were anti - cmv antibody negative at first screening, 687 (77.0%) were in the first trimester of pregnancy (group a), 131 (14.7%) in the second trimester (group b), and 74 (8.3%) in the third (group c). Three hundred and seventy - four of the women of group a (54.4%) were also screened in the second trimester, and 258 (37.6%) were also screened in the third (table 2). Of these, two became positive for igm (confirmed by elfa) and igg, one in the second trimester (0.3%), and one in the third (0.4%), for a mean seroconversion rate of 0.32% . Of the 131 women of group b, 64 (48.9%) were also screened in the third trimester; there were no cases of seroconversion (table 2). Of the 1899 women who were anti - cmv igg antibody positive (and negative for igm) at first screening, 1606 (84.6%) were in the first trimester of pregnancy (group a), 184 (9.7%) in the second trimester (group b), and 109 (5.7%) in the third (group c). One hundred and fifteen of the women of group a (7.2%) were also screened in the second trimester, and 66 (4.1%) were also screened in the third . Of the 184 women of group b, 20 (10.9%) were also screened in the third trimester (table 2). Nineteen of the 28 igm - positive samples at elisa (67.9%) were confirmed as being igm - positive by means of elfa, and 13 (46.4%) showed low or moderate igg avidity . The 19 elfa - confirmed cases (17 first screened in the first trimester, and the two seroconversions) included six (31.6%) with a high degree of igg avidity, five (26.3%) with moderate avidity, and eight (42.1%) with a low degree of avidity (table 3). Table 4 shows the results of the confirmatory igm elfa and igg avidity tests by trimester of pregnancy . In particular, of the five cases showing moderate avidity, one was recorded in our files as having been seronegative for both igg and igm four and a half months before (i.e., about two months before conception), and another was the case of seroconversion in the third trimester after being seronegative in the first and second . In the remaining three cases, the only data available were those of the initial positive sample, but the general practitioner of one of these women, who was contacted after the detection of igm positivity, reported symptoms compatible with ongoing cmv infection . No symptoms were reported by the general practitioners in any of the other two cases nor additional information was available . All of the 13 cases with low or moderate igg avidity were therefore considered as having primary infection: 11 (84.6%) occurring in the first trimester, one (7.7%) in the second, and one (7.7%) in the third (both seroconversions). Unfortunately there were no data regarding the transmission of infection to the fetus because the igm - positive women were all referred for further investigations to reference centers throughout the area . The cumulative incidence of cmv infection (new cases observed during pregnancy) calculated on the basis of the 13 cases with low or moderate igg avidity was 1.4% (95% ci: 0.971.83), and the risk of infection during pregnancy, calculated on the basis of all of the women (seronegative and seropositive), was 0.5 100 pregnancies (95% ci: 0.240.76). The differences in incidence and risk by age was not statistically significant . To take into account the loss to follow - up within the groups and different admission to the study of women during different trimester, there were also calculated the density incidence as cases / pregnant woman - trimester and the correlated risk for all women (seronegative and seropositive): they were, respectively, 0.8% (95% ci: 0.471.13) and 0.4% (95% ci: 0.170.63). The overall prevalence of anti - cmv igg antibodies in our pregnant women was 68.3% (95% ci: 66.670.0), without any significant differences between age classes . As first pregnancies in italy generally occur later than they did in the past, the majority of women have already recovered from primary infection by the time they reach childbearing age and almost certainly by the time of their first pregnancy . Moreover, in this study 95% of the women had an age between 21 and 40 years while age classes of 20 or less than 20 and over 40 years were under - represented; so this could be a further cause of the lack of difference in seroprevalence . On the basis of the results of the igg avidity test, the cumulative incidence of cmv infection was 1.4% (95% ci: 0.971.83%), the density incidence was 0.8% (95% ci: 0.471.13), and the risk of infection was 0.5% (95% ci: 0.240.76%) without any significant differences by age . Seroconversion or clinical data indicating acute infection were available for three of the five cases with moderate avidity in this study, thus moderate avidity was considered as a potential marker of acute infection . Moderate and low igg avidity were considered together, and both were included in the calculation of incidence . However, the incidence may be an underestimate because only about half of the seronegative women underwent further screening in the second trimester and about one - third in the third, and so some cases of seroconversion may have been missed . For the same reason, the proportion of primary infections (84.6%) occurring in the first trimester may be overestimated; however, assuming the same rate of seroconversion among the seronegative women who did not undergo further screening, the majority of primary infections occurred in the first trimester . The fact that 84.6% of the primary infections occurred in the first trimester may have been due to different behaviors before the pregnancy was recognized, whereas greater care during pregnancy may lead to less exposure . The fact that there were no differences related to the age of the women indicates the same type of behavior at different ages . It is therefore important to start screening in the first trimester of pregnancy, when there is a greater risk of infection and in order to have initial findings to compare with subsequent follow - up . In the absence of baseline data, the presence of igg without igm in women undergoing their first screening in the third trimester raises doubts as it may be the result of a previous infection occurring at any time in life before the pregnancy; however, although this is statistically the most probable situation, the possibility of an infection occurring in the first trimester with the subsequent loss of igm cannot be excluded . Finally some limitations of the study must be taken into account as no outcome data for newborns, substantial loss to follow - up, and limited testing of igg positive women for reinfections or reactivations . However, for the latter two cases, as there are no official recommendations, the follow - up was performed at the discretion of the general practitioner with compliance of pregnant woman who, above all, must pay for cmv antibody screening . In conclusion, although screening is not recommended by any public health system (including italy's) because of its cost / benefit ratio, it is actually adopted by many general practitioners in our area . Such screening provides an opportunity to identify seronegative women who can be counselled about using appropriate hygienic measures to prevent infection, especially in relation to their behavior with children, who are a major source of infection . Furthermore, the screening identified primary infections in pregnant women who could be referred to reference centers to check for prenatal infection . Amniocentesis, funicolocentesis, ultrasonography, and magnetic resonance imaging can all be used to detect infection and allow the planning of appropriate interventions (e.g., antiviral therapy, termination of pregnancy). Although some authors consider that screening is not justified on the grounds of its economic cost, the imperfect nature of congenital infection prognostic criteria, the risk of spontaneous abortions induced by invasive tests such as amniocentesis, and the few data concerning effective treatments during pregnancy, it is unthinkable to deny pregnant women appropriate information concerning the health of their unborn child as this raises a number of ethical and legal questions . The incidence and risk of cmv infection in pregnancy found in our area, therefore, support the use of serological screening, certainly in the first trimester when the risk of infection is higher and, in the case of seronegative women, possibly also one screening in the second trimester and one in the third.
Bladder cancer is the second most commonly diagnosed genitourinary malignancy in the usa, with an estimated 74,000 newly diagnosed cases and 16,000 deaths in 2015 . The incidence of bladder cancer rises with age, peaking between age 50 and 70 years, and is three times more common in men than in women . Commonly accepted risk factors for bladder cancer include cigarette smoking, occupational exposure to aniline dyes, benzidene compounds, analgesic abuse (phenacetin) and chronic irritation, such as indwelling catheters . Of all newly diagnosed cases of bladder cancer, about 70% present as non - muscle invasive bladder cancer (nmibc), it comprises a heterogeneous group of patients and includes pathological stage ta (confinement to the epithelium or mucosa), t1 (invasion of the subepithelial connective tissue or lamina propria) and cis (tis: flat, high - grade, non - papillary carcinomas confined to the urothelium). Of all newly diagnosed nmibc, 70% present as stage ta, 20% as t1 and 10% as cis . Approximately 50 - 70% of nmibc will recur and roughly 1020% will progress to muscle (i.e., muscularis propria) invasive disease . In patients with low - grade ta disease, patients with high - grade ta tumors had a progression - free survival of 61% and a disease - specific survival of 74%, whereas patients with t1 disease had a progression - free survival of 44% and a disease - specific survival of 62%, providing support to the view that grouping all patients with nmibc as one disease is misleading as one patient's prognosis can be quite different from that of another patient . When considering a patient's prognosis with nmibc, it is necessary to consider not only clinical and pathological factors but also take into account the potential effect of the intravesical treatment received and molecular alterations present in the tumors . Table 1 summarizes the available predictive models to predict recurrence and progression in patients with nmibc . The most important risk factor for progression is grade, not stage, because patients with high - grade tumors progress with similar frequency regardless of whether they were invasive (t1) or non - invasive (ta). Millan - rodriguez et al . Evaluated a cohort of 1529 primary nmibc patients treated with transurethral resection (tur) and random bladder biopsy and identified prognostic factors for recurrence, progression and disease - specific mortality (median follow - up: 40 months). Multivariate analysis demonstrated that the main prognostic factors of recurrence were multiplicity, tumor size> 3 cm, presence of cis and treatment with bacillus calmette guerin (bcg). The prognostic factors for progression were grade 3 disease, multiplicity, tumor size> 3 cm, cis and treatment with bcg . Furthermore, the prognostic factors for mortality were presence of grade 3 disease and cis . Prediction of disease recurrence and progression in patients with non - muscle invasive bladder cancer in another study, millan - rodriguez et al . Risk groups were classified as: low risk (grade 1 stage ta disease and a single grade 1 stage t1 tumor)intermediate risk (multiple grade 1 stage t1 tumors, grade 2 stage ta disease and a single grade 2 stage t1 tumor)high risk (multiple grade 2 stage t1 tumors, grade 3 stages ta and t1 disease and any stage disease associated with cis). Low risk (grade 1 stage ta disease and a single grade 1 stage t1 tumor) intermediate risk (multiple grade 1 stage t1 tumors, grade 2 stage ta disease and a single grade 2 stage t1 tumor) high risk (multiple grade 2 stage t1 tumors, grade 3 stages ta and t1 disease and any stage disease associated with cis). The rates of recurrence, progression and mortality were 37%, 0% and 0% in the low - risk group, 45%, 1.8% and 0.73% in the intermediate - risk group and 54%, 15% and 9.5% in the high - risk group . The relative risks of recurrence, progression and mortality in the low - risk versus intermediate - risk and high - risk groups were 1.37, 2.84 and 1, and 1.87, 24.76 and 14.69, respectively . The european organization for research and treatment of cancer (eortc) developed risk tables to predict recurrence and progression in patients with nmibc using clinical and pathologic information from 2596 patients enrolled in seven clinical trials that utilized prophylactic treatments after tur . The median follow - up was 3.9 years and 78% of patients had received intravesical treatment, mostly with chemotherapy . In their study cohort, 80% of patients had a maximum tumor diameter <3 cm and 56% of patients had pta tumors . The eortc risk tables [tables 2 and 3] for recurrence and progression were based on the scoring system derived from the following six clinical and pathological factors: number of tumorstumor sizet categorytumor grade (who 1973)prior recurrence ratepresence of concurrent cis . Weights used to calculate eortc risk tables recurrence and progression scores probability of recurrence and progression according to the eortc risk tables total score based on these eortc risk tables (electronic calculator is available at http://www.eortc.be/tools/bladdercalculator/), the probability of recurrence varied from 15% to 61% at 1 year and from 31% to 78% at 5 years . The probability of progression varied from 0.2% to 17% at 1 year and from 0.8% to 45% at 5 years . For their recurrence and progression models, harrell's concordance indices at 1 year were 0.66 and 0.74, respectively . Of note, only 6.6% (171 of 2596) patients received bcg for six induction instillations . In addition, patients with high - grade disease did not have a second tur or receive maintenance bcg therapy . Bcg is currently the most effective intravesical therapy for nmibc, especially in intermediate- and high - risk tumors . A meta - analysis of 24 randomized trials (n = 4863) showed that bcg significantly reduces the risk of progression to muscle - invasive disease after tur in patients with nmibc who receive maintenance bcg . The patient's response to bcg at 3 or 6 months is an important prognostic factor to predict subsequent tumor recurrence and progression . Because the eortc risk tables were generated using nmibc patient's clinical and pathological information, where majority of the patients did not receive bcg induction and/or bcg maintenance therapy, the eortc risk tables tend to overestimate patient's risk for recurrence and progression after bcg therapy . The spanish club urolgicoespaol de tratamientooncolgico (cueto) group developed a scoring model using information from 1062 patients treated with intravesical adjuvant bcg therapy to predict risk of recurrence and progression . Patients had bcg induction as weekly instillations for 6 weeks and six additional instillations repeated at 2-week intervals as a maintenance therapy . By the end of the study, 4.2% of patients received fewer than six instillations, 22.5% of patients received six to nine instillations and 73.3% of patients received more than 10 instillations . The risk tables for recurrence and progression were based on the scoring systems derived from seven clinical and pathological factors: agegendernumber of tumorsrecurrent tumort categorytumor grade (who 1973)presence of concurrent cis . Tumor grade (who 1973) presence of concurrent cis . Based on the cueto risk tables, the probability of recurrence varied from 8.24% to 41.79% at 1 year to 20.98% to 67.61% at 5 years . The probability of progression varied from 1.17% to 13.97% at 1 year to 3.76% to 33.57% at 5 years . For their recurrence and progression models, harrell's concordance indices at 1 year were 0.636 and 0.687, respectively . Using the cueto risk tables, the calculated risk of recurrence were lower than that obtained by the eortc risk tables . The lower risks in the cueto tables is likely attributable to using bcg, which is a more effective intravesical therapy . Although the eortc and cueto risk tables are the two best - established predictive multivariate models for recurrence and progression risk calculation in patients with nmibc, limitations include retrospective analysis, use of the 1987 tnm classification and use of the who 1973 grading system . In addition, repeat tur for high - grade cancer and immediate instillation of chemotherapy, such as mitomycin, was not routinely performed after tur; hence, both models are prone to overestimate the risk of recurrence and progression in patients treated with current standard of care . A restaging tur is routinely performed these days in patients with high - grade nmibc, especially for high - grade t1, as the rate of residual tumor is high and 30% of tumors are upstaged when muscle is absent in the first obtained specimen . Photodynamic diagnosis with blue light and narrow - band imaging have been shown to reduce residual tumor rates by roughly 20% and to improve recurrence - free survival of nmibc patients compared with white light cystoscopy . A meta - analysis of seven randomized trials (n = 1476) confirmed the effectiveness of single immediate intravesical instillation of chemotherapy in treated patients compared with tur alone, reporting a 39% decrease in the odds of recurrence . The efficacy of intravesical mitomycin c can be optimized by administering a dose of 40 mg at a concentration of 2 mg / ml in water, complete bladder emptying just before dose administration, fluid restriction and oral bicarbonate to alkalinize the urine . This approach improved the recurrence - free rate at 5 years from 24.6% to 41% and increased the interval to tumor recurrence from 11.9 to 29 months . Using a multi - institutional cohort of 4689 patients with nmibc, xylinas et al . Showed that both the eortc and cueto risk tables exhibit a poor discrimination for disease recurrence and progression . These models overestimated the risk of disease recurrence and progression in high - risk patients . Additional factors not included in the eortc or the cueto models could be added to new prognostic models to enhance their usefulness . Bladder neck involvement, prostatic urethra involvement, lymphovascular invasion and depth of lamina propria invasion, i.e., whether the tumor is superficial to, into or beyond the muscularis mucosae (t1a, t1b, t1c), have been identified as risk factors for progression in patients with nmibc . In the prognostic factor meta - analysis of 33 studies of high - grade t1 bladder cancer patients (n = 8880), the highest impact risk factor for progression and cancer - specific survival was depth of invasion (t1b / c) into the lamina propria . In this meta - analysis, several other previously proposed factors, i.e. Lymphovascular invasion, associated cis, non - use of bcg, tumor size> 3 cm, gender, multiple tumors and older age, also predicted progression . Nmibc patients with progression to muscle - invasive disease tend to have worse prognosis compared with patients with primary muscle - invasive disease, and it underscores the need to significantly improve risk stratification and earlier definitive treatment for high - risk nmibc . Risk groupings and risk tables provide average predictions, which may or may not apply to the patient interested in knowing individualized risk . Nomograms have been proposed as a method that avoids the arbitrary division of patients into risk groups . Developed nomograms to estimate the risk of disease recurrence and progression in patients with nmibc using a large international cohort . Nomograms based on age, gender, urine cytology and dichotomized nmp22 had accuracy of 0.811 for recurrence of any bladder cancer; 0.772 for recurrence of grade 3 ta or t1 or cis and 0.774 for recurrence of stage t2 bladder cancer after bootstrap validation . However, this nomogram had several limitations, including failure to incorporate established predictors, such as pathologic grade and stage, number and pattern of previous recurrences, time since the original diagnosis and prior use of intravesical therapy . Furthermore, the performance of the nomograms varied significantly among the institutions, emphasizing the need for external validation prior to its clinical use . Artificial neural networks (ann) are algorithms that can be trained to identify complex patterns between input variables and outcomes in the data sets and then apply these patterns to new cases . They have a theoretical advantage over conventional statistics as they are not limited by predefined mathematical relationships between input variables and outcomes; thus, they are able to model complex non - linear parameters . Qureshi et al . Used ann to predict bladder cancer recurrence and progression within 6 months of diagnosis in a small cohort of about 100 patients with nmibc . The accuracy of ann in predicting stage progression and recurrence was 80% and 75%, respectively . However, there was no significant difference between the ann and the clinicians predictions of progression and recurrence in the patients with nmibc . On restricting the validation subset to patients with t1g3 tumors in relation to stage progression, ann accuracy was better than predictions of clinicians . Several investigators have compared ann and other machine learning techniques with standard statistical approaches to predict outcomes and did not find improvement in predictive accuracy with ann and other machine learning techniques over standard statistical approaches . Most of the currently available predictive tools assume that nmibc is pure urothelial carcinoma and does not consider the impact of variant histology on prognosis prediction . Variant histology such as micropapillary, sarcomatoid and plasmacytoid has been shown to be associated with poor prognosis, and early cystectomy is generally advocated in such cases . Small cell carcinoma of the bladder is considered a systemic disease and chemotherapy followed by tailored local therapy is recommended for patients with non - muscle invasive small cell carcinoma of the bladder . However, spaliviero et al . Reported not significantly worse outcomes in conservatively managed t1 micropapillary bladder cancer patients compared with patients treated with early radical cystectomy . Given the conflicting findings on the impact of variant histology in smaller studies, the association of variant histology with prognosis deserves further evaluation in larger studies . For molecular biomarkers to be of clinical use, they should be able to increase the predictive accuracy beyond the standard clinical and pathological parameter models . Several investigators have attempted to use molecular biomarkers as prognostic factors to predict outcomes in patients with nmibc . However, molecular biomarkers have shown mixed results so far and are not sufficiently validated to be used in clinical practice at this time . It is becoming clear that superficial low - grade cancers and invasive or high - grade cancers harbor distinctive genetic defects: the low - grade, non - invasive papillary tumors are characterized by activating mutations in the h - ras gene and fibroblast growth factor receptor 3 (fgfr3) gene and the high - grade invasive tumors are characterized by structural and functional defects in the p53 and retinoblastoma protein (rb) tumor - suppressor pathways . The deletion of both arms of chromosome 9 occurs frequently in bladder cancer during the earliest stages of tumorigenesis . However, these chromosomal aberrations do not seem to distinguish between the two tumor development pathways . Tumor invasion and progression in the bladder seems to be a multifactorial process, promoted by micro - environmental changes that include the up - regulation of n - cadherin, the down - regulation of e - cadherin, the overexpression of matrix metalloproteinases 2 and 9, an imbalance between angiogenic and anti - angiogenic factors and increased synthesis of prostaglandin . In patients with t1 bladder cancer, nuclear overexpression of p53 protein has been reported to have a higher probability of disease progression . In a meta - analysis, p53 was a predictor for recurrence, progression and mortality in patients with bladder cancer . However, investigators of this meta - analysis had concerns of overestimating findings because of publication and reporting bias, and suggested that current evidence is not sufficient to conclude whether changes in p53 act as markers of outcome in patients with bladder cancer . Hernandez et al ., in a prospective cohort of 772 patients with nmibc, showed that fgfr3 mutations are prevalent in low - grade ta and that these mutations are independent predictors of recurrence in patients with low - grade ta tumors . Van rhijn et al . Showed that molecular grading, the grading system based on fgfr3 and mib-1 (ki-67) status, is an independent predictor for recurrence, progression and disease - specific survival . Van rhijn et al . Validated the utility of molecular grading as a prognostic factor to predict outcomes in patients with nmibc . The addition of molecular grade to the multivariable model for progression increased the predictive accuracy from 74.9% to 81.7% . Evaluated nmp22 for detecting recurrence and progression in patients with nmibc in a large multi - institutional international cohort . There was no clearly defined nmp22 cut - off that could indicate higher risk disease, but there was a continuum of risk for recurrence and progression . The international consensus panel on cytology and bladder tumor markers evaluated the prognostic utility of molecular markers for bladder cancer . Molecular markers were classified into six groups, i.e. Microsatellite - associated markers (e.g. Fish, loh), proto - oncogenes / oncogenes (e.g. Her-2/neu, h - ras, bcl-2, mdm-2, fgfr-3, c - myc) tumor suppressor genes (e.g., p53, rb), cell cycle regulators (e.g., p21, p27, ki-67, cyclin - d1, cyclin - e), angiogenesis - related factors (vegf, cox-2, tsp-1) and extracellular matrix adhesion molecules (e.g. E - cadherin, mmps, timps, cd44, u - pa). The panel concluded that although certain biomarkers, such as ki-67 and p53, appear to be promising in predicting recurrence and progression in patients with bladder cancer, the data are still heterogeneous . The panel recommended that identifying definitive criteria for test positivity, a clearly defined patient population, standardization of techniques used to evaluate markers and clearly specified endpoints and statistical methods will help to bring accurate independent prognostic indicators into the clinical management of patients with bladder cancer . Nmibc comprises of a heterogeneous group of patients and includes pathological stage ta, t1 and cis . Patients with low - grade ta disease have very low risk of progression while patients with t1 disease with concurrent cis have a much higher risk of progression, approaching 50% . The eortc and cueto risk tables are the two best - established predictive models for recurrence and progression risk calculation in patients with nmibc . However, both the eortc and the cueto risk tables exhibit a poor discrimination for prognostic outcomes and overestimate the risk of disease recurrence and progression in high - risk patients in external validation . Additional prognostic factors such as depth of lamina propria invasion should be added to new prognostic models to enhance their usefulness . Molecular biomarkers such as ki-67, fgfr3 and p53 appear to be promising in predicting recurrence and progression, but need further validation prior to using them in clinical practice . Future research should focus to enhance the predictive accuracy of the risk assessment tools by incorporating additional prognostic factors such as depth of lamina propria invasion and molecular biomarkers after rigorous validation in multi - institutional cohorts.
Personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies in order to achieve the best medical outcomes for individual patients, thus improving public health . Despite the variety of clinical, morphological, and molecular parameters used to classify human malignancies, patients receiving the same diagnosis can have markedly different clinical courses and treatment responses . Since there is no simple way to determine who will have an adverse reaction, the current system of one - size - fits - all- diagnoses is simply not good enough . An increasing number of studies have demonstrated sex differences in drug reactions to the same drug treatment . Migeon implied that males and females responded differently to drug treatments and that sex plays a key role in cancer . In addition, females are historically less studied subjects due to the complication of estrous cycle, and therefore such studies would further benefit women's health and promote public health . Recent advancements in biotechnology have accelerated the search for molecular biomarkers useful in the diagnosis and treatment of disease . Molecular biomarkers of disease risk and status are critical to an accurate treatment by identifying patients most likely to benefit from particular drugs or experience adverse reactions . Because medicine is always practiced on individuals rather than populations, the goal is to change the assignment of therapies from a population - based approach to an individualized approach . Gene - expression data can be used to identify patients with a good disease prognosis, thereby preventing some patients from unnecessary therapies and toxicity . For example, gene - expression profiling was used to predict clinical outcomes in pediatric patients with acute myeloid leukemia and to find genes whose aberrant expression leads to a poor prognosis . Thus, accurate classification of prognosis of patients leads to efficient cancer treatment and prolonged survival of patients . Classification algorithms are needed for biomedical decision making in clinical assignment of patients to treatment therapies based on individual risk factors and disease characteristics . Since many of those genes are not relevant, feature selection is a commonly addressed problem in classification [3, 4]. The goal of gene selection is to identify a set of genes which plays an important role in the classification . A common approach is to select a fixed number of the highest ranked genes based on t - test - like statistics, some discrimination measures [6, 7], or classification algorithms including support vector machines (svm) [8, 9] and random forest (rf) [10, 11]. Development of a biomarker classifier involves two distinct components: (1) a procedure for building a classifier and (2) validation / evaluation procedure for estimating the error rate of biomarker . The most important consideration is the validation / evaluation of a biomarker classifier to assess whether it can accurately and unbiasedly predict new samples based on a set of selected features . Two methods are commonly used to develop and assess performance of a classifier: the split - sample procedure and cross - validation procedure . In the split - sample procedure, the sample dataset is randomly split into two subsets: a training set for model building and a test set for performance assessment . That is, the training dataset is used for building the biomarker classifiers and the test set is used for evaluating the classifier . However, samples are often insufficient to split into two sample sets of approximately equal size for model building and model testing, and a cross - validation (cv) procedure is commonly used for performance assessment [12, 13]. A cv can be regarded as a generalization of the split - sample method . It involves repeatedly splitting the data into a training set containing most of the samples and applying the prediction rule to the test set of the remaining samples to estimate the prediction accuracy rate . The prediction accuracy is the average accuracy of the numerous training - test partitions . For model building, given that differences in the biology of lung cancer and other diseases exist between men and women [14, 15], investigations to identify genomic biomarkers for clinical assignment of therapies on an individual patient basis are crucial . In this paper, the ratio of between - group to within - group sums of squares (bw ratio,) gene selection algorithm is used to obtain a feasible set of influential genes via variable importance ranking procedure in the training phases of 20 trials of 10-fold cv within leave - one - out cross - validation (loocv) procedure as illustrated below . The genes with largest bw ratios are ranked high as significant genes . In the development of biomarker classifier, the predictive accuracy of the classifier must be evaluated on a separate set of data . To derive an unbiased accuracy estimate, a nested cross - validation procedure, 20 trials of 10-fold cv within loocv, is used in this paper . In other words, in each loocv, 90% of the wherefore genes are selected only using learning data sets of size (n 1) each, and in the evaluation / validation step a never - touched and left - out single observation in loocv is used to assess the predictive performance of selected genes . In this way, each test case is never used for gene selection . To avoid a bias due to partitions of data, if the performance is assessed using the very same data that are used for developing the classifier, this obviously leads to a biased down estimate of classification error . In other words, if one applies a method to the original data with 20 trials of 10-fold cv only as a way of building the classifier and selects a set of genes and then on that very same data calculates the classification error, it clearly leads to a biased upward estimate of classification accuracy . Three publicly available data sets of interest in this paper are pediatric acute myeloid leukemia (aml), b - cell chronic lymphocytic leukemia (b - cll), and primary cutaneous melanoma . The data sets are downloaded from the brb - arraytools data archive for human cancer gene expression located at the website http://linus.nci.nih.gov/~brb/dataarchive_new.html . Sex differences in disease rates or in rates of adverse reactions to treatment are common, which we intend to exploit to obtain sex - specific biomarkers from gene - expression data . We hypothesize that genomic biomarkers developed from the sex - specific application of classification algorithms will further improve class prediction accuracy . The summary of our algorithm to find sex - specific predictive / prognostic genomic biomarkers for efficacy or toxicity in individualized treatment of patients for serious diseases is as follows . In each loocv trial, firstly, the data is partitioned into a test data set with one observation and the remaining data as the learning data set . The learning data set is further separated into male and female patients' learning data sets . Thus, this process will be applied n times, where n represents the total number of patients . Secondly, within each trial of loocv, twenty trials of 10-fold cv are conducted for each set of male and female patients in the learning data set . At each 10-fold cv step, two sets of top - ranked genes, one for male patients (smi) and one for female patients (sfi), are obtained via the bw ratio to differentiate types of patients such as diseased versus nondiseased . This can be done by applying variable importance ranking approach in order to extract most influential genes and by combining a measure of importance in each gene . A score taking the average of the measure in cross - validation (cv) is prioritized in the list of genes according to variable importance . Thirdly, the mutually exclusive sets of male - specific genes and female - specific genes are obtained . Fourthly, within each loocv trial, after the sets of potential sex - specific genes are determined in the second and the third steps, they are fitted to a model with diagonal linear discriminant analysis (dlda;) using each learning data set for male and female patients . Finally, the performance is measured with the test data with one observation in each loocv trial . This method is implemented in r. the r code is available upon request . Within each trial of loocv, each set of top - ranked genes for male and female patients is obtained in the second and the third steps as follows . First, in order to build genomic biomarkers, 20 trials of 10-fold cv are applied to each learning data set for males and females, where 90% were randomly selected without replacement as a set for each trial of cv . Next, for each trial of 10-fold cv, the bw ratio was applied to this 90 percent learning set and the top 25 ranked genes were selected in each process with the target endpoint of a dataset . The bw ratio for a gene j is defined as (1)bwj=iki(yi = k)(xkjxj)2iki(yi = k)(xijxkj)2, where x-j=ixij / n indicates the average value of a gene j across all the training samples, x - kj=i(yi = k)xij / nk indicates the average value of a gene j for a class k, and i indicates an observation . Here this criterion has been shown to be reliable for variable selection from high - dimensional data sets [6, 12]. Next, to avoid selection bias from a pattern of selection of learning samples, we repeat the entire process 20 times by shuffling samples at every 10-fold cv . In order to obtain the variable importance ranking, 200 sets of top 25 ranked genes were combined so that the maximum possible rank score of a gene would be 200 . A set of genes that has been selected at least once (rank score> 0) was obtained separately for males and females . These most influential genes used in the classification process are identified in order to extract a feasible set of sex - specific genes . For both male and female learning data sets, the final product of the 20 trials of 10-fold cv described in each loocv is the sets of genes selected for male and female patients in the learning data set smj = {g1,, gxmj} and sfj = {g1,, gxfj}, j = 1, 2,, n, respectively . The sets smj and sfj contain prognostic / predictive genes for male and female patients, respectively . There would be n sets of selected genes . Within each trial of loocv, genes that are commonly identified between male - specific and female - specific genes finally, the test sample with one observation is tested using these sex - specific genes in each loocv . At the end of the entire loocv, each selected gene has a combined variable importance score that is less than or equal to n. to verify sex - specific biomarkers, we consider the following four different cases: we classify the outcome of (1) male patients with a set of male - specific genes smj,(2) female patients with a set of male - specific genes smj,(3) male patients with a set of female - specific genes sfj, and (4) female patients with a set of female - specific genes sfj . We anticipate that data with a set of male - specific genes have higher predictable power to predict male patients than the data with female - specific genes . Similarly, data with female - specific genes have higher predictable power to predict female patients than the data with male - specific genes . Upon completion of loocv, the performance of sex - specific biomarkers is obtained . In order to validly evaluate the performance of a gene set selected by the proposed method, cv utilizes resampling without replacement of the entire data set to repeatedly develop classifiers on a training set and to evaluate these classifiers on a separate test set and then averages the results over the resamples . In this section, we apply the proposed algorithm to the following genomic data sets to find the most meaningful sex - specific predictive / prognostic genomic biomarkers for improving individualized treatment of patients and for evaluating the biomarkers from the proposed algorithm . Current chemotherapy enables a high percentage of pediatric patients with aml to enter complete remission (cr), but a large number of them experience relapse (r) with resistant disease . Because of the wide heterogeneity of aml, predicting a patient's risk for treatment failure or relapse at the time of diagnosis is critical for the optimal treatment . This gene - expression data set consists of 54 aml pediatric patients (<15 years old) with an oligonucleotide microarray containing 12,566 probe sets and it is also available at ftp://ftp.ncbi.nih.gov/pub/geo/data/soft/gds/gds1059.soft.gz . Patients with cr for more than 3 years are classified as having a good prognosis, while patients experienced relapse within 1 year of the first cr are considered as having a poor prognosis . In this data set, there are 28 patients with cr and 25 patients with r. since a five - month male patient experienced induction failure (no cr achievement) within 3 months of the start of treatment is considered neither cr nor r, we exclude this subject . Therefore, there are 32 male patients and 21 female patients in this data set . With the prognostic endpoint (r / cr), the average accuracy of 66% (sd 3.0%) for pediatric patient classification was obtained when no gene selection was introduced . When a set of 200 genes was selected in a learning phase of each cv, the average accuracy of 68.0% (sd 3.0%) was achieved . When a set of 20 genes was selected in a learning phase of each cv, the average accuracy of 71.0% (sd 5.0%) was obtained . Since it appeared to be no substantial difference between accuracy and the number of genes selected, a feasible set of 25 genes in the learning phase of each cv was collectively ranked by the bw ratio to find sex - specific biomarkers . At the end of loocv trials, a set of male - specific genes that were selected at least 75% of the time in the entire loocv were 1882_g_at (mecom: mds1 and evi1 complex locus), 37902_at (cryz: crystallin, zeta (quinone reductase)), 38789_at (tkt: transketolase (wernicke - korsakoff syndrome)), 39105_at (vasp: vasodilator - stimulated phosphoprotein), 40844_at (ctr9: ctr9, paf1/rna polymerase ii complex component, homolog (s. cerevisiae)), 36981_at (srp9: signal recognition particle 9 kda), 36338_at (luzp1: leucine zipper protein 1), 31870_at (cd37: cd37 antigen), 1624_at (rap1gds1: rap1, gtp - gdp dissociation stimulator 1), and 39142_at (nudt21: cleavage and polyadenylation specific factor 5, 25 kda). These ten top - ranked male - specific genes were selected as potential male - specific genomic biomarkers to classify male patients into r / cr . Among them 38789_at (tkt: transketolase) and 36338_at (est) were also included in the thirty - five genes associated with prognosis of pediatric aml identified by yagi et al . . In order to select a feasible set of sex - specific biomarkers a cut - off criterion of 75 percent is used . Since every dataset may have a different sample size, the number of genes selected is given by the percentage, which is a rank score of the selected genes greater than 75% of the sample size in our case . For example, if a sample size is 100, then genes that have rank scores greater than 75 have been selected . Similarly, nine top - ranked genes for classifying female patients into r / cr were 40601_at (tm2d1: tm2 domain containing 1), 36330_at (ccbl1: cysteine conjugate beta- lyase; cytoplasmic (glutamine transaminase k, kynurenine aminotransferase)), 40586_at (eef1e1: eukaryotic translation elongation factor 1 epsilon 1), 36648_at (crsp9: cofactor required for sp1 transcriptional activation, subunit 9, 33 kda), 32351_at (gpr20: g protein - coupled receptor 20), 1718_at (arpc2: actin - related protein 2/3 complex, subunit 2, 34 kda), 38622_at (mtg1: mitochondrial gtpase 1 homolog (s. cerevisiae)), 36496_at (impa2: inositol(myo)-1(or 4)-monophosphatase 2), and 38337_at (znf193: zinc finger protein 193). These nine top - ranked genes which were selected at least 75% of the time in the entire loocv were considered as potential female - specific genomic biomarkers to classify female patients into r / cr . Data with male - specific genes showed higher prediction accuracy (71.9%) to classify male patients than the accuracy (43.8%) to classify male patients from data with female - specific genes . Similarly, data with female - specific genes showed higher prediction accuracy (76.2%) to classify female patients than the accuracy (61.9%) to classify female patients from data with male - specific genes . As shown in table 1, sensitivity and specificity were also higher in using sex - specific genes for each sex . Genomic aberrations and mutational status of the immunoglobulin variable heavy chain (vh) gene have been shown to be among the most important predictors for outcome in patients with b - cll . B - cll is the most common leukemia in the western world, and due to its clinical heterogeneity (wide range of life expectancy) and correlations to genomic aberrations, it is important to predict a patient's vh mutation status at the time of diagnosis for optimal treatment . In addition, the study presented by haslinger et al . Suggested that the genomic signature for vh mutational status might be sex related . The gene - expression data consists of 100 b - cll patients with an oligonucleotide microarray containing around 12,000 probe sets, and it is available at http://linus.nci.nih.gov/~brb/dataarchive_new.html . Patients are classified as either vh - mutated or unmutated (m / nm). There are 62 males (33 m and 29 nm) and 38 females (18 m and 20 nm), with a total of 51 mutated and 49 unmutated patients . In step 1, for each data set with male only and female only patients using the target endpoint (i.e., vh - mutated (m) versus unmutated (nm)), we separately selected and ranked 25 potential prognostic genes for males and for females in every cv trial and separately combined ranks of these genes for males and females during the learning phase of 20 trials of 10-fold cv within each loocv trial . In every loocv trial, we prioritized and combined the final top - ranked 200 genes from the male patients result (sm) and 200 genes from the female patients result (sf) as explained in section 3.1 . At the end of the entire loocv trials, a set of potential sex - specific genes are obtained for each sex by prioritizing and combining n sets of top - ranked 200 genes . After deletion of overlapped genes in both males and females, eleven potential male - specific genes were obtained to classify male patients into m / nm . They were 41209_at (lpl: lipoprotein lipase), 41755_at (cobll1: cobl - like 1), 39878_at (pcdh9: protocadherin 9), 38211_at (zbtb20: zinc finger and btb domain containing 20), 39488_at (pcdh9: protocadherin 9), 36886_f_at (kir2dl3: killer cell immunoglobulin - like receptor, two domains, long cytoplasmic tail, 3), 32140_at (sorl1: sortilin - related receptor, l(dlr class) a repeats - containing), 33535_at (p2rx1: purinergic receptor p2x, ligand - gated ion channel, 1), 39967_at (ldoc1: leucine zipper, downregulated in cancer 1), 32842_at (bcl7a: b - cell cll / lymphoma 7a), and 36899_at (satb1: special at - rich sequence binding protein 1 (binds to nuclear matrix / scaffold - associating dna's)). For female patients, only five genes were selected at least 75% of the time in the entire loocv trial as potential female - specific genomic biomarkers to classify female patients into m / nm . They were 33745_at (phkg2: phosphorylase kinase, gamma 2 (testis)), 38152_at (loh11cr2a: loss of heterozygosity, 11, chromosomal region 2, gene a), 34142_at (pde8a: phosphodiesterase 8a), 39593_at (fgl2: fibrinogen - like 2), and 217_at (klk2: kallikrein 2, prostatic). To verify the sex - specific genomic biomarkers, we considered the performance of four different cases in the loocv trials as described in section 2 . Data with male - specific genomic biomarkers showed higher accuracy (67.7%) to classify male patients into m / nm than the accuracy to classify male patients into m / nm from data with female - specific genomic biomarkers (40.3%). However, female data with female - specific genes showed prediction accuracy similar to random guess close to 50% . Therefore, there is insufficient evidence to support that the selected female - specific genes were female - specific genes (see table 2) with 38 female patients in this data set . Although cutaneous melanoma represents a small subset, it is the most life - threatening neoplasm of the skin, and its incidence and mortality have been increasing worldwide . The key underlying molecular events have not been clearly elucidated, which may explain why no target has been developed and why almost no clinical benefits from new therapies have been clearly demonstrated in patients with melanoma since the late 1970s . Additionally, gene - expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors . This gene - expression data set was collected from 83 patients corresponding to the training data set and 17 patients corresponding to the validation data set . The probes are from tumor tissue and from reference tissue that are differentially labeled by the incorporation of cyanine 3 (cy3) and cyanine 5 (cy5), respectively . Set, the endpoint was patient prognosis and survival along with tumor stages, defined as follows . In stage i, cure rates are excellent with surgical removal, since they are the least likely to spread . In stage ii, melanomas can be cured, but the success rate lags behind that of stage i because a small number of cancer cells may have spread to distant sites . In stage iii, since the tumor has started to metastasize (the spreading of a disease from one organ or part to another nonadjacent organ or part), the survival rate for these stages is lower than the earlier ones . Stage iv is associated with metastasis beyond the regional lymph nodes to distant sites in the body, such as the lung, liver, or brain, or to distant areas of the skin . Based on the tumor size, descriptions, and number of lymph nodes the stages are categorized in two classes . A class of high survival and small tumor size (hs / st) is defined and composed of stages 1 and 2 . The second is defined as low survival and non - small tumor size (ls / nst), which is composed of stages 3 and 4 . Tables 3 and 4 show the distribution of the primary cutaneous melanoma data based on sex, the defined clinical endpoint hs / st and ls / nst for the training data and validation data . Among 83 patients in the training dataset, there are 27 males (12 hs / st and 15 ls / nst) and 56 females (30 hs / st and 26 ls / nst). There are 42 cases of hs / st and 41 cases of ls / nst in total . After preprocessing the data the final gene count is 4641 genes . Among 17 patients in the validation data set, there are 8 males (1 hs / st and 7 ls / nst) and 9 females (1 hs / st and 8 ls / nst). There are 2 cases of hs / st and 15 cases of ls / nst in total . Since the validation set was separately provided, the sex - specific genes were selected via 20 trials of 10-fold cv in the learning set . The following ten male - specific genes were selected at least 75% of the time during 20 trials of 10-fold cv: a_23_p128263 (prb1: proline - rich protein bstni subfamily 1), a_23_p83838 (ca8: carbonic anhydrase viii), a_24_p212990 (mgc70863: similar to rpl23ap7 protein), a_23_p333650 (rad9b: rad9 homolog b (s. cerevisiae)), a_32_p125251 (n / a), a_23_p108835 (ypel5: yippee - like 5 (drosophila)), a_32_p150856 (loc407835: mitogen - activated protein kinase kinase 2 pseudogene), a_23_p11936 (ubxn11: ubx domain protein 11), a_24_p169976 (n / a), and a_32_p3998 (znf600: zinc finger protein 600). For female patients, the following eight female - specific genes were selected from 20 trials of 10-fold cv: a_23_p69497 (clec3b: c - type lectin domain family 3, member b), a_24_p118884 (n / a), a_23_p152420 (kiaa0182), a_24_p265177 (phc3: polyhomeotic - like 3 (drosophila)), a_23_p251421 (cdca7: cell division cycle associated 7), a_23_p211738 (ubp1: upstream binding protein 1 (lbp-1a)), a_23_p47377 (hsd17b12: hydroxysteroid (17-beta) dehydrogenase 12), and a_32_p113646 (cdna flj45341 fis, clone brhip3009672). Using a given validation set, the sex - specific genes were verified . As shown in the confusion matrix in table 5, there was no difference in the performance from female data with female genes compared with the performance of female data with male genes . However, there was only one misclassification for male patients with male genes as shown in table 6, while there were four misclassifications for male patients with female genes as shown in table 7 . Therefore, we conclude that there exists evidence of male - specific genes in a classification of primary cutaneous melanoma . Large inter individual differences in benefit from chemotherapy highlight the need to develop predictive genomic biomarkers for selecting the right treatment for the right patient . Inappropriate chemotherapy can result in the selection of more resistant and aggressive tumor cells . To date, no reliable genomic biomarkers have been developed to provide the physician with prechemotherapy information to accurately predict the efficacy of a specific therapy . We proposed a procedure to find sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm using variable importance ranking via combination of 20 trials of 10-fold cv and loocv . The proposed procedure was applied to data sets obtained from the brb arraytools data human cancer archive . However, the issue arose out of there being not enough samples, and the data was unbalanced (i.e., more males than females or more positives (disease patients) than negatives (nondisease patients)). While patient's sex information was not specified for many of the publicly available data sets adding to that the difficulty of searching for data, we found the following three genomic data sets that had sex information: (1) pediatric patients with aml, (2) b - cell chronic lymphocytic leukemia (b - cll), and (3) primary cutaneous melanoma . In one application, pediatric patients with aml were classified by the algorithms as having either a good or poor prognosis, in terms of the likelihood of induction failure or relapse within one year of the first complete remission, based on gene - expression profiles . If this were brought into clinical application, a patient with a confidently predicted good prognosis might want to elect out of adjuvant chemotherapy and its associated debilitating side effects . With current rule - based decisions, the overall average accuracy of this data set with a variable selection from pooled patients (males and females) was about 71.0% . However, using male - specific genes found by the proposed procedure, the accuracy was improved to about 72% as we found in the model validation studies . Similarly, using female - specific genes found by the proposed procedure, the average accuracy was improved to about 76% (see table 1). In the b - cell chronic lymphocytic leukemia (b - cll) dataset we found male - specific prognostic genomic biomarkers associated with b - cell chronic lymphocytic leukemia and its average classification accuracy was improved to about 68% . There was no substantial evidence to find female - specific prognostic genomic biomarkers in this data set . Similarly, male - specific predictive genomic biomarkers associated with a classification of primary cutaneous melanoma were found with the classification accuracy of about 88% . The scope of our paper was to find sex - specific genomic biomarkers, if any, imbedded in the data instead of finding genomic biomarkers from the data . If commonly identified genes were kept in the proposed procedure, our procedure was not sex - specific genomic biomarker classifier involving two populations (males and females) any more but rather it became genomic biomarker classifier involving one combined population . In fact, even though commonly identified genes were kept, it did not necessarily improve the classification accuracy . For a counterexample, for the pediatric aml data of yagi et al ., the accuracy for female patients with female - specific genes by keeping the commonly identified genes was 62%; however, the accuracy without commonly identified genes was 76% . For the male patients with male - specific genes by keeping the commonly identified genes the accuracy was 59%, but the accuracy without the commonly identified genes was 72% . For the related note, the accuracy of this data can be found anywhere between 59% and 66% with gene preprocessing and between 58% and 71% with gene selection . It is not an easy task to find sex - specific genes, let alone verifying and proving that they are indeed sexspecific . We have presented a procedure for finding sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm . The procedure is shown to have good sensitivity and specificity in the sense that the sex - specific genes obtained can improve prediction accuracy in classification of individual patient's prognosis . The proposed procedure to discover predictive and prognostic sex - specific genomic biomarkers for individualized treatment of diseases can play a critical role in developing safer and more effective therapies that replace one - size - fits - all drugs with treatments that focus on specific patient needs.
In the last decade, preimplantation genetic diagnosis (pgd) has become an important alternative to prenatal diagnosis for couples at high risk of transmitting inherited disorders to their children . Fluorescence in situ hybridization (fish) has been offered for gender selection in x - linked diseases (1, 2), chromosomal translocations (3 - 5), aneuploidy, and recurrent implantation failure (6, 7). Procedures using polymerase chain reaction (pcr) has been applied for single gene defects including x - linked diseases (8 - 11). Ornithine transcarbamylase (otc; ec 2.1.3.3) is placed in the mitochondrial matrix where it contributes to the detoxification of nitrogenous wastes through the urea cycle, and catalyzes the synthesis of citruline from carbamyl phosphate and ornithine . The otc gene is located on the short arm of the x chromosome within band xp21.1 (12). It spans 73 kb, has an open reading frame of 1,062 nucleotides, and contains 10 exons and 9 introns (13, 14). Otc deficiency (mim-#311250) is an x - linked and co - dominant metabolic disorder with partial penetrance in females (15). The phenotype of otc deficiency is extremely heterogeneous, ranging from asymptomatic adult hemizygous males to acute neonatal hyperammonemic coma in the first week of affected male babies . Approximately 80% of heterozygous females are asymptomatic and remaining 20% show clinical severity similar to males with partial deficiencies (16, 17). There are more than 341 mutations known to cause otc deficiency, all of which are specific for the individual families . Mutations have been found in all exons and introns with a relative paucity of mutations in the sequence encoding the leader peptide (exon 1 and a part of exon 2) and in exon 7 (17, 18). To our knowledge, only two cases have been reported where specific pgd for otc deficiency were carried out using multiplex or duplex - nested pcr assay and healthy babies devoid of the otc mutation were born (19, 20). In this study, we describe the successful pregnancy and birth after pgd for otc deficiency with simultaneous analyses of fish and duplex - nested pcr in korea . We have applied a duplex - nested pcr for the amplification of both the causative mutation and the y chromosome specific sry gene, and fish for aneuploidy screening of chromosome x, y and 18 . A couple was referred to our center after therapeutic termination of the second pregnancy following prenatal diagnosis for otc deficiency . In the first pregnancy, a male baby was born but died at 10 days after birth due to liver dysfunction accompanying hyperammonemia . Molecular genetic analysis of the couple's otc gene was performed by pcr and direct sequencing at seoul asan hospital and revealed a single base substitution (r320x) in the exon 9 of the female partner . In the second pregnancy, the fetus was identified to be an affected boy by pcr and direct sequencing on a chorionic villus sample, and the pregnancy was therapeutically terminated . In order to avoid a pregnancy with an affected baby, the couple was counseled for pgd in cheil general hospital . In order to determine the efficiency of single cell pcr and allele drop - out (ado) rate, primers used for the detection of the causative mutation and y chromosome specific sry gene were tested using single lymphocytes (table 1). Ovarian stimulation, in vitro fertilization, and blastomere biopsy were done as previously described (4, 5). After the blastomere biopsy procedure, the embryos were washed several times and transferred to the g2.2 medium (vitrolife sweden ab, kungsbacka, sweden). Each blastomere was washed twice through two drops of g2.2 medium and transferred into sterile 0.2 ml pcr tubes containing 5 l of alkaline lysis buffer . The nested - duplex pcr analysis was performed by previously described protocol (6). Mutation analysis by restriction fragment length polynorphism (rflp) with bcli restriction enzyme was carried out only for positively amplified pcr products of blastomeres . The fish analysis for chromosome x, y and 18 was done as previously described and the manufacture's protocol (4, 5). The embryos with the normal genotype were selected and transferred on day 4 after fertilization . All 10 exons and exon - intron boundaries of the otc gene were screened by pcr followed by direct dna sequencing analysis . The heterozygous otc gene with r320x nonsense mutation (cgatga) and wild type was detected in the exon 9 of the female partner by rflp (fig . 1) and direct sequencing (data not shown). This r320x nonsense mutation was associated with acute neonatal hyperammonemia in their first pregnancy and reduced enzyme activity by 10 - 15% (21). The efficiency and accuracy of the duplex - nested pcr analysis were tested on single lymphocytes from the heterozygous female partner and normal male partner . Pcr on single lymphocytes resulted in 90.6% (58/64) amplification rate, whereas none of the negative controls showed a positive band . After ovarian hyperstimulation, a total of 18 oocytes were retrieved and 17 oocytes were injected using intracytoplasmic sperm injection in order to prevent dna contamination from inseminated sperm . Among a total of 11 embryos, two blastomeres per embryo from 9 embryos were biopsied and simultaneously analyzed by duplex - nested pcr and fish, and one blastomere per embryo from 2 embryos by only duplex - nested pcr . As a result of duplex - nested pcr and rflp analysis with the bcli restriction enzyme, ten showed successful amplification for the exon 9 of the otc gene, and the amplification rate was 90.9%, almost same as that of the single lymphocyte test . There were two unaffected males, five affected males, two unaffected females, and one heterozygous female (fig . 2 and table 2). On the other hand, out of nine blastomeres analyzed by fish, only six blastomeres were identified as chromosomally normal embryos (fig . However, among total four unaffected embryos (embryo 1, 2, 3, and 8) diagnosed by pcr, only two embryos (embryo 3 and 8) were identified as chromosomally normal by fish . The other two embryos (embryo 1 and 2) were shown to have trisomy 18 and monosomy 18, respectively . Only one male embryo (embryo 8) was transferred, and another female unaffected embryo (embryo 3) was cryopreserved at blastocyst stage on day 5 after fertilization . This pgd cycle resulted in a singleton pregnancy and the genotype of the fetus was confirmed to be a normal male by amniocentesis at 16 weeks of gestation . The genotype of the baby for the exon 9 of otc gene and level of ammonia were confirmed as normal . Most pgd cycles for x - linked disorders such as duchenne muscular dystrophy and fragile x syndrome involved the selection of female embryos which were diagnosed as normal or heterozygous . These gender selections were supported by couples at risk of x - linked recessive conditions . Ado for affected allele could lead to yielding an affected heterozygous baby, even if the female embryo was diagnosed as normal by pgd for otc deficiency . Gender selection with fish for y chromosome - specific loci is more reliable because amplification failure could occasionally occur in single cell pcr . Thus, our protocol with pcr and fish in this study provides higher accuracy for the selection of genetically and chromosomally normal embryos in the pgd for x - linked co - dominant defects . We applied duplex - nested pcr and fish analysis in two blastomeres from each embryo, which were more than 6-cell stage . Biopsy of more than a quarter of blastomeres from each embryo could interfere with developmental potential of embryos . Thus, single blastomere biopsy was applied to less than 6-cell stage embryos in this study . Sequential pcr and fish analysis in one blastomere (cell recycling) was reported and could be used to detect the aneuploidy, but the rate of ado after pcr seems to be higher using this method (22, 23). A duplex - nested pcr analysis was developed for the simultaneous diagnosis of a r320x mutation and a gender selection, and fish analysis was used to detect aneuploidy for chromosome x, y and 18 . As a result of duplex nested pcr and fish analysis, concordant results have been obtained from two blastomeres at the sex locus, and the amplification efficiency was 100% (9/9 except two blastomeres, that were not analyzed by fish analysis) for sry . And the amplification rate was 90.9% (10/11) for exon 9 of the otc gene . Several strategies have been developed to decrease amplification failure and ado in single cell analysis of pgd . These included the development of sequential first and second polar body analysis (24), improvement of the pcr condition by increasing the denaturation temperature and time (19), using a more powerful lysis method (25 - 28) and the use of fluorescent pcr for mutations or linked markers and multiplex pcr (29 - 32). As mentioned above, we have also endeavored to optimize single cell pcr conditions, which included longer initial denaturation time, higher denaturation temperature, and the selection of taq polymerase, the concentration of mgcl2, and lysis methods . The incidence of ado was 5 - 15% in many pgd laboratories (27, 28). At a preliminary experiment on single lymphocytes in this study, the ado rate for exon 9 of the otc gene was about 13.0% when using duplex - nested pcr . We could not absolutely exclude the possibility of ado in the diagnosis of unaffected female embryo owing to the relatively higher ado . Thus, fish was simultaneously applied for the aneuploidy screening and sex selection to prevent the pregnancy of heterozygous female . In this study, we report the successful pgd and delivery of a healthy boy in a couple at risk of transmitting the otc deficiency . This was carried out by the simultaneous analysis of the duplex - nested pcr for a r320x mutation in the otc gene underlying the otc deficiency and y chromosome - specific loci, and fish for aneuploidy screening of chromosome x, y and 18 . This strategy could provide higher accuracy for the selection of genetically and chromosomally normal embryos in pgd for single gene defects.
Musculoskeletal stiffness and limited range of motion (rom) can restrict functional joint movement, particularly during ankle supination following neurological diseases1 . Ankle dorsiflexion rom restrictions impair dynamic balance and gait, which may contribute to secondary injuries; reduced ankle rom and tight calf muscles can cause poor gait, inefficient energy use, impaired balance, and an increased risk of falls2 . Musculoskeletal transformations manifesting as abnormal joint stiffness and limited rom result in numerous restrictions in the function and joint movements of patients3 . Limited passive ankle dorsiflexion rom during knee extension can alter foot positioning and result in compensatory foot movements, leading to an abnormal gait, which can result in ankle sprains and lower extremity overuse injuries4 . Studies have reported that balance - training programs that alter the somatosensory input, such as the use of an unstable surface to induce equalized weight distribution, can be helpful in hemiplegic patients5 . Kim et al . Used an ankle board with a slope in their center to achieve efficient ankle stretching and increased dorsiflexion6 . They suggested that stretching exercises using their device resulted in a greater increase in foot pressure compared to that with stretching exercises using existing inclined boards during ankle dorsiflexion stretching6 . Therefore, we investigated the effects of an unstable inclined board on the active and passive ankle rom in patients with ankle stiffness . The study included 10 female patients (mean age, 22.8 5.3 years; height, 158.9 3.0 cm; weight, 61.2 4.7 kg) with ankle stiffness caused by diseases or medical conditions . The patients were selected based on ankle stiffness scores obtained using a symptom checker tool . The patients were assigned randomly to two groups (both n = 5) and their height and weight were measured . The patients were not cognitively impaired and were capable of independent walking for 20 m without difficulty . Patients were excluded if they had a history of or current neurological conditions . Before participating, the purpose and methods of the study were explained to the patients, and all provided informed consent, according to the principles of the declaration of helsinki . Active and passive ankle dorsiflexion were measured using a standard 31.75 4.45 cm clear plastic goniometer, with one increment, a 360 scale, and a bubble level at each end . Group 1 performed the ankle dorsiflexion stretching exercise using a wooden inclined board, and group 2 performed ankle dorsiflexion stretching exercises using an air - cushioned inclined board that provided an unstable surface . Rubber sheets were attached to the inclined boards to prevent falls due to sliding . The patients performed ankle dorsiflexion stretching exercises for 5 min, with a 10-min rest between each set, five times a day for 1 week . The active and passive ankle dorsiflexion angles were measured bilaterally using the goniometer before and after the study . Independent and paired t - tests were used to assess within- and between - group differences in ankle dorsiflexion, with spss for windows (spss, chicago, il, usa). The amount of active and passive dorsiflexion did not differ between the two groups before the exercises, with active dorsiflexion angles of 18.2 6.4 and 19.3 4.2 and passive dorsiflexion angles of 23.4 5.5 and 22.6 5.7 in groups 1 and 2, respectively (both p> 0.05). The stretching exercises significantly (p <0.05) increased the active and passive ankle dorsiflexion angles in both groups compared to those at baseline . The active dorsiflexion angle was significantly increased (p <0.05) in group 2 compared to that in group 1 after the exercises . The active dorsiflexion angles before and after the exercises were 18.2 6.4 and 22.1 5.0 in group 1 and 19.3 4.2 and 28.1 6.9 in group 2, respectively . The passive dorsiflexion angles did not differ significantly between the groups after the exercises (p>0.05) and were 23.4 5.5 and 32.2 5.3 in group 1 and 22.6 5.7 and 35.1 6.0 in group 2 before and after exercising, respectively . The air - cushioned inclined board provided an unstable surface for performing ankle dorsiflexion stretching exercises . The results showed that ankle stretching exercises using the unstable inclined board significantly increased the active dorsiflexion angle compared to that with exercises using a wooden inclined board . We believe that the use of the air - cushioned inclined board provides somatosensory input and subsequently stimulates active ankle dorsiflexion by promoting automatic postural control . Sensory proprioception of the ankle and sensory compression of the plantar are important in the control of sway7 . An increase in the number of somatosensory inputs affects the muscles, and the use of an unstable support surface result in many different somatosensory inputs that limit compensatory action7 . Performing cognitive tasks in a standing posture reduces internal focus and induces external focus, which promotes automatic postural control8 . Stretching improve the flexibility of tendons, and changes in the mechanical condition of tendons depend on the type of stretching performed9 ., the passive dorsiflexion angles did not differ significantly between the two groups after ankle stretching exercises . However, stretching exercises significantly increased the active and passive ankle dorsiflexion angles in both groups compared to those at baseline . These results suggest that passive ankle exercises using a wooden inclined board only stretch muscle and tissue, while exercises using an unstable inclined board stretch muscle and tissue, and stimulate activation of the ankle dorsiflexor . Active ankle dorsiflexion was more effectively improved with ankle stretching exercises using an unstable inclined board than with exercises using a wooden inclined board.
Renal tubular involvement in sjgren's syndrome (ss) usually manifests with fanconi syndrome, distal (type 1) renal tubular acidosis (rta), nephrogenic diabetes insipidus, and hypokalemia ., we describe a case a young female with ss who presented with features of gitelman syndrome and hypokalemic paralysis . A 29-year - old female presented with weakness of both upper and lower limb weakness since 15 days . She had a history of dry eyes and dry mouth for the past 6 months . On examination, laboratory evaluation showed hemoglobin - 9.5 g / dl, platelet 2.95 lakh / mm . Blood urea nitrogen was 7 mg / dl, serum creatinine was 0.65 mg / dl, serum sodium was 134 meq / l, serum potassium was 1.81 meq / l and serum chloride was 84.3 urine chloride (spot) was 36.7 meq / l, urine creatinine (spot) 23.5 mg / dl, and urine calcium (spot) 1.23 mg / dl . This patient satisfied the american european consensus criteria for diagnosis of primary sjgren's syndrome (ss). Ten days after admission, she was discharged on spironolactone and prednisolone . On follow - up this patient presented with muscle weakness and her laboratory reports showed hypokalemia and metabolic alkalosis with normal blood pressure . Her urinary chloride levels were above 20 meq / l, which ruled out extra - renal causes of metabolic alkalosis . The presence of hypomagnesemia and absence of hypercalciuria (urinary calcium creatinine ratio <0.2) ruled out bartter syndrome . Hypokalemic alkalosis, high urinary chloride, hypomagnesemia, absence of hypercalciuria in background of no history of diuretic use favored the diagnosis of gitelman syndrome . Tubular involvement in ss is usually distal tubular dysfunction, type i (distal) renal tubular acidosis and nephrogenic diabetes insipidus . Proximal tubular abnormalities are less frequent, and rarely fanconi's syndrome has been reported in patients with ss . Acquired gitelman syndrome is relatively rare . To the best of our knowledge, only five cases of acquired gitelman syndrome have been reported in english literature so far . Among these five cases, four had ss, one was a case of chronic sialoadenitis . Acquired gitelman syndrome secondary to ss presenting with hypokalemic weakness is very rare . Only two cases have been reported so far . Though gitelman syndrome is an inherited disorder, given the paucity of reports, we believe that ss presenting as acquired gitelman syndrome may be relatively rare . Acquired gitelman syndrome should be considered in differential diagnosis of renal involvement in patients with ss.
Inadequate organization has been identified as an important cause of inappropriate courses of treatment for patients in the health care system [1, 2]. A response to this challenge has been to focus on integrated health care and co - ordination between the actors involved in the process [37]. An illustrative example of this can be found when looking at frail elderly patients on their discharge from hospital . Studies show that readmission can be diminished by increased collaboration between hospital, district nurses and general practitioners [8, 9]. Follow - up home visits by the district nurse and general practitioner to frail elderly patients after discharge have shown promising results [10, 11]. The aim of follow - up home visits to elderly and frail persons is to enable them to remain in their own home, avoid readmission to hospital, admission to nursing homes or other forms of sheltered housing and to improve the functional ability and general wellbeing of the elderly . A follow - up home visit comprises a home visit by a general practitioner (gp) and district nurse within about one week after discharge from hospital . The visit is expected to last approximately one hour, a relatively long period compared to other types of doctor - patient contact . This visit can be supplemented with two subsequent home visits or visits to the gp if this is considered necessary . The latest study from denmark has shown that the readmission rate in the intervention group was 40% while it was 53% in the control group . At the same time, studies have shown that there are indications that patients prefer primary care follow - up to specialist care . These results are overall convincing and, as a result, on - going work is being done by health authorities to test and further develop follow - up home visits as an initiative, including a programme in copenhagen carried out from 20082011 . The copenhagen programme was conducted as a randomized controlled trial which implies strict procedures for the implementation process . A total of approximately 300 patients from bispebjerg hospital (a major hospital in copenhagen) were included in the intervention and control group . Approximately 100 follow - up home visits to patients in the intervention group have been made . In the remaining approximately 200 cases in the intervention group it has not been possible to carry out a follow - up home visit, first of all because gps could not meet the requirements . In this feasibility study gps were not carefully selected for the programme as they were in an earlier study, and, as a result, more gps chose not to take part in the programme . The effect of the copenhagen programme will be reported in 2012 by the danish institute of health services research . This paper will focus on the implementation of follow - up home visits using a qualitative methodological approach . We know some of the general dynamics in the area but we do not know more precisely what the practical limitations of the home visitor's programmes are and how these limitations could make a difference to the outcomes assessed [14, 15]. Why do so few gps take part in the follow - up home visit programme? Why is the documented effect of such visits not motivation enough for gps to encourage them to do so? Addressing such questions could help us understand how to design and implement these visits in order to fully exploit the potential benefits of the intervention . Moreover, such results could generate useful hypotheses which could be addressed in future trials attempting to examine the effects of home visiting programmes . The aim of this study is to explore one particular aspect of follow - up home visits: the motivation and rationale of local care providers to invest time and effort in follow - up home visits . What is the main motivation of gps, district nurses and hospital staff to take part in a cross - sectoral activity like follow - up home visits? The case study is placed in a danish setting . In order to understand the organizational dynamics involved a short description of the setting is needed: the danish health care system is mainly a public system based on general taxation, and characterized by universal access to health care services . Gps have a central position in the system since they have a kind of gatekeeper function, they are usually the first professional confronted with the patient's problems and they see it as their responsibility to guide patients through the system . Gps are self - employed, general practices are small and typically privately owned; only a minority of gps are organized in health centres . Home care organizations (district nurses) have occupied an ever more central role since a major administrative reform in 2007 gave the local level the main responsibility for preventive health care . Co - ordination between organizational units in primary and secondary health care is determined by agreements between regional and municipality authorities, which include procedures for such cases as when frail and elderly people are discharged from hospital . In general, the danish system is considered quite integrated, in particular because the climate of trust in denmark makes co - operation possible [7, 16]. As a result, follow - up home visits and other cross - sector interventions, such as follow - home arrangements and case managers have been quite regularly implemented on an experimental basis as a possible alternative to hospital - based measures . The interventions have shown promising results but studies in the area also point to problems in connection with implementing the interventions [7, 8, 10, 12, 17, 18]. Empirical data were gathered to develop hypotheses and theoretical concepts . At the same time, however, a rough theoretical framework was used as an underlying foundation in the study to ensure that a relevant focus and approach were applied . Resource dependency theory tells us that network formation can largely be explained by an organization's need to reduce costs or gain resources and power . Organizations only work together when administrative and economic structures are designed which makes co - operation a profitable pursuit . Organizations thus typically follow their own agenda rather than involve themselves in cross - sectoral activities . In the health sector some support can be found for this basic claim . Studies have shown that network formation in general is limited and poor co - ordination predominant in the area [13, 21]. Resource dependency theory also tells us, however, that organizations need to reduce uncertainty . To reduce uncertainty, organisations tend to co - operate with organisations they trust and depend on to achieve common goals [19, 22]. Hence, in order to understand co - operation you also need to look at the question of trust and the quality of inter - organizational networks, as done in inter - organizational network theory . This perspective has shown us that we need to have a comprehensive view of the factors which make health sector organisations work together . Cross - sectoral programs and inter - organizational activities are not based purely on partners' own needs and resources [23, 24]. Motivation plays a key role in inter - organizational theory [22, 25]. Working together is a challenging task; it is typically regarded as uncertain whether it will bring benefits equivalent to the resources invested . Motivation and commitment to the process are therefore needed among the actors involved in order to keep things moving and overcome the uncertainties inherent in the process . What motivates organizations to work together? The literature points to two aspects in particular: the object needs to be seen as highly relevant and benefits should be obvious for all involved [22, 25]. This paper is based on an evaluation report made by one of the authors for health and social care, city of copenhagen . The interviews were structured around the following questions: to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant?how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits? Are the benefits obvious? To which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant? How do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits? Two focus groups and seven individual interviews were conducted in march may 2010, giving a total of 23 respondents . The focus group approach was chosen because the interaction between some of the key respondents (hospital staff, district nurses) was considered to be of importance, since a key goal in the study was to understand the shared views on collaboration among these key actors in the programme . One focus group interview was conducted with seven employees at bispebjerg hospital who were involved in the follow - up home visit programme . Recruitment criteria were designed in order to include a suitable spread of respondents, i.e. To ensure that members of all staff groups directly involved in the project were represented . We ensured that the project co - ordinator at the hospital responsible for the selection of patients for the programme was represented in the focus group . At the same time we also ensured that a doctor and nurses from the most relevant wards were represented . Another focus group interview was conducted with nine district nurses who had been involved in the project . Nurses from different districts in copenhagen were selected for the focus group in order to ensure that different ways of organising follow - up home visits were represented . Gps were not as motivated to take part in the research project as district nurses and hospital staff, so a less time - consuming method than focus groups was considered appropriate . As it turned out, in - depth interviews proved to be highly relevant for gps because gps had divergent and less firm views about follow - up home visits than other groups in the study and the methodological design allowed these views to be fully expressed . Five individual interviews were held with gps involved in the project . Both gps with a positive attitude towards follow - up home visits and gps with a less positive attitude were recruited to ensure that a wide range of attitudes was included in the study . These recruitment criteria were based on data registered by the city of copenhagen showing which gps had refused to participate in follow - up home visits and which had agreed . At the same time we ensured that the gps represented covered different districts in copenhagen . Two individual interviews were held with employees from referrals in the city of copenhagen home nursing services . There is a purchaser - provider split in the city of copenhagen, and the referrals purchase while the district nurses provides . The two referrals came from different districts and had different positions in the organization, ensuring that the most important experiences from this organization were covered . The focus group interviews took place in a neutral meeting room arranged by the project manager, and the in - depth interviews took place in the gps' offices . The focus group interviews lasted between 90 and 120 minutes, and the in - depth interviews between 25 and 60 minutes . Both were recorded with a digital voice recorder and transcribed verbatim . The interview transcripts were analyzed under the two headings which form the basic empirical questions in the study: relevance of collaboration and benefits of collaboration . Quotes from the interviews were grouped under each heading in order to find the quotes which were most illustrative of the views of gps, district nurses and hospital staff . To ensure reliability, results were presented and discussed in a steering group with representatives from the main professional groups involved in the programme (gps, district nurses and hospital staff). A guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme . Gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case . The main elements of the guide are: a medication review, a general health assessment (including a number of relevant tests), and an assessment of the need for follow - up arrangements (home care, rehabilitation etc . ). The gps generally see the follow - up home visit as a relevant type of contact . A gp said: it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp ., it can give the patient extra resources when you visit the patient on the patient's home ground . The district nurses also generally see the follow - up home visits as a relevant type of contact . The gps find that the medication review is an especially important element in follow - up home visits . A gp said: we should be able to see what type of medication the patient has . But the patient may have been given some additional medicine by the hospital that we are not aware of, and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes . At the same time the gps also stress that follow - up home visits are not only about reviewing medication: the purpose of a follow - up home visit is first of all about making a patient review . It is not only the medicine we should look at, but the patient in this sense follow - up home visits are very relevant . The district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient: the guide creates a good structure for the home visit . The doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are . The two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . As a result, the relationship between the gp, the district nurse and the patient is typically not further developed after the first home visit . At the hospital the general view was also that the content of the follow - up home visit was relevant . At the same time, however, several respondents expressed the need for co - ordination between the hospital and the prime sector: the guide is quite voluminous . We have already made a lot of the tests at the hospital so there is no need to repeat these at home . You should make sure that the gp has read the discharge summary before the follow - up home visit the target group of the programme is elderly and frail patients just out of from hospital . A number of specific criteria have been formulated: the patient has to be 65 years or older, been in specific wards at bispebjerg hospital, have returned to his or her own home, have experienced deteriorating health, have a limited social network, and experienced many readmissions . At the hospital a nurse at the hospital expressed it this way: it is typically this type of patient that we see again and again: deteriorating health and a small social network . The gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients . Other patients would have benefited from a follow - up home visit but they were not included in the programme gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged, ideally within three days . This form of communication works fine but is, according to gps and district nurses in the study, not sufficient when it comes to elderly and frail patients: it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged . A follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc . A gp said: the hospital has contact with the patient at the time of discharge . In the present situation there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group . It is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems: if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital . But it is often difficult to find out who is responsible and it is difficult to sort out the problem (gp). The problem, according to several respondents, is that nobody is in charge of this transition phase and, as a result, communication problems occur: we need an anchor with overall responsibility for the patient when the patient is discharged from hospital . A fax from the hospital to the home nursing services is not enough (doctor, hospital). In the city of copenhagen the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them . It is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having: follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility . The study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and, as a result, they could not be very outreaching . Today i find we often do not hear what is going on in the daily life of our patients . The district nurses know more what is going on than we do . The health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits, and other forms of information have also been used . These activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them . Such information is essential since follow - up home visits are new to many gps . The study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low: i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . In these cases i received a reprimand and was told that it was certainly not his [the gp's] duty to participate in such a visit the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened . A gp said: you get to talk that is the biggest gain, to get the dialogue . We work in two parallel organizational systems . By not having the dialogue we as gps miss some observations . The gps want, however, more information about the efficacy of follow - up home visits . Gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health: if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits . Information could be better (gp). At the hospital the staff involved in the programme a nurse at the hospital said: follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment . It is really nice to know that there is this form of support when patients get home . Communication about the implementation and results of follow - up home visits could, however, be better according to hospital staff . Doctors and nurses at the hospital miss feedback from the follow - up home visits, as illustrated in the following quote: at meetings we have often asked for the good story. It would make it much more meaningful for us if we were told that mrs . That is the most annoying that we are not told what the results of follow - up home visits are a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme . Gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case . The main elements of the guide are: a medication review, a general health assessment (including a number of relevant tests), and an assessment of the need for follow - up arrangements (home care, rehabilitation etc . ). The gps generally see the follow - up home visit as a relevant type of contact . A gp said: it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp ., it can give the patient extra resources when you visit the patient on the patient's home ground . The district nurses also generally see the follow - up home visits as a relevant type of contact . The gps find that the medication review is an especially important element in follow - up home visits . A gp said: we should be able to see what type of medication the patient has . But the patient may have been given some additional medicine by the hospital that we are not aware of, and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes . At the same time the gps also stress that follow - up home visits are not only about reviewing medication: the purpose of a follow - up home visit is first of all about making a patient review . It is not only the medicine we should look at, but the patient in this sense follow - up home visits are very relevant . The district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient: the guide creates a good structure for the home visit . The doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are . The two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . As a result, the relationship between the gp, the district nurse and the patient is typically not further developed after the first home visit . At the hospital the general view was also that the content of the follow - up home visit was relevant . At the same time, however, several respondents expressed the need for co - ordination between the hospital and the prime sector: the guide is quite voluminous . We have already made a lot of the tests at the hospital so there is no need to repeat these at home . You should make sure that the gp has read the discharge summary before the follow - up home visit the target group of the programme is elderly and frail patients just out of from hospital . A number of specific criteria have been formulated: the patient has to be 65 years or older, been in specific wards at bispebjerg hospital, have returned to his or her own home, have experienced deteriorating health, have a limited social network, and experienced many readmissions . At the hospital a nurse at the hospital expressed it this way: it is typically this type of patient that we see again and again: deteriorating health and a small social network . The gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients . Other patients would have benefited from a follow - up home visit but they were not included in the programme gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged, ideally within three days . This form of communication works fine but is, according to gps and district nurses in the study, not sufficient when it comes to elderly and frail patients: it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged . A follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc . A gp said: the hospital has contact with the patient at the time of discharge . There is not much direct communication between the hospital and the gp after the discharge of a patient in the target group . It is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems: if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital . But it is often difficult to find out who is responsible and it is difficult to sort out the problem (gp). The problem, according to several respondents, is that nobody is in charge of this transition phase and, as a result, communication problems occur: we need an anchor with overall responsibility for the patient when the patient is discharged from hospital . A fax from the hospital to the home nursing services is not enough (doctor, hospital). In the city of copenhagen the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them . It is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having: follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility . The study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and, as a result, they could not be very outreaching . Today i find we often do not hear what is going on in the daily life of our patients . The district nurses know more what is going on than we do . The health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits, and other forms of information have also been used . These activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them . Such information is essential since follow - up home visits are new to many gps . The study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low: i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . In these cases i received a reprimand and was told that it was certainly not his [the gp's] duty to participate in such a visit the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened . A gp said: you get to talk that is the biggest gain, to get the dialogue . The gps want, however, more information about the efficacy of follow - up home visits . Gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health: if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits . The staff involved in the programme is very positive towards follow - up home visits . A nurse at the hospital said: follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment . It is really nice to know that there is this form of support when patients get home . Communication about the implementation and results of follow - up home visits could, however, be better according to hospital staff . Doctors and nurses at the hospital miss feedback from the follow - up home visits, as illustrated in the following quote: at meetings we have often asked for the good story. It would make it much more meaningful for us if we were told that mrs . That is the most annoying that we are not told what the results of follow - up home visits are the analysis illustrates that it is a big challenge to co - ordinate activities across organizational boundaries and motivate actors to participate in cross - sectoral health programs . Resources clearly matter (fees for gps etc .) But the paper also points to other key factors which need to be taken into consideration when trying to motivate partners to engage actively in a cross - sectoral programme like a follow - up home visit programme . In the following these key factors will be discussed under three sub - headings: the focus of collaboration, a partnership of equals, and the will to co - operate . The analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits . The guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient's overall situation are followed by all professionals involved . As a result, gps and district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards, a type of procedure unusual for the health sector . Collaboration requires partners to subscribe to common standards, and the fixed structure of follow - up home visits is a powerful way of defining these standards . The medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient's intake of medicine . The study indicates that the gp gains a very realistic picture of the patient's medicine intake during the home visit, a picture the gp could not as easily have gained in a different setting . As a result, communication between the gp and the district nurse about a patient's medicine is typically improved in the treatment following the visit . This is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [6, 23]. The results also showed that follow - up home visits aimed at the right target group, and this motivated care providers to take part in the intervention . Studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits, and that is a notion shared by most gps and district nurses in the program . Thus, the results indicate that the object at the centre of these efforts the focus of collaboration needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits . Inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [18, 26, 27]. Providers in the health sector typically have different incentives, different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts . They are responsible for contacting the gp, organizing the visit, and co - ordinating the activities following the visit . The results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care, a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [6, 8]. This is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results . Gps generally did not feel they had a central role in follow - up home visits and, as a result, felt no responsibility for them . Moreover, follow - up home visits break with gps' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse . This is time - consuming and, from a narrow cost - benefit analysis, probably not as rewarding as other activities in general practice . As a result, a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part . Hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but, despite this essential role, they do not see how they contribute to the overall goals of the programme . The interviews showed that hospital staff lacks information about the actual visits, and how this type of intervention fits into the treatment given at the hospital . The main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could, as a result, focus more explicitly on what is most needed . This form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and, as a result, increase the motivation to make these visits . It is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated . If the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers . There needs to be a shared responsibility a partnership of equals to engage all partners in a cross - sectoral activity like follow - up home visits . Research has shown that an interorganizational relationship is at its most effective when it is in equilibrium, a state in which the organizational network is balanced in terms of roles and functions [22, 29]. Research has also shown, however, that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes . For gps in particular the partnership of equals in follow - up home visits is clearly not equal enough . They are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating . The interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals . Gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice . Danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units, like health centres, and it typically requires an extra effort to motivate gps to take part in a larger scheme, like a follow - up home visit programme . The results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership . Hospital staff is given no feedback after follow - up home visits, and need a clearer notion of how this type of intervention fits into the treatment given at the hospital . As hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular, the need in the secondary health sector for information about the treatment given in the primary sector is growing . District nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . In order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values, a recommendation also found in other inter - organizational studies in the area [20, 26, 31]. Basically, the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . In particular there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach . Follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . In particular, there has been a lack of opportunity and incentives for collaborative partners to learn by working together . The learning potentials in integrated care for older people are big, and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated . The analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits . The guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient's overall situation are followed by all professionals involved . As a result, gps and district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards, a type of procedure unusual for the health sector . Collaboration requires partners to subscribe to common standards, and the fixed structure of follow - up home visits is a powerful way of defining these standards . The medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient's intake of medicine . The study indicates that the gp gains a very realistic picture of the patient's medicine intake during the home visit, a picture the gp could not as easily have gained in a different setting . As a result, communication between the gp and the district nurse about a patient's medicine this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [6, 23]. The results also showed that follow - up home visits aimed at the right target group, and this motivated care providers to take part in the intervention . Studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits, and that is a notion shared by most gps and district nurses in the program . Thus, the results indicate that the object at the centre of these efforts the focus of collaboration needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits . Inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [18, 26, 27]. Providers in the health sector typically have different incentives, different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts . They are responsible for contacting the gp, organizing the visit, and co - ordinating the activities following the visit . The results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care, a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [6, 8]. This is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results . Gps generally did not feel they had a central role in follow - up home visits and, as a result, felt no responsibility for them . Moreover, follow - up home visits break with gps' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse . This is time - consuming and, from a narrow cost - benefit analysis, probably not as rewarding as other activities in general practice . As a result, a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part . Hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but, despite this essential role, they do not see how they contribute to the overall goals of the programme . The interviews showed that hospital staff lacks information about the actual visits, and how this type of intervention fits into the treatment given at the hospital . The main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could, as a result, focus more explicitly on what is most needed . This form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and, as a result, increase the motivation to make these visits . It is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated . If the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers . There needs to be a shared responsibility a partnership of equals to engage all partners in a cross - sectoral activity like follow - up home visits . Research has shown that an interorganizational relationship is at its most effective when it is in equilibrium, a state in which the organizational network is balanced in terms of roles and functions [22, 29]. Research has also shown, however, that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes . For gps in particular the partnership of equals in follow - up home visits is clearly not equal enough . They are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating . The interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals . Gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice . Danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units, like health centres, and it typically requires an extra effort to motivate gps to take part in a larger scheme, like a follow - up home visit programme . The results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership . Hospital staff is given no feedback after follow - up home visits, and need a clearer notion of how this type of intervention fits into the treatment given at the hospital . As hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular, the need in the secondary health sector for information about the treatment given in the primary sector is growing . District nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . In order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values, a recommendation also found in other inter - organizational studies in the area [20, 26, 31]. Basically, the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . In particular there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach . Follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . In particular, there has been a lack of opportunity and incentives for collaborative partners to learn by working together . The learning potentials in integrated care for older people are big, and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated . The findings support a basic claim in inter - organizational theory that other factors than resources matter if we want to understand what drives efforts at collaboration . This analysis shows that the focus of collaboration needs to be clearly defined and agreed upon, there needs to be a high degree of equality between the professionals involved, and there has to be a will to co - operate based on a shared understanding of values and learning potentials . One lesson for current policy is that motivational factors need to be addressed in future collaborative programs in order to fully exploit the potential health benefits . The follow - up home visit programme in copenhagen suffered from a lack of motivation among key actors (especially gps) to participate and, as a result, readmissions to hospital can be expected to be higher than anticipated prior to the programme . Susanna bihari axelsson, senior lecturer, associate professor nordic school of public health, box 12133, nya varvet building 25, se-402 42 gteborg sweden janne seemann, assistant professor, phd, department of sociology and social work, aalborg university, denmark j.d.h . Van wijngaarden, dr, assistant professor, institute of health policy and management, erasmus university rotterdam, p.o.
Alzheimer s disease (ad) is a progressive neurodegenerative disorder starting with mild memory loss and manifesting in severe cognitive decline . Neuropathologically, the disease is characterized by an extensive deposition of amyloid (a) peptides in extracellular amyloid plaques and by intracellular neurofibrillary tangles (nfts) of hyper - phosphorylated tau protein . Sporadic late - onset ad is the most common form of dementia in the elderly and is strongly associated with age . In rare cases (<5%), ad is inherited and results in an early disease onset . One genetic risk factor for late - onset ad that has been reported throughout many studies is apolipoprotein e (apoe) which physiologically functions as a ligand in receptor - mediated endocytosis of lipoprotein particles [2, 3]. Several single nucleotide polymorphisms in apoe lead to alterations in the coding sequence and result in three common isoforms called apoe2, apoe3 and apoe4 with the e4 allele being an ad risk factor and e2 being protective [46]. Strong evidence suggests that apoe influences ad via its effect on a metabolism; however, the details of this process have to be fully elucidated . Positional cloning studies of familial ad (fad) cases have identified mutations in three genes, amyloid precursor protein (app), presenilin 1 and 2 (ps1, ps2), which are tightly linked to the generation of a peptides . Proteolytic processing of amyloid precursor protein (app) by bace (-site app cleaving enzyme) is followed by cleavage by -secretase containing presenilins and results in the release of a peptides of different length . A40 and a42 (40 or 42 amino acids, respectively) are the most prominent a peptides and since the c - terminus has important implication for toxicity, the longer a42 peptide exhibits stronger neurotoxic properties . The fad - linked mutations cause an elevated production of amyloidogenic a, which is the basis for the amyloid hypothesis of ad, stating that a peptides are the key - players in neuronal dysfunction and subsequent neurodegeneration in ad . Recently, it has been suggested that the soluble oligomeric forms of a42 are the potentially harmful species and that these oligomers affect various important cellular mechanisms, resulting in decreased functionality and survival of neuronal cells . However, a peptides are not exclusively generated under pathological conditions as they are part of the normal cellular metabolism and a significant load of amyloid plaques is also observed in brains of healthy aged individuals [10, 11]. This challenges the amyloid hypothesis of ad and up to date the nature of the disease - relevant alteration in aged neurons remains unclear . Although alois alzheimer already described protein deposits in the brain of demented patients, the question why distinct proteins that are potentially causative for the disease accumulate in discrete brain regions in the course of aging remains unanswered . Today, we know that the progressive deposition of misfolded and aggregation - prone proteins in defined regions of the nervous system is not unique to ad, but a well - described characteristic of several neurodegenerative disorders, including parkinson s disease (pd), huntington s disease (hd) and amyotrophic lateral sclerosis (als). Recently, it has been proposed that the accumulation of disease - relevant proteins in aggregates might actually even be considered as a protective mechanism to dispose these proteins and remove them from the cellular metabolism [13, 14]. Generally, within the crowded cellular environment, proteins are always at risk for denaturation . Changing genetic and environmental factors, as during pathology or aging, challenge the integrity of the cellular protein homeostasis (proteostasis). Therefore, the stability and metabolism of the proteome needs to be controlled carefully and is carried out by a complex network of several hundred evolutionarily conserved proteins . This network, including molecular chaperones, the ubiquitin proteasome system (ups) and the autophagy system is stringently regulated and indispensable for the maintenance of proteostasis . All main players of this network are ubiquitously expressed and consequently also present in neuronal tissue . Molecular chaperones, most be prominently represented by the heat shock protein 70 (hsp70) family and their regulators, are responsible for correct folding and refolding of proteins as well as for the transport of misfolded proteins to the protein - degradation systems . The chaperone network is controlled by the activity of transcription factors, e.g. Heat shock transcription factor 1 (hsf1), which rapidly adjusts the cellular chaperone capacity during stress conditions . The ups is the major protein - degradation machinery of the cell and controls the metabolism of cytosolic proteins and the degradation of misfolded proteins . Proteins that are destined for proteasomal degradation become specifically tagged with the protein ubiquitin in a well - organized enzymatic cascade . Interestingly, also a ubiquitin - independent way of proteasomal degradation has been described recently that mostly involves small and misfolded proteins . As an additional mechanism for protein degradation the major autophagic systems described so far are macroautophagy and chaperone - mediated autophagy (cma). Macroautophagy is a complex process that involves the formation of a membrane structure, the autophagosome, which engulfs bulk cellular material (proteins and organelles) and subsequently fuses with the lysosome . Cma involves the constitutively expressed heat shock cognate 70 (hsc70) that targets client proteins with a specific consensus motif directly to the lysosome . In fact, hsp70 is also an important factor to assure selectivity of macroautophagy of degradation - prone proteins in a distinct pathway, mediated via the co - chaperone bcl2-associated athanogene 3 (bag3). Aging and disease challenge the proteostasis network and the accumulation of instable proteins results in wide - spread protein aggregation [21, 22]. Our discussion presented here attempts to integrate two major factors that are associated with neurodegeneration, protein homeostasis and aging . Most importantly, we focus on the role of specific pathways which are needed to restore and maintain proteostasis during aging and their interplay with a special focus on impaired proteostasis in ad, which may add to a novel view and understanding of ad pathogenesis . Since recently many excellent reviews on various aspects providing in depth discussions have been presented, we are concentrating here on the integration of these different aspects . Therefore, we frequently refer to review work with the recommendation of further reading rather than trying to fully cover the discussed issues by their original publications . Cells permanently encounter the problem to maintain the integrity and functionality of the proteome . Within the crowded cellular environment, the correct conformation of proteins must be controlled and misfolded and irreversibly damaged proteins must be efficiently refolded or removed . Central players of the protein homeostasis system are molecular chaperones that sense misfolded proteins and, when refolding fails, direct them to the protein - degradation pathways . Molecular chaperones are specified as proteins that interact with and participate in folding or refolding of non - native proteins . Therefore, chaperones help unfolded proteins to achieve their functional conformation without being present in the final structure [15, 2327]. They exert a multitude of activities, including de novo folding, refolding of denatured proteins, transport to subcellular compartments, oligomeric assembly and disposal by proteolytic degradation [28, 29]. Different classes of chaperones work together to form co - operative networks and are often termed heat shock proteins (hsps), because they are sensitively up - regulated under stress conditions in which the amount of misfolded proteins is increased . Chaperones, which are involved in protein folding and refolding, such as hsp70, hsp90 and chaperonin (hsp60), promote folding activity through atp / cofactor - regulated binding and release cycles [15, 31]. They recognize short hydrophobic amino acid stretches of misfolded proteins and can co - operate with atp - independent chaperones, e.g. Small hsps (shsps), to prevent protein aggregation . Hsp70 proteins are central players in proteostasis control and increasing hsp70 levels prevent protein aggregation in various disease models that are based on the expression of aggregation - prone proteins [32, 33]. The activity of hsp70 is atp dependent and is controlled by chaperones of the hsp40 family as well as nucleotide exchange factors (nefs) [34, 35]. After atp hydrolysis, a nef binds to the atpase domain of hsp70 and catalyzes the nucleotide exchange, which results in substrate release . Thus, chaperone binding to the hydrophobic region of misfolded proteins transiently blocks aggregation and the subsequent atp - dependent release allows controlled folding of the client protein . Hsp40/dnajs, hsc70-interacting protein (hip), bcl2-associated athanogene 1 (bag1) and bag3 are prominent co - chaperones that control the atp - dependent activity of hsp70 . Dnaj proteins generally enhance the atpase activity of hsp70, but their action can lead to different fates of their bound substrates . Dnajb1 is an example for a dnaj protein that supports substrate folding, whereas dnajb2-bound clients are degraded by the proteasome . The co - chaperone hip stabilizes the adp - bound state of hsp70 and co - operates with hsp70 in protein folding . Other hsp70-binding cofactors are the ubiquitin ligases carboxy terminus of hsc70-interacting protein (chip) and parkin, which provide a link between hsp70, co - chaperones and the ups, resulting in client ubiquitination and degradation by the proteasome [34, 38, 39]. Recently, it has been shown that bag3 interacts with hsp70 and directs the chaperone and its substrates into the autophagic pathway, providing an association of hsp70, co - chaperones and autophagy . An extensive description of the hsp70 machinery, the exact structure - function relationship between hsp70 and its various cofactors, has been excellently reviewed elsewhere . The functional variability of the chaperone system that is exerted by a multitude of single proteins highlights the complexity of this proteostasis network . However, limited knowledge exists on the exact composition and capacity of the chaperone system within a cell or a certain tissue . A general view assumes that total levels of cellular chaperones exceed the actual requirements and that a sufficient amount of chaperones is free of clients . Thus, the network has excess capacity to initially deal with sudden additional chaperone requirements, as exhibited during stress conditions . An opposite view proposes that the capacity of the chaperone network is always closely titrated to the actual demand of chaperone activity and that the system is otherwise rapidly adapted to conditions of increased requirements . The analysis of the chaperone capacity of neuronal and muscular tissue of caenorhabditis elegans (c. elegans) demonstrates that these tissues indeed display different folding activities and that neurons are particularly sensitive to protein denaturation during heat stress . However, the adaptation of the chaperone network during aging is critical as it is well acknowledged that protein aggregation and disruption of proteostasis are characteristic for aged cells . The periodical application of mild heat stress decreases mortality in drosophila melanogaster and c. elegans, which is mediated by hsp70 activity and the overexpression of hsp70 in c. elegans increases the life span of the nematode [4345]. Several reports have analyzed chaperone protein or mrna levels in aged cells and found increased or basal amounts, whereas the stress - mediated induction of chaperone expression is impaired . The transcription of chaperone genes in response to stress conditions is controlled by the transcription factor hsf1, which shows an impaired dna - binding potential in aged cells . A reduced activity of hsf1 in c. elegans results in a shortened life span and, conversely, the enhanced expression of the transcription factor increases the life span . Activity is also essential for the extended life span of the extremely long - lived daf-2/insulin / igf-1 receptor mutants of c. elegans [47, 48]. Thus, hsf1 and chaperone activity can promote longevity, demonstrating a clear association of chaperones, proteostasis and aging . Some of the factors mentioned so far are involved in linking chaperone functions with cellular protein - degradation pathways, the ups and autophagy, for the removal of misfolded proteins . Besides protein aggregation, one factor that induces ubiquitination is protein damage caused by free radical oxygen species (ros) and oxidative stress . Most likely, irreversible oxidation may activate chaperones and the ups to induce protein repair of misfolded proteins and lead to ubiquitination and protein degradation . During aging, in particular, the mitochondrial respiratory chain is strongly linked to the production of ros and as one consequence may cause protein dysfunction, apoptosis, necrosis, aging and disease [49, 50]. Protein oxidation leads to a change in protein conformation and function and chaperones may sense such changes and in turn activate the ups as a quality control system . Depending on the degree of oxidation, irreversible oxidation and loss of protein function may lead to degradation and/or accumulation of damaged proteins and to the formation of so - called aggresomes, which display a high autophagic activity [51, 52]. The ups is a complex enzymatic pathway that starts with the ligation of ubiquitin, a 76-amino - acid - long and highly conserved protein, to other cellular proteins and thus labels them for degradation . Initially the c - terminal end of ubiquitin is activated by atp - dependent phosphorylation and formation of a thiol ester via an activating enzyme, e1 . It is then transferred to a thiol group of an ubiquitin - carrier protein, e2 . The e3 ligase directs ubiquitin from e2 to an -amino group of the target protein [53, 54]. The c - terminus of an additional ubiquitin protein can be ligated onto one of the seven lysine residues within the attached ubiquitin molecule . For degradation via the proteasome ubiquitin linkages between either the c - terminus and lysine residues k48 or k63 are the major recognition signals for proteasomal degradation . Ubiquitin chains also occur among other lysine residues, whereas ubiquitin extension via k6 is associated with dna repair, k11 with endoplasmatic reticulum - associated protein degradation and cell cycle regulation, k27 with ubiquitin fusion degradation, k29 with lysosomal degradation and k33 with kinase modification . Monoubiquitination can modify the activity of the protein transport machinery and when attached to transmembrane proteins can serve as a sorting signal to direct their movement between different cellular compartments [5659]. The polyubiquitinated proteins destined for degradation are processed by the multienzymatic proteasome complex: first they become deubiquitinated and then degraded by the 26s proteasome, a system that is composed of various proteasome subunits . The eukaryotic 20s proteasome core complex consists of four heptameric rings comprising two classes of seven non - identical but homologous subunits . The outer rings contain alpha - type subunits with gating function for substrate entrance and product release, while the beta - subunits exhibit peptide hydrolyzing activity . The 19s regulatory complex binds to the 20s catalytic core in a flexible manner and consists of six atpases, forming a ring at the entrance of the core and exerting chaperone - like activity . The ups is the major degradation system in the cell for the degradation of short - lived, misfolded and defective proteins . An accumulation of polyubiquitinated proteins is reported for numerous neurological disorders, which suggests that ups dysfunction plays a prominent role in the pathogenesis of neurodegenerative diseases and moreover in aging . Since proteasomal activity decreases during aging over all, the degradation demand of the cell increases, which results in an age - associated induction of the autophagy pathway that delivers substrates for degradation ultimately to lysosomes [20, 63]. Autophagy is negatively regulated via the evolutionarily conserved enzymatic mammalian target of rapamycin complex 1 (mtorc1). In turn, if mtor activity is inhibited by rapamycin, the autophagy pathway is switched on . Rapamycin exerts its stimulatory effect on autophagy by preventing mtor phosphorylation at ser-2448 and thereby subsequently blocks mtor signaling . So far, many different autophagic systems have been described including macroautophagy and cma that form the main pathways . Macroautophagy is a multi - step process by which cytosolic material is sequestered into a double - layered membrane structure, the autophagosome, and is delivered to the lysosome for degradation . It is a highly orchestrated process and involves autophagy - related (atg) proteins . More than 30 genes have been identified in genetic analyses of yeast mutants which have defects in autophagic function . After autophagy stimulation through mtor inhibition, the formation of phagophores (autophagosome precursors / pre - autophagosomal structures) is initiated in a not yet completely understood mechanism . A subset of atg proteins is required for autophagosome formation: first, the atg9 system including atg9, the atg1 kinase complex (atg1 and atg13), atg2 and atg18; second, the phosphatidylinositol 3-oh kinase (pi(3)k) complex consisting of vacuolar protein sorting (vps)34, vps15, beclin 1/atg6 and atg14 and third, the ubiquitin - like protein (ubl) system composed of two ubl proteins (lc3/atg8 and atg12), an e1 enzyme (atg7), two analogues of e2 ubiquitin - conjugating enzymes (atg10 and atg3), an lc3/atg8-modifying protease (atg4), the protein target of atg12 attachment (atg5) and atg16 . Beclin 1/atg6 regulates autophagic trafficking and membrane trafficking in a variety of physiological and pathological processes . Microtubule - associated protein 1 light chain 3 (lc3/atg8) is commonly used as a marker for autophagic activity . With its e1-like activity, atg7 converts cytosolic lc3-i to the membrane - associated lc3-ii, which then is conjugated to phosphatidylethanolamine . Lc3-ii binds the inner and outer membrane of the forming autophagosome and provides a physical link between the autophagosome and lc3 interacting proteins, such as sequestosome 1 (p62/sqstm1) or neighbor of brca1 gene 1 (nbr1). These so - called autophagy receptors can simultaneously bind to ubiquitinated proteins and lc3, employing their lc3-interacting motif and ubiquitin - associated domain . The identification of autophagy receptors, such as p62/sqstm1 and nbr1 has provided a molecular link between ubiquitination and autophagy . Thus, p62/sqstm1 and nbr1 are possibly also important bridging factors between ups and autophagy . Through self - oligomerization, which is stimulated by ubiquitin binding, p62/sqstm1 sequesters ubiquitinated substrates in form of inclusion bodies . These inclusions are then specifically engulfed by the autophagosome membrane by recruiting lc3 [69, 70]. While macroautophagy is considered as a rather unspecific robust degradation process, cma is a highly selective lysosomal pathway that removes a distinct subset of proteins containing a pentapeptide lysosome - targeting motif . These substrates can on the one hand directly be translocated into the lysosome after docking to the lysosomal receptor lysosomal - associated membrane protein 2a or on the other hand unfolded by a chaperone complex containing hsc70 and the co - chaperones bag1, hip, hsp70hsp90 organizing protein (hop) and hsp40/dnajb1 . These co - chaperones provide a direct link to the ups and the folding and refolding activity of molecular chaperones . In the course of aging, a variety of proteins tend to aggregate and impair the ups directly . Therefore, these proteins, most of which are ubiquitinated, cannot be handled by the proteasome and have to be degraded by different protein clearance pathways . Recently, it has been shown that the cellular protein quality control (pqc) of polyubiquitinated proteins by proteasomal and autophagic systems is regulated by the hsp70 co - chaperones bag1 and bag3, respectively . Proteasome activity decreases in an age - dependent manner in a cell model of replicative senescent human fibroblasts and is associated with an increased autophagic activity in aged cells . This correlates with decreased levels of bag1 and increased levels of bag3 in aged cells, an age - related bag1/bag3 switch that serves as an induction control of the macroautophagic pathway (bag3-mediated selective macroautophagy) and may be considered as a backup of the ups in pqc ([40, 72]). Therefore, the expression shift from bag1 to bag3 during aging, but also upon acute stress (e.g. Proteasome inhibition, oxidative stress), can be considered as a physiologically important adaptive response [20, 40]. The bag3-mediated selective pathways have been shown to be involved in a variety of disease causing processes . In a model for hd, bag3 in concert with hspb8 facilitates the disposal of polyq43-huntingtin by stimulating macroautophagy and this complex is also involved in z - disc maintenance in flies, mice and men [73, 74]. Additionally, the transport of target proteins to the aggresome, a compartment with high autophagic activity, has been shown to be mediated by bag3 . This aggresome - targeting pathway involving bag3 and hsp70 is distinct from other before - described mechanisms as it does not depend on substrate ubiquitination . Taken together the molecular chaperone machinery, the ups and the autophagy system are involved in pqc and maintaining proteostasis within aging and disease to prevent protein misfolding and aggregation . A summary of the main routes for protein degradation is shown in fig . 1 . As aggregated proteins are found in ad patients and aging is a prevailing risk factor for ad it is important to further characterize how these pathways are regulated and how misregulation possibly contributes to the pathology of ad.fig . 1the ubiquitin proteasome system (ups) and various autophagy routes as the main pathways for protein degradation (regular turnover) and of misfolded and aggregated proteins (and mitochondria) the ubiquitin proteasome system (ups) and various autophagy routes as the main pathways for protein degradation (regular turnover) and of misfolded and aggregated proteins (and mitochondria) ad is characterized by the accumulation of intracellular a (oligomers), extracellular high molecular weight deposits of a peptides (fibrilliar forms), and the intracellular aggregation of hyper - phosphorylated tau . Indeed, modification of the cellular proteostasis and the metabolism of a and tau have been proven to cause neuronal dysfunction and cell death . The malfunctioning proteostasis control results in the accumulation of misfolded and aggregated proteins, which is a general hallmark of aging and neurodegeneration, as observed in pd, hd, als and ad . Aging in particular is characterized by decreasing proteostasis capacity and increasing protein damage which are in combination a major challenge for the cell due to the inability to maintain metastable proteins in folded states . To counteract a cascade of protein destabilization caused by metastable proteins which escaped the pqc the proteostasis network including molecular chaperones, the ups and autophagic pathways must manage the increasing burden of protein misfolding to maintain proteome stability . Since aging is the major risk factor of ad [7880], the age - dependent loss of proteostasis might be an important contributor to the pathology of ad . An overview of proteins that affect proteostasis in ad is shown in table 1.table 1proteins affecting proteostasis in alzheimer s diseaseproteinfunctionreferencea40/42decreases proteasome activity, modulates autophagy through mtor signaling[99102, 104, 143146]tauinhibitory effect of tau aggregates on proteasome activityhsf1induces app gene during stress[81, 82]grp78 (er isoform of hsp70)modulates app maturation and reduces a40/42 secretion[84, 85]hsps (hsp22, 27, 70, 90)small hsps (hsp22, hsp27) bind to fibrillar amyloid plaques and inhibit their fibrillarisation; hsp70, hsp90 inhibit early stages of amyloid aggregation[88, 89]chipe3 enzyme for phosphorylated tau[93, 117]bag1regulates proteasomal degradation of tau together with hsp70ps1increases production of a42, essential for lysosomal proteolysis and autophagy by enabling the acidification of lysosomes required for protease activation[163, 165]ubb+1potently inhibits the degradation of polyubiquitinated substrates and therefore induces neuronal cell death[120, 121]uch - l1dub needed for proteasomal degradation of client proteins, oxidized and down - regulated in ad[124, 125]ubqln1ubqln1 activity is necessary to regulate the production of app and app fragments[127, 128]mtorinhibitory and/or activating modulation of mtor through a42 but not a40[143146]beclin1beclin1 is down - regulated in ad brains and consequently increases app levels and its metabolites[161, 162]a amyloid beta, app amyloid precursor protein, hsf1 heat shock transcription factor 1, grp78 glucose - related protein 78, hsp heat shock protein, chip carboxy terminus of hsc70-interacting protein, bag1 bcl2-associated athanogene 1, ps1 presenilin 1, uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1, ubqln1 ubiquilin 1, mtor mammalian target of rapamycin, dub deubiquitination enzyme, er endoplasmic reticulum proteins affecting proteostasis in alzheimer s disease a amyloid beta, app amyloid precursor protein, hsf1 heat shock transcription factor 1, grp78 glucose - related protein 78, hsp heat shock protein, chip carboxy terminus of hsc70-interacting protein, bag1 bcl2-associated athanogene 1, ps1 presenilin 1, uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1, ubqln1 ubiquilin 1, mtor mammalian target of rapamycin, dub deubiquitination enzyme, er endoplasmic reticulum the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements (hse) via the transcription factor hsf1 . Interestingly, promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [81, 82]. As app is a membrane - associated protein it maturates in the endoplasmic reticulum (er) and the golgi apparatus . There, the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 (grp78, the er isoform of hsp70) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [84, 85]. The cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing . In addition to the er and golgi - associated hsps, the cytosolic chaperones are as well linked to app and the a metabolism . The shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization . Moreover, hsp70 and hsp90 inhibit early stages of a aggregation [88, 89]. Hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [90, 91]. In ad brains, furthermore, extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1 day and decreased the amount after 3 days in vitro . These observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance . Besides extracellular a accumulation, the second major hallmark of ad is the intracellular aggregation of tau . Post - translational modifications of tau, e.g. Hyper - phosphorylation, affect the conformation of the protein and promote the aggregation state . These post - translational modifications impact the interaction of tau with microtubules, and thus, there may be specific forms of tau that are preferred chaperone substrates relative to others . Hsp27, hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [9294]. Hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [94, 95]. Protein levels of soluble tau positively correlate with hsp27, hsp40, hsp90, alphab - crystallin and chip levels in ad brains . Vice versa hsp protein levels are inversely correlated with levels of tau oligomers, which are an intermediate of tau filaments . In different cellular models, it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding . Tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [97, 98]. Collectively, these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . The ubiquitin proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro . A40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity . A42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [99102]. It still has to be elucidated which form of a is now affecting proteasome function . There is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . In a cell free proteasome activity assay, it has been shown that indeed a40 and a42 oligomers, but not monomers, decreased proteolytic activity of the proteasome in a dose - dependent manner . There is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes . Consistent with these results, it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level . So far, it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . In cultured neurons, it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . In normal and ad brain, a42 has also been shown to accumulate predominantly in multivesicular bodies, indicating that these structures are the possible interaction site of a and the proteasome in vivo . Further supporting the influence of the ups on a metabolism thus, a decrease in proteasome activity would increase -secretase activity and thereby a production . This illustrates a clear association between a peptides and the proteostasis network, highlighting the importance of chaperones and the ups in a metabolism . The protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [108111]. Furthermore, mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau, which are the recognition signals for degradation via the ups [112114]. It has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity, suggesting an inhibitory interaction between tau aggregates and proteasome activity . In addition, in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome . In contrast, non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity . These data show that different aggregation states of tau modify its turnover via the proteasome . It has been shown that tau co - immunoprecipitates with chip, an e3 ubiquitin ligase, that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [93, 116, 117]. In ad, the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip . In addition to phosphorylation, tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau . Moreover, changes in the combination of proteasome subunits have been reported in ad brain, resulting in an altered proteasome activity . Taken together, these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function . Ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein (ubb+1). Overexpression of this mutant, which contains a c - terminal amino acid extension, leads to an impairment of the proteasome and induces neuronal cell death [120, 121]. Oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome . It is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 (uch - l1) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [122, 123]. Uch - l1 functions as a deubiquitination enzyme (dub) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation . Interestingly, when overexpressed, uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [124, 125]. Several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis . For example, tau seems to be a substrate of the ups and of the autophagic system in vivo . Recently, it has been shown that truncated tau (tauc) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups . In addition, tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery, which in turn interacts with the ups - related proteins bag1 and chip, both known to be involved in tau degradation . Taken together, these data indicate that decreased ups function may be involved (or even partially causative) for ad pathogenesis . This view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 (ubqln1). Ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [127, 128]. Interestingly, mutations in another member of the ubiquilin family, ubiquilin 2 (ubql2), are associated with the motor neuron disease als, which supports a general role of the ups in neurodegeneration . In fact, already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase . These structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles (avs) [131, 132]. These histological changes reflect either an increased synthesis of components of the lysosomal system, a disturbed clearance of avs, or both, as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [133135]. A general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition . Neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system, because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission . Furthermore, post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime, cellular aging and disease progression, which highlight the importance of an effective degradation system in this particular cell type . Besides changes of the autophagic system on a histological level, also molecular regulators of autophagic degradation are altered during ad progression . Genes that represent negative regulators of autophagy are repressed, whereas positive modulators are more likely to be up - regulated in the brain of ad patients . However, enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease, resulting in an accumulation of undegraded proteins . The most prominent physiological modulator of mtor signaling is caloric restriction (cr) meaning the energy content of food is reduced without compromising the supply of essential nutrients . Attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes, but may also rely on cytoprotective effects of mtor - mediated autophagic activity [141, 142]. Interestingly, a42, but not a40, affects mtor signaling and is as well affected by mtor modulation itself . The discussion, if a silences or enhances mtor signaling is still ongoing: some studies show inhibitory effects of a on mtor signaling, while others show an activation [143146]. In a murine neuroblastoma cell line (n2a) and hippocampal slice cultures exposed to a42, in appswe / ps1, a double - transgenic mouse model for ad, and in lymphocytes of ad patients, the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [144, 145]. Furthermore, in the tg2576 app - overexpressing mice, the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation . In contrast, cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin . Similar results have been seen in triple transgenic ad mice (3tg - ad mice), combining a and tau - pathology of ad, which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment . Interestingly, the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients, but not in others . The beneficial effect of rapamycin treatment and, therefore, autophagy stimulation on learning and memory is also reported for a further ad mouse model . These animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin . Interestingly, already in 1992, haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments (app - ctfs) which are found in lysosomes . Later, alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease, where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs, indicating an impaired lysosomal flux . This suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism, which does not directly involve macroautophagy . The accumulation of sphingolipids, which characterizes lysosomal lipid storage disorders, has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity . Thus, sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . Pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner . Consequently, after inhibition of autophagy, a significant accumulation of a peptides and app - ctfs has been observed . The upcoming data support the involvement of autophagy in the clearance of a and app - ctfs . The autophagy receptor p62/sqstm1 shows a reduced expression in ad patients, as well as in 3tg - ad - expressing mice . This might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter, which leads to reduced p62/sqstm1 transcription [151, 152]. Because guanine is the nucleobase which is most susceptible to oxidation, the p62/sqstm1 promoter is exceptionally prone to oxidative stress, as it is rich of gc regions [153, 154]. Interestingly, tau is degraded via the autophagic - lysosomal system, whereby tau aggregates are cleared via macroautophagy . As tau possesses two putative cma - targeting motifs, soluble tau is most likely degraded via cma . Hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice, indicating an interplay of oxidative stress and the clearance of protein aggregates in ad . Another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [157160]. Beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment . In addition, mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes . Moreover, the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux . However, the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [161, 162]. Recently, it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a . Ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . In neurons lacking ps1, the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane . This subunit is needed for correct function of the proton pump, which is responsible for lysosomal acidification . -secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er, which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . As a result, v01a is entrapped in the er and cells loose lysosomal function . In addition, an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines . The first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease . Taken together, all these findings suggest an important role for autophagy in the pathogenesis of ad . The two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements (hse) via the transcription factor hsf1 . Interestingly, promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [81, 82]. As app is a membrane - associated protein it maturates in the endoplasmic reticulum (er) and the golgi apparatus . There, the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 (grp78, the er isoform of hsp70) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [84, 85]. The cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing . In addition to the er and golgi - associated hsps, the cytosolic chaperones are as well linked to app and the a metabolism . The shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization . Moreover, hsp70 and hsp90 inhibit early stages of a aggregation [88, 89]. Hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [90, 91]. In ad brains, furthermore, extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1 day and decreased the amount after 3 days in vitro . These observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance . Besides extracellular a accumulation, the second major hallmark of ad is the intracellular aggregation of tau . Post - translational modifications of tau, e.g. Hyper - phosphorylation, affect the conformation of the protein and promote the aggregation state . These post - translational modifications impact the interaction of tau with microtubules, and thus, there may be specific forms of tau that are preferred chaperone substrates relative to others . Hsp27, hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [9294]. Hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [94, 95]. Protein levels of soluble tau positively correlate with hsp27, hsp40, hsp90, alphab - crystallin and chip levels in ad brains . Vice versa hsp protein levels are inversely correlated with levels of tau oligomers, which are an intermediate of tau filaments . In different cellular models, it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding . Tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [97, 98]. Collectively, these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . The ubiquitin proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro . A40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity . A42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [99102]. It still has to be elucidated which form of a is now affecting proteasome function . There is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . In a cell free proteasome activity assay, it has been shown that indeed a40 and a42 oligomers, but not monomers, decreased proteolytic activity of the proteasome in a dose - dependent manner . There is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes . Consistent with these results, it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level . So far, it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . In cultured neurons, it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . In normal and ad brain, a42 has also been shown to accumulate predominantly in multivesicular bodies, indicating that these structures are the possible interaction site of a and the proteasome in vivo . Further supporting the influence of the ups on a metabolism thus, a decrease in proteasome activity would increase -secretase activity and thereby a production . This illustrates a clear association between a peptides and the proteostasis network, highlighting the importance of chaperones and the ups in a metabolism . The protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [108111]. Furthermore, mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau, which are the recognition signals for degradation via the ups [112114]. It has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity, suggesting an inhibitory interaction between tau aggregates and proteasome activity . In addition, in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome . In contrast, non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity . These data show that different aggregation states of tau modify its turnover via the proteasome . Particularly intriguing it has been shown that tau co - immunoprecipitates with chip, an e3 ubiquitin ligase, that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [93, 116, 117]. In ad, the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip . In addition to phosphorylation, tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau . Moreover, changes in the combination of proteasome subunits have been reported in ad brain, resulting in an altered proteasome activity . Taken together, these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function . Ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein (ubb+1). Overexpression of this mutant, which contains a c - terminal amino acid extension, leads to an impairment of the proteasome and induces neuronal cell death [120, 121]. Oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome . It is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 (uch - l1) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [122, 123]. Uch - l1 functions as a deubiquitination enzyme (dub) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation . Interestingly, when overexpressed, uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [124, 125]. Several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis . For example, tau seems to be a substrate of the ups and of the autophagic system in vivo . Recently, it has been shown that truncated tau (tauc) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups . In addition, tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery, which in turn interacts with the ups - related proteins bag1 and chip, both known to be involved in tau degradation . Taken together, these data indicate that decreased ups function may be involved (or even partially causative) for ad pathogenesis . This view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 (ubqln1). Ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [127, 128]. Interestingly, mutations in another member of the ubiquilin family, ubiquilin 2 (ubql2), are associated with the motor neuron disease als, which supports a general role of the ups in neurodegeneration . In fact, already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase . These structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles (avs) [131, 132]. These histological changes reflect either an increased synthesis of components of the lysosomal system, a disturbed clearance of avs, or both, as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [133135]. A general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition . Neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system, because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission . Furthermore, post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime, cellular aging and disease progression, which highlight the importance of an effective degradation system in this particular cell type . Besides changes of the autophagic system on a histological level, also molecular regulators of autophagic degradation are altered during ad progression . Genes that represent negative regulators of autophagy are repressed, whereas positive modulators are more likely to be up - regulated in the brain of ad patients . However, enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease, resulting in an accumulation of undegraded proteins . The most prominent physiological modulator of mtor signaling is caloric restriction (cr) meaning the energy content of food is reduced without compromising the supply of essential nutrients . Attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes, but may also rely on cytoprotective effects of mtor - mediated autophagic activity [141, 142]. Interestingly, a42, but not a40, affects mtor signaling and is as well affected by mtor modulation itself . The discussion, if a silences or enhances mtor signaling is still ongoing: some studies show inhibitory effects of a on mtor signaling, while others show an activation [143146]. In a murine neuroblastoma cell line (n2a) and hippocampal slice cultures exposed to a42, in appswe / ps1, a double - transgenic mouse model for ad, and in lymphocytes of ad patients, the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [144, 145]. Furthermore, in the tg2576 app - overexpressing mice, the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation . In contrast, cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin . Similar results have been seen in triple transgenic ad mice (3tg - ad mice), combining a and tau - pathology of ad, which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment . Interestingly, the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients, but not in others . The beneficial effect of rapamycin treatment and, therefore, autophagy stimulation on learning and memory is also reported for a further ad mouse model . These animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin . Interestingly, already in 1992, haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments (app - ctfs) which are found in lysosomes . Later, alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease, where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs, indicating an impaired lysosomal flux . This suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism, which does not directly involve macroautophagy . The accumulation of sphingolipids, which characterizes lysosomal lipid storage disorders, has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity . Thus, sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . Pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner . Consequently, after inhibition of autophagy, a significant accumulation of a peptides and app - ctfs has been observed . The upcoming data support the involvement of autophagy in the clearance of a and app - ctfs . The autophagy receptor p62/sqstm1 shows a reduced expression in ad patients, as well as in 3tg - ad - expressing mice . This might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter, which leads to reduced p62/sqstm1 transcription [151, 152]. Because guanine is the nucleobase which is most susceptible to oxidation, the p62/sqstm1 promoter is exceptionally prone to oxidative stress, as it is rich of gc regions [153, 154]. Interestingly, tau is degraded via the autophagic - lysosomal system, whereby tau aggregates are cleared via macroautophagy . As tau possesses two putative cma - targeting motifs, soluble tau is most likely degraded via cma . Hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice, indicating an interplay of oxidative stress and the clearance of protein aggregates in ad . Another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [157160]. Beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment . In addition, mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes . Moreover, the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux . However, the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [161, 162]. Recently, it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a . Ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . In neurons lacking ps1, the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane . This subunit is needed for correct function of the proton pump, which is responsible for lysosomal acidification . -secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er, which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . As a result, v01a is entrapped in the er and cells loose lysosomal function . In addition, an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines . The first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease . Taken together, all these findings suggest an important role for autophagy in the pathogenesis of ad . There is quite some evidence that in ad, the proteostasis network is impaired . Based on pathological analysis, ad mouse models, in vitro and cellular investigations a molecular link between chaperones, the ubiquitin proteasome system, autophagy pathways and pathogenetic mechanisms of ad can be suggested . Deregulation and changes of chaperones and proteasome activity might have serious implications for aging as well as for age - associated diseases . Autophagy pathways are key mechanisms and of vital importance for cellular function and, especially, for cell survival under adverse conditions . Consequently, an effect of proteostasis control on neurodegeneration or, vice versa, of neurodegeneration on proteostasis is reasonable . As with other pathomechanisms that are investigated in the search for the cause of ad, it is still open whether an impairment of proteostasis is an upstream or downstream event during ad onset and progression . Experimental approaches employing mouse models clearly demonstrate that stabilization or induction of proteostasis can be neuroprotective . Whether this can be translated into the human condition and, most importantly, whether supporting proteome integrity can be a real target for pharmacological intervention for the prevention and treatment of ad is currently still open . Also, one has to consider that, e.g. The stimulation of general autophagy may in the long term lead to the stimulation of proliferation of non - neuronal cells, eventually resulting in tumor formation . Indeed, a permanent autophagy induction is known as one cellular mechanism that promotes the escape of cells from proliferation control . As beneficial it may be to support autophagy in post - mitotic neurons that are confronted with disturbed proteostasis and an impairment of proteasome function, this stimulation should target specific autophagy pathways, such as selective chaperone - mediated macroautophagy involved in the degradation of disease - associated protein aggregation . Although there are obviously still many questions to be answered to understand the role of proteostasis in ad (and also in other age - associated neurodegenerative disorders), it is a big step forward in ad research to consider a possible role of pathogenetic pathways that are not directly linked to the usual suspects a and tau . A better understanding of how the proteostasis network is regulated might help to identify targets which lead to prevention of the deleterious loss of neuronal cells and tissue in ad.
The union rate and hardware - related complications remain a major concern in these patients, especially following multilevel spinal fusion surgeries . Developing an adjunct therapy that essentially supplements the mainstay surgical management is of great importance to these patients . Multitudinous research to gain insight into use of teriparatide (134 recombinant human parathyroid hormone) for enhancing bone formation in osteoporosis is readily available in the literature . Its action as a direct anabolic agent in bone tissue, consistently increases bone mineral density and thus improves skeletal architecture . Since its availability in market, indications are unravelling and is being attempted in many conditions including fracture healing, dental stability, hypoparathyroidism, and hypocalcaemia . Another evolving field of interest is the role of teriparatide for enhancing bone graft union and osseo - integration of implants . It is said to accelerate bone graft union and reduce the incidence of pedicle screw loosening after lumbar spinal fusion surgeries . We intended to study this effect and analyze if teriparatide can be an effective adjunct therapy to achieve solid posterolateral fusion mass and reduce the incidence of subsequent screw loosening after multilevel instrumented lumbar fusion surgeries in elderly patients . An open - labeled, nonplacebo controlled, retrospective, single center study is formulated by shortlisting 62 elderly patients (male 9; female = 53) between the age group of 65 to 78 years (mean age standard deviation [sd] = 70.1 3.7), who underwent instrumented lumbar fusion for various degenerative pathologies over a period of time . All of them had received multilevel lumbar fixation (3 levels or 4 vertebral segments) with pedicle screw constructs and postero - lateral intertransverse fusion with a combination of local bone graft and allograft . The prime indication for surgery was chronic unresolving low back pain, with or without neurological symptoms, associated with any one of the following conditions: a)multisegmental spinal instability (spondylolisthesis grade 1),b)multilevel degenerative spondylosis with or without segmental instability, andc)degenerative scoliosis with or without segmental instability . Multisegmental spinal instability (spondylolisthesis grade 1), multilevel degenerative spondylosis with or without segmental instability, and degenerative scoliosis with or without segmental instability . We excluded: a)those patients operated with oligo - level spinal fusions (2 levels or 3 vertebral segments).b)those with 1 or more osteoporotic vertebral compression fractures among the fused levels.c)those with previous surgery in the same level.d)those in whom infection or tumor was an indication for surgery.e)those with 1 or more potential data missing . Those patients operated with oligo - level spinal fusions (2 levels or 3 vertebral segments). Those with 1 or more osteoporotic vertebral compression fractures among the fused levels . Those with previous surgery in the same level . Those in whom infection or tumor was an indication for surgery . Those with 1 or more potential data missing . Selection was irrespective of individual bone mineral density (bmd) status, as bmd scoring was not done as a part of routine preoperative evaluation in patients undergoing multilevel spinal fusions . Our sample included elderly patients 65 years who are expected to have detoriating osteogenesis potential . Associated medical comorbidities included diabetes mellitus (dm) and bronchial asthma (ba) in 25 and 8 patients, respectively . A mixture of fresh frozen allograft and local bone chips were used as graft . Local bone consisted of spinous process, lamina, and facets from the decompression site . Occasionally, additional interbody cages were used at insufficient discs especially at the caudal end of the construct to increase stability . Following surgery, patients were given an option of postoperative teriparatide use and opting to it was solely based on their own decision . In total, 30 patients who accepted for long - term daily use of teriparatide were in the experimental group and remaining 32 patients were in the control group . Teriparatide (self - administered; 20 mcg sc injection) was started for those patients in the experimental group from the first postoperative day, once daily using delivery device . The control group received similar standardized and accepted treatment protocols as those in the experimental group except for the use of postoperative teriparatide . All patients were instructed to take calcium and vitamin d supplements every day following surgery . Outcome analysis was done using radiological data which included antero - posterior (ap), conventional lateral view, and dynamic stress lateral view radiographs taken at 12 months follow - up (figs . 1 and 2). (c) mri images showing severe degeneration of disc levels and canal stenosis from l1l5 . (d) 12 months follow - up ap view x - ray which was considered as solid fusion (lenke type 1). Ap = antero - posterior, mri = magnetic resonance imaging . Radiological outcome illustration of a random case from the control group . (a) (b) preoperative dynamic stress (extension and flexion) lateral view radiographs showing degeneration from l3s1 segments . (c) mri images showing severe degeneration of disc levels and canal stenosis from l3s1 . (d) 12 months follow - up ap view x - ray showing bilateral signs of pseudo - arthrosis interpreted as nonunion (lenke type 4). They divide postero lateral fusion mass into 4 grades based on the appearance in an ap radiograph . Grade a represents definitive fusion with bilateral thick bony masses, grade b represents probable fusion with unilateral thick bony mass and thin bony mass on other side, grade c represents no probable fusion with thin bony mass on the one side and pseudo - arthrosis on the other side, grade d represents no fusion with thin bony mass on both the sides showing obvious pseudo - arthrosis or resorption of graft bilaterally . We interpreted grade 1 and grade 2 of lenke's criteria as solid fusion which is explained as unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebra showing continuous trabeculation in an ap radiograph . Although most of the patients had long fixations, segmental cobb's angle change involving the upper and lower ends of the construct as seen in dynamic (flexion and extension) stress lateral views will also be considered as nonunion . Screw loosening was determined when there was occurrence of radiolucency (halo sign) around the pedicle screws in either ap or lateral view radiographs . Change in the screw position noticed in dynamic stress lateral view radiographs will additionally be considered as screw loosening, which may occur at the cephalic and caudal ends of the construct . Results were tabulated and statistical analyses were carried out using graph pad prism 5 (graphpad software inc ., san diego, ca). Analyses were done using unpaired t test for continuous variables and fisher's exact test for categorical variables . Probability values of less than 0.05 were considered statistically significant . Written informed consent was obtained from all patients including acceptance of daily usage of teriparatide delivery device from those in the experimental group . This study was approved by the institutional review board of chang gung memorial hospital with irb registry no - 201600800b0 and was performed in accordance with the ethical standards stated in the most recent version of the 1964 declaration of helsinki . Sixty - two elderly patients (age = 6578 years; mean = 70.11 years; male = 9, female = 53) are divided into 2 groups based on teriparatide administration following surgery . Group 1 or the experimental group (n = 30; mean age sd = 69.8 3.8 years) was those in whom teriparatide was administered and group 2 (n = 32; mean age sd = 70.4 3.6 years) was the control group . The presence of concomitant medical comorbidities in group 1 (dm = 14; ba = 4) and group 2 (dm = 11; ba = 4) was matched . The demographic variation in characteristics of the groups is statistically insignificant (table 1). Surgical parameters of the experimental group (fusion levels = 137; pedicle screws = 269) and the control group (fusion levels = 144; pedicle screws = 283) had no statistically significant variance . Duration of teriparatide administration averaged 7.4 2.4 months in the experimental group . Follow - up data were available for a mean duration of 25.4 months for those in the experimental group and 27 months for those in the control group . Even though follow - up was done for a longer duration, analyzing outcome further after 1 year may not be related to teriparatide use . Considering the duration of administration of teriparatide, radiological outcome analysis was decided to be done at 1 year follow - up . None of our patients missed the 1st year follow - up and required x - rays were available in our database for all the patients . Radiological outcome analysis was done using ap, lateral, and dynamic stress lateral view radiographs taken at 12 months follow - up . Two or more observers concurred stating that there was unilateral or bilateral bridging bone formation across the transverse process of adjacent vertebras showing continuous trabeculation confirming solid fusion in 20 patients (66.7%) of the teriparatide group and 16 patients (50%) in the control group . Nonunion was noted in 10 patients (33.3%) of teriparatide group and 16 patients (50%) in the control group . The 16.7% advantage procured with teriparatide use was not statistically significant (p = 0.20) (table 2). Screw loosening was calculated comparing the number of screws showing signs of loosening to total number of screws in each group . The incidence of screw loosening was 13.4% in the teriparatide group and 24.4% in the control group . This difference was statistically significant with p = 0.001 (table 2). Elderly patients with chronic unresolving symptoms due to degenerative spine disease are prone to have poor surgical outcomes . They also predispose for more hardware - related complications, especially following long spinal fusions . Considering the possibility of poor outcomes, treating these conditions cannot be ignored but rather a potential solution needs to be developed which effectively supplements the mainstay surgical management . It is said to play a reasonable role in treatment of osteoporosis . Apart from being used in osteoporotic individuals, insights into its action on many other conditions are unraveling . Animal studies yielded promising results for teriparatide usage to enhance postero - lateral fusion mass in rat models . In human studies relating to teriparatide, most authors studied its effect only in osteoporotic individuals . In our sample, patients were between 65 and 78 years of age and are more susceptible to have poor bmd . Patients had comorbidities such as dm and bronchial asthma that have negative influence over bmd . Dm is said to reduce bmd at a higher rate in elderly women, even if the baseline bmd was high . Along with postero - lateral fusion, additional interbody cages were used especially at very insufficient discs often seen in the caudal end of the construct . This step also allows the graft to access the pluripotent stem cells of the bone marrow . Since a large amount of graft is required for long fusions, there usually is a lack of host bone . Static and dynamic radiographs are used for radiological assessment of spinal fusion by many authors, but computerized tomography (ct) is considered more accurate . Most of the radiographic criteria developed to assess fusion mass consider movement in dynamic radiographs as a main component for assessment . They lack reliability as the absence of motion does not confirm fusion, especially in the case of long instrumented levels . Based on ap radiographs, we considered grade a (definitive fusion; bilateral thick bony mass) and grade b (probable fusion; unilateral thick bony mass) described by lenke et al as solid fusion . Fusion masses that did not favorably appear to have definitive or probable fusion were considered as nonunion . We also considered change in segmental cobb's angle, visualized in dynamic stress lateral view radiographs as an additional criterion to interpret as nonunion . Formation of a radiolucent zone (halo sign) around the pedicle screws seen in conventional ap / lateral view radiographs and change in screw position seen in dynamic stress lateral view radiographs were considered definitive indicators for screw loosening . Teriparatide is said to have potential to increase the quality of lumbar spine bone marrow and pedicle cortex and thus reduces the incidence of pedicle screw loosening . It is even believed to increase the insertional torque of pedicle screws when administered from at least 1 month prior to surgery . Long - term teriparatide use can be associated with significant improvement in bone microarchitecture and trabeculation . It is also said that; more than 6 months of daily injection facilitates effective bone union following postero - lateral fusion . Considering this, we administered teriparatide for a mean duration of 7.4 months and none of the patients had any drug induced complications . Most of the similar studies in literature included only short segment postero - lateral fusions . Our study is foremost to discuss the effect of teriparatide on multilevel posterolateral fusions in elderly patients . According to our preliminary results, the percentage of patients achieving solid fusion following teriparatide use was found to be more than that of the control group . Besides that, use of teriparatide effectively reduced the incidence of subsequent screw loosening which proved to be statistically significant . Our study evaluates the radiological outcome following postoperative teriparatide administration in patients undergoing multilevel instrumented lumbar fusion surgery . Even though percentage of solid fusion was more among patients who used teriparatide, the difference was not statistically significant . However, teriparatide was more significantly influential in reducing the incidence of subsequent pedicle screw loosening . Being a retrospective study, there are few methodological shortcomings that may have influenced our outcome . The lack of preoperative bmd data, variation in surgical indications, and differential treatment durations of teriparatide administration may have biased the sample . Use of ct as an evaluation tool to assess bone graft union could have been more empirical . Considering the selected sample size, the study may be underpowered to draw potential conclusions.
According to the world health organization's recent update, diabetes, hypertension, and obesity are one of the top five continuing risk factors for cardiovascular deaths in the world . Obesity is increasing substantially and is one of the major contributors of disease prevalence due to its pathophysiological link to other cardiovascular risks such as hypertension and diabetes . It is estimated that, in 2010, 6.4% of adults would have diabetes mellitus affecting 285 million in the world and it will increase to 7.7% by 2030, affecting 439 million adults . Of special note is that there will be a 67% increase in the prevalence of diabetes in developing countries from 2010 to 2030 . Metabolic syndrome (ms) is a constellation of overweight / obesity, hypertension, and disturbances of lipid and carbohydrate metabolism . The definition of ms was debated for a long time to produce a standardized clinical criterion . The world health organisation describes ms as the presence of type 2 diabetes or impaired glucose tolerance with any two of the following characteristics: obesity, high levels of triglycerides, low levels of high - density lipoprotein, and hypertension . The international diabetes federation (idf) takes central obesity as a prerequisite for the diagnosis of ms with the association of any two of the other factors, that is, high blood pressure, abnormal blood glucose, high levels of triglycerides, and low levels of high - density lipoprotein . Also, the idf has derived specific reference values for central obesity for different ethnicities . The national cholesterol education programme (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel, or atp, iii), the national heart, lung and blood institute, and the american heart association have released a report on the criteria for diagnosing and managing ms . The panel describes ms as the presence of any three of the following: abdominal obesity, dislipidemia (high levels of triglycerides, low hdl), increased blood pressure, and elevated fasting glucose . For the purpose of this paper, the atp iii and idf's definitions are used and compared . Each component of ms is a known risk factor for the development of type 2 diabetes, atherosclerosis, and coronary artery disease (cad). People with ms are 310 times more likely to develop cardiovascular disease commensurate with a high risk of morbidity and mortality [5, 6]. Central obesity, one of the components of ms, predicts the occurrence of diabetes and overall cardiovascular risk . The ncep atp iii states that ms is equal to cigarette smoking as a contributing factor for premature cardiovascular disease . The prevalence of metabolic syndrome is increasing all over the world with different regions having individual clusters of epidemic risk factors [6, 8], and in particular there is evidence of a high prevalence of ms and diabetes in south asians . Substantial increase in the prevalence of type 2 diabetes in asia in recent years has raised serious concerns about cardiovascular consequences for these populations [5, 10]. However, in developing countries, many of these subclinical conditions are not diagnosed until the onset of complications such as myocardial infarction or stroke . It is essential to initiate early detection of these chronic diseases in underdeveloped countries in asia, such as nepal, so that preventative action can minimize the consequences . This study aims to establish the prevalence of hypertension, diabetes, obesity, and metabolic syndrome in the participants of a major health screening programme in nepal . This study also aims to establish the relationship between the components of ms and lifestyle of the participants . Nepal is one of the poorest countries of the world at the 136th position of human development index . The subjects were the participants of the programme for detection and management of chronic kidney disease, hypertension, diabetes and cardiovascular disease, a community - based screening programme in eastern nepal . In this community - based programme a series of community awareness programmes were conducted in a specific locality with the help of local leaders, medical students, and community volunteers . Various screening centres such as permanent centers (in health clinics, community centers, etc .) And temporary screening centers (in schools, clubs, houses of worship, and private homes) were used to screen the population . Each center used a group of five to seven people as community volunteers and consisted of a local leader (priest, administrator, school teachers, and local political leaders), a laboratory technician, and nurse . Medical students (approximately 100 in number) and nursing students (around 25) assisted the community volunteers . Prior to screening, the community volunteers went from door to door to record the number of family members residing permanently and to inform the members of the family, about the need of the project . All people of 20 years were invited to come to a predefined place in very close vicinity to their house . Pregnant or menstruating women at the time of analysis, people with a fever or acute illness, and those who had recently engaged in heavy exercise were excluded . The research team also collected general information on the participants' demographic data, diet, smoking, alcohol consumption, and physical activity . The data recorded included family and medical history for kidney disease, high blood pressure, diabetes, cardiovascular disease and any current medication or treatment . Blood pressure was measured by the auscultatory method with a random zero mercury sphygmomanometer and standard cuff (12 34 cm). The blood pressure measurement was taken in the seated position, quietly in a chair with feet on the floor and an arm support at the heart level . Hypertension was defined according to the guidelines of the seventh report of the joint national committee on prevention, detection, evaluation and treatment of high blood pressure, that is, systolic blood pressure 140 mm hg or diastolic blood pressure 90 mm hg and/or concomitant use of antihypertensive medications . Body weight and height were assessed with all subjects standing without shoes and heavy outer garments to the nearest 0.1 kg and 1 cm, respectively . Waist circumference was measured over light clothing at a level midway between the lower rib margin and the iliac crest in centimetres rounded up to nearest 0.5 cm . Abdominal obesity is defined as an abdominal circumference> 102 cm (40 in) in males and> 88 cm (35 in) in females for ncep criteria and> 90 cm in males and> 80 cm in females for idf criteria for south asians . Plasma glucose concentration was determined by the glucose oxidase - peroxidase method (vitalab selectra-2, merck, germany). The diagnosis of diabetes was defined by either casual plasma glucose 200 mgdl associated with symptoms of diabetes and on fasting samples plasma glucose 126 mgdl . Individuals with self reported, prior physician - diagnosis of diabetes were classified as having previously diagnosed diabetes . Serum lipids that include total cholesterol, high - density lipoprotein (hdl), low - density lipoprotein (ldl), and triglycerides (tg) were also measured (vitalab selectra-2, merck, germany). The results from any person having a history of hypertension or found to have hypertension were verified by qualified doctors . The biochemical tests were completed in semiautomatic analysers (microlab 300, vital scientific, the netherelands). Epidata refers to a group of applications used in combination for creating documented data structures and analysis of quantitative data . In this study, the idf and ncep atp iii's criteria for metabolic syndrome were used to calculate and compare the frequency of metabolic syndrome . The relationships between the prevalence of cardiovascular risk factors, demographic details, lifestyle, and physiological test results were analysed using the spearman correlation test . Further, the differences in the categorical variables were examined using chi - squared test . Odds ratios (ors) and their 95% confidence interval were calculated using binary logistic regression (for gender and age) and multinomial logistic regression (for life style factors). In total, 14,425 people, aged 20100 (mean age 41.4 15.1), were included in the study . Among them, the percentage of education level is illustrated in accordance to the number of years in education (15 years the participants were divided into four categories according to their work: labourer / farm, office, house, and none / unknown . Participants' physical activities were defined according to the time spent every day on physical activity as> 60 min, 3060 min, <30 min / day, and none . Abdominal obesity was observed in 11.5% (n = 1607/14002) of the participants as per ncep criteria (mean waist circumference: male107.38 6.19 cm, female94.84 5.84 cm) and in 34.7% (n = 5006/14418) of the participants as per idf criteria . According to the revised bmi, 10.6% (n = 1534/14423) were underweight (bmi <18.5), 28.2% (n = 4065/14423) were overweight (bmi = 2224.9), and 32.5% (n = 4689/14423) were obese (bmi> 25). Diabetic prevalence was 6.3% (889/14008) of which 4.8% (n = 673/14008) were under treatment . A figure of 12.3% (n = 1718/14009) had a family history of diabetes . Hypertension was observed in 33.9% (n = 4894/14422) of the participants (mean systolic 138.72 18.03 mm hg and mean diastolic 93.09 8.45 mm hg). Only 12.9% (1812/14009) were previously diagnosed, and 8.5% were receiving treatment for hypertension . A history of coronary artery disease was present in 1.6% (n = 218/14007), and 1% (n = 142) were under treatment for ischemic heart disease or stroke . Table 2 shows the goodness of fit for the prevalence of obesity, hypertension, and diabetes . Prevalence of hypertension showed no difference from these data, and obesity showed only a small difference . The percentages of the participants who had abnormal lipid profile that includes total serum cholesterol, serum ldl cholesterol, serum hdl cholesterol, serum triglycerides are listed in table 3 . There were 2191 sets of data eligible to meet the criteria for metabolic syndrome . Ms was observed in 22.5% (n = 494/2191) of the participants according to the idf criteria and 20.7% (454/2191) according to the ncep criteria . The percentages of individual ms risk factors among the total participants and the participants with ms are illustrated in figures 1 and 2 . Generally, among the total participants and the specific participants with ms, the presence of abnormal lipids was higher than the other factors defining ms . However, the presence of abdominal obesity was higher among ms participants using idf criteria (figure 2). The females had a higher prevalence of ms than males . According to the ncep criteria, the age groups 4160 and 6180 had a higher prevalence of ms than the lower age group . According to idf criteria, the age groups 4160 and 2040 had a higher prevalence of ms . The participants who worked at home had a high incidence of ms according to both the criteria used . The sedentary group had a higher incidence of ms than the participants who were physically active . The univariate correlations between cardiac risk factors are shown in table 5, and the chi - squared independence of them in the metabolic syndrome prevalence is listed in table 6 . The prevalence of ms (ncep scores) had a significant positive relationship with education levels and physical activity . There were significant positive correlations between physical activity and the three individual ms components: high glucose (r = 0.03, p <.01), high bp (r = 0.04, p <.01), and low hdl (r = 0.23, p <.01). There was no correlation between physical activity and the other two ms components: high triglyceride (r = 0.003, p>.05) and abdominal obesity (r = 0.003, p> the ncep scores had a positive correlation between the family history of diabetes (r = 0.83, p <.01) and hypertension (r = 0.115, p <.01). Although a number of these correlations show high levels of significance, the common variance is extremely low, suggesting that the sample size is having a major impact on the significance . As a result of this table 7 lists the chi - squared independence, odds ratios, and confidence intervals in the association between age, gender, and specific lifestyle factors in metabolic syndrome prevalence . Gender, age, education level, and physical activity show a positive association with the prevalence of metabolic syndrome . It is important to observe the prevalence of diabetes, hypertension, and obesity individually and also the combination of risk factors as metabolic syndrome to predict the risk of cardiovascular disease . Any association between lifestyle factors and these risk factors would provide the opportunity to encourage a change in lifestyle to promote lower levels of subsequent cvd . The large number of poorly educated people and the large number of school dropouts could be linked to the disease prevalence . The prevalence of hypertension and metabolic syndrome in poorly educated people was large when compared with the educated participants . Though the results are not generalized, the relationship between education levels and the prevalence of hypertension agrees with earlier studies [19, 20]. These found that education levels significantly influence the knowledge of hypertension and the awareness of cardiovascular risk . This suggests that there is a need to improve the awareness of health and use education to prevent or reduce the risk of ms and cardiovascular risks in these groups . The office workers had a lower prevalence of ms (ncep scores) than the other groups . A considerable number of office workers (64%) undertook regular physical activity of more than 30 min / day . The home workers education levels and physical activities were comparatively lower than the other work groups . These findings clearly show that education and physical activity have an influence on the prevalence of ms . Most of the females were home workers (75.5%), and their education was comparatively lower than the males . The amount of physical activity involved in home workers is unknown, but the results suggest it is less than that undertaken by other workers . Asian populations continue to modernize, and levels of physical activity are declining as (i) home and work place jobs become more automated and sedentary and (ii) transportation is more readily available . The prevalence of ms among the participants who had no physical activity was surprisingly no different than others . This may be due to a higher than average number of missing values in these data (2191/14425 complete data to meet the criteria for ms) or to other unknown socioeconomic factors . Controversially, there was a high prevalence of ms among people who regularly ate fruit and vegetables . . Found that a higher intake of macronutrients such as fruits and vegetables is associated with general obesity . However, it is not clear how the vegetables and fruits were eaten, for example, overcooked, processed, and so forth . The exact quantity of the dietary intake was not recorded as it was not the primary area of focus of the study . In these populations, these tend to report a low intake of mono - unsaturated fats (mufas), n-3 polyunsaturated fats (pufa), and transfatty acids (mostly related to widespread use of vanaspati, a hydrogenated oil). The healthy traditional plant - based diets are being replaced by cheaper calorie dense high - fat foods . These changes are resulting in a rapid increase in the prevalence of obesity throughout asia and the subsequent development of ms . Ness and powles also found in their review that many studies were reporting the null or negative effects of fruit and vegetable intake on the prevalence of cardiovascular diseases . However, the correlations found in those studies were generally low, as seen in our study . Further, they suggest that a food - based analysis would complement the nutrient - based analysis to clarify these issues . In nepal, the regular diet in addition to fruits and vegetables, that is, such as rice, which is high in carbohydrates, and the methods of cooking may be dietary causes of metabolic syndrome . The age groups 4060 had a large prevalence of ms in this study . Also, it is important to note that this middle - aged group had a high incidence of overweight or general obesity and abdominal obesity . The other age groups had a lower prevalence of ms than the 4060 years old, yet it was still relatively high . Inadequate maternal nutrition in pregnancy, low birth weight, and childhood obesity may be important factors for the development of metabolic syndrome and diabetes . Specifically in children and young individuals, a high intake of n-6 pufa is correlated with hyperinsulinaemia . In adults, unger described metabolic syndrome as a failure of the system of intracellular lipid homeostasis which prevents lipotoxicity in organs of overnourished individuals . In this study in addition to low levels of hdl, the hdl particles are small, dense, and dysfunctional in south asians . Hypertriglyceridaemia is a direct reflection of an insulin resistance condition, and it is interrelated to the low hdl concentrations in developing endothelial dysfunction . In nepal, a high number of the participants had abdominal obesity and were overweight / obese, according to their bmi . The bmi is a simple useful measure for overall abnormal weight, yet not a standard measure for obesity . Bmi cannot differentiate between whether the condition was due to unusual muscular development or the accumulation or distribution of fat in the body [26, 27]. Despite the low prevalence of general body obesity compared to western countries, metabolic syndrome is growing into a significant public health problem in asia . This may be mainly due to the large number of people with central obesity, a feature which was also observed in this study . The higher prevalence of ms in females is also more likely to be due to a higher incidence of abdominal obesity . Abdominal obesity is an important factor because metabolic syndrome and increased abdominal fat are related to a reduction of adiponectin, an adopicyte - derived hormone with antiatherogenic and anti - inflammatory properties . The abdominal adipose tissue results in release of free fatty acids directly in the portal veins and altered lipid levels in the blood . Further abdominal adiposity increases insulin secretion, and it would be exaggerated by decreased hepatic clearance leading to hyperinsulinemia . Thus abdominal obesity is an important indicator of cardiovascular disease due to its link to dyslipidemia, hyperinsulinemia, hypertension, and impaired fibrinolytic capacity . State that if the ncep's criteria were applied to the asian population, it might underestimate the prevalence of metabolic syndrome and the risk of cardiovascular disease . Idf's specific reference values for abdominal obesity make a substantial difference to the prevalence of ms between the two criteria . The idf's cut - off points for south asians' waist circumference are lower than the ncep's general cut - off points (90 cm versus 102 cm in men and 80 cm versus 88 cm in women). Another study on chinese population also found a large increase in the prevalence of metabolic syndrome using idf criteria compared with ncep criteria . However, in our study both definitions demonstrated a higher prevalence of metabolic syndrome (20.722.5%) in nepal when compared with the studies done in other southeast asian countries such as thailand (1218% using ncep definition) and india (18.3% using idf definition). These findings suggest the need for specific attention to control the disease prevalence in nepal . Although data were prospectively collected, they may not be generalizable outside of eastern nepal . The results did not show substantiate relationship between smoking histories, diet, family history of cardiovascular, and metabolic syndrome . Abdominal obesity, with the revised reference values, is an important risk due to its physiological relationship to the other ms risk factors . The ms prevalence may be due to lack of awareness and unhealthy lifestyles, so health education and more preventive measures should decrease the prevalence of ms and cardiac risks in nepal.
Balance is very important in daily activities of the elderly and greatly affects their quality of life . Due to age - related changes, balance ability deteriorates . Osteoarthritis is a degenerative disease that causes severe pain in elderly patients with low pain thresholds . Research has shown that it results in a high disability score, low quality of life, and limited range of motion, in addition to weak muscle strength, poor balance ability, and high risk of falls1,2,3,4,5 . As osteoarthritis causes severe pain and functional disorders, in general, the clinical treatment is focused on pain management and amelioration of related disorders . The treatments include muscle strength training, electrical stimulation, manual therapy, aquatic therapy, balance therapy, and pharmacotherapy6 . As balance ability and falls are closely related in the elderly, exercise therapies need to focus on the maintenance of such ability7 . Horseback riding exercise is one such therapy . In research conducted by arajo et al . 8 where elderly individuals performed horseback riding exercise for eight weeks, the researchers reported that the exercise significantly improved the participants balance and muscle strength . Unlike other interventions, horseback riding exercise can arouse the interest of patients and motivate them while improving their balance, bone density, muscle strength, and mental state9,10,11 . According to some studies, horseback riding exercise increased the static muscular contraction of the rider against the movement of the horse and improved the muscle strength in the quadriceps femoris and knee flexors of young riders12, 13 . Most people do not indulge in horse riding because of where they live (i.e., urban rather than rural areas) or the costs and risks involved in horse riding . Hence, simulator horseback riding exercise equipment using virtual reality has been developed for use at home and in small spaces . Previous studies reported that horseback riding exercise significantly increased oxygen intake, minute ventilation, met, calorie consumption, and muscle14, 15 . Although various therapeutic approaches have made with horseback riding exercise in the previous studies there is a lack of research on the effects of horseback riding exercise equipment on the balance and gait abilities of elderly patients with osteoarthritis . Experimental procedures were performed in accordance with the protocols established by the institution of animal care and use committee (iacuc) at the daegu university . Include criteria was aged 65 years or over (table 1table 1.general characteristics of the subjectsvariablessubjects (n=15)gender (m / f)6/9age (years)76.5 7.7height (cm)160.6 9.7weight (kg)54.7 9.8mean sd), and with a history of none falls in previous year, lower pain for perform experiment, and visual or auditory damage, nervous system or of the vestibular organ problems, who ca nt understand the contents of the experiment were excluded . After providing information study all subjects received a participation agreement . In this study, the subjects, while providing conventional physical therapy, 8 weeks three times a week the movement of the horse riding equipment, was carried out for 30 minutes . Before performing horse riding simulation exercise, subjects were educated about exercise program through a research assistant s demonstration . In this study, used the slimrider (shinhwa, mx-0004se, korea), which is a horse riding simulator . While performing a horse riding equipment exercise, subjects were effort to maintain the correct posture . Exercise intensity was increased gradually according to the state of the subjects while performing the exercise, the research assistant were always observed to prevent falling . In order to evaluate the balance of the elderly, it was using the frt (functional reach test) and sbbsf (short form berg balance scale). The spss for windows (version 18.0) was used for data analysis of the study . In order to compare the differences between before and after, it was analyzed using the paired t - test . In this study, sfbbs scores and range of frt increased significantly after horse riding simulator exercise (table 2table 2.comparison of measurement values at pre - test and post - testvariablesprepostsfbbs (score)18.6 5.822.3 4.4*frt (cm)19.7 9.526.3 7.5mean sd . Osteoarthritis causes severe pain in elderly patients who have a low threshold for pain and high disability scores, thereby resulting in a low quality of life, increased risk of falls, and poor balance ability1, 2, 6 . In the present study, the static, dynamic, and functional balance abilities of osteoarthritis patients who undertook horseback riding exercises on exercise equipment were measured using a frt and sfbbs . The findings are in accordance with those of study that stroke patients performed horseback riding exercise for 35 min five times a week for eight weeks and arajo8 in which elderly patients performed horseback riding exercise for eight weeks . They are also in accordance with the results of han jun - young16 in a study of stroke patients who performed horseback riding exercise for 12 weeks that an eight - week - long horseback riding exercise significantly improved the bbs scores of osteoarthritis patients . In general elderly, the average bbs score is 20.60 4.0817 . In the present study, after eight weeks of the exercise, the average score was 22.3 4.4, higher than the average . The results suggest that horseback riding exercise equipment can improve dynamic and functional balance abilities . Furthermore, according to the frt results, the static balance ability of the participants improved . These results are in line with the research findings of yasuhiro et al.18 and kim sung - gil11 where the frt scores improved significantly after 12-week - long and 8-week - long horseback riding exercise, respectively . An increase in the frt score is considered an improvement in static balance ability . Based on the findings of the present study, horseback riding exercise equipment seems to improve the static balance ability of the elderly.
, medulloblastomas can be classified in 4 different molecular subtypes: wnt, shh, group 3 and group 4 . Each of these molecular subtypes has specific underlying molecular features, gene expression, demographic characteristics and prognosis . Sonic hedgehog (shh) medulloblastomas are characterized by activation of the hedgehog (hh) signaling pathway and are often driven by mutations in hh pathway components . Here we focus on shh medulloblastoma and discuss how cell senescence shapes the natural history and molecular evolution of this childhood cancer . For many years, it was thought that tp53 mutations were infrequent in medulloblastomas and that p53 signaling was dispensable for medulloblastoma tumor suppression . However, recent studies reported that tp53 mutations are frequent in human wnt (with a rate of 16%) and shh primary medulloblastomas (with a rate of 13% to 21%, depending on the study) and are indicators of poor prognosis exclusively in shh medulloblastoma . Moreover, tp53 mutations have recently been identified as a key event in the pathogenesis of medulloblastoma recurrence . Although the specific roles of p53 in medulloblastoma are still largely unknown, the presence of recurrent tp53 mutations suggests that p53 signaling plays an important role in driving medulloblastoma tumorigenesis . Human genomic studies also established that tp53 mutations co - exist with mutations or amplifications of hh signaling components such as shh, smo, sufu, gli2 and mycn . These studies, however, do not establish the sequential order of genetic events that lead to medulloblastoma formation and how these mutations correlate with presumptive histopathological stages of medulloblastoma . Nevertheless, patients with li - fraumeni syndrome, caused by germ - line mutations in tp53, offer an opportunity to study how medulloblastomas arise at the genetic level . Li - fraumeni patients are cancer - prone and sometimes develop medulloblastoma; therefore, at least for this subset of patients, tp53 mutations are the first genetic event leading to medulloblastoma formation (fig . Notably, most li - fraumeni medulloblastomas belong to the shh subgroup, indicating that tp53 mutations specifically predispose to shh medulloblastoma . Because cerebellum granule cell precursors (gcps) are the cells of origin of shh medulloblastoma moreover, these shh medulloblastomas seem to be the result of chromothripsis, a massive genome rearrangement caused by chromosomal shattering likely occurring in a single event . These chromothriptic events likely lead to mutations in components of hh signaling, such as gli2, boc and mycn amplifications . Since hh signaling is the most important mitogenic pathway for gcps, it is therefore expected that acquisition of hh pathway mutations efficiently causes shh medulloblastoma in li - fraumeni patients . (a) in li - fraumeni patients with germ - line tp53 mutations, cerebellum gcps experience chromosomal instability . Chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn . (b) in ptch1 mice, loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia . Spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . Proliferation and senescence levels during medulloblastoma formation are indicated . (a) in li - fraumeni patients with germ - line tp53 mutations, cerebellum gcps experience chromosomal instability . Chromothriptic events lead to massive chromosomal rearrangements and high levels of amplification in hh signaling genes such as gli2 and mycn . (b) in ptch1 mice, loss of heterozygosity of the ptch1 wild - type allele leads to the formation of preneoplasia . Spontaneous p53 mutations or p16ink4a inactivation leads to senescence evasion and progression to advanced medulloblastoma . Proliferation and senescence levels during medulloblastoma formation are indicated . Although p53 knockout mice or mouse models of li - fraumeni syndrome do not develop medulloblastoma, elegant studies have demonstrated that the inactivation of p53 together with other dna repair factors such as as xrcc4, ligase iv, xrcc2 and brca2 leads to shh medulloblastoma . Interestingly, those medulloblastomas also harbored spontaneous mutations in hh signaling components such as ptch1, mycn and gli2 . This result not only highlights how important it is to maintain genomic stability to prevent shh medulloblastoma formation, but it also indicates that hh signaling activation seems to be necessary for medulloblastoma formation even when p53 and dna repair mechanisms are absent . In summary, germ - line p53 mutations in both mouse and human cause genomic instability and lead to mutations in hh signaling components, conducing to medulloblastoma formation (fig . This is an interesting paradigm showing that hh signaling mutations happen subsequent to tp53 mutations in li - fraumeni syndrome . Half of the shh medulloblastomas with tp53 mutations have a germ - line (li - fraumeni) origin and are potentially explained by the mechanism described above . However, it is not known how the other shh medulloblastoma cases (including the ones with somatic tp53 mutations and the ones without tp53 mutations) develop and the temporal order in which they acquire their mutations . Another unresolved, yet related, question is whether advanced medulloblastomas arise in a step - wise manner from subclinical precancerous lesions . For many epithelial cancers, the availability of preneoplastic lesions allowed the establishment of tumor progression models with sequential histopathological stages and the molecular changes that characterize them . However, the problem for understanding brain tumor development lies in the inability to detect and obtain precancerous lesions; therefore, genome sequencing of advanced brain tumors only offers a snapshot of the mutations present in advanced tumors but does not show the order in which mutations are acquired during tumor progression . To address this question, we used ptch1 heterozygous mice, a well - established model of shh medulloblastoma (fig . 2). Ptch1 mice develop preneoplastic lesions with high frequency, but only a fraction of those animals develop advanced medulloblastoma . Therefore, we hypothesized that an unidentified tumor suppressive mechanism might restrain the progression of medulloblastoma preneoplasia into advanced tumors . We found that apoptosis levels are the same between preneoplastic lesions and advanced medulloblastoma, eliminating apoptosis as an essential tumor suppressor in this model of shh medulloblastoma . Surprisingly, when we looked at cell senescence, we found that while preneoplastic lesions display high numbers of p21 and p16 positive cells (which are effectors and markers of cell senescence and cell cycle arrest), advanced medulloblastomas have very low levels of senescence . These high levels of senescence are paralleled by lower levels of proliferation and correlate with loss of heterozygosity (loh) of the ptch1 wild - type allele in preneoplastic lesions (fig . 1b), suggesting that high levels of hh signaling may contribute to cell senescence . Using laser capture microdissection, we found that one - third of all advanced medulloblastomas acquired p53 mutations that were not present in preneoplastic lesions, supporting the notion that p53 mutations allow senescence evasion and medulloblastoma progression (fig . Moreover, we found that engineered p53 mutations prevent cellular senescence and accelerate medulloblastoma formation, showing that p53 mutations lead to senescence evasion . Further supporting the idea that senescence evasion is necessary for medulloblastoma progression, advanced tumors without p53 mutations display low expression of p16 due to promoter methylation . In summary, we found that, contrary to li - fraumeni syndrome (where tp53 germ - line mutations lead to hh signaling mutations), hh signaling hyperactivity leads to cell senescence in preneoplastic lesions and creates selection pressure for the inactivation of p53 or p16, which allows the progression from preneoplasia to advanced medulloblastoma (fig . Figure 2.medulloblastoma formation in ptch1 mice . During postnatal development, granule cell precursors (gcps) of the cerebellum, the cells of origin of shh medulloblastoma, are located in the external granule - cell layer (egl). After their proliferation, most gcps differentiate, populate the internal granule - cell layer (igl), and disappear from the egl after the second postnatal week in the mouse . Loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . While most preneoplastic lesions disappear, some of them progress to advanced medulloblastoma . Medulloblastoma formation in ptch1 mice . During postnatal development, granule cell precursors (gcps) of the cerebellum, the cells of origin of shh medulloblastoma, are located in the external granule - cell layer (egl). After their proliferation, most gcps differentiate, populate the internal granule - cell layer (igl), and disappear from the egl after the second postnatal week in the mouse . Loh of the ptch1 wild - type allele causes a clonal expansion and leads to the formation of preneoplasia . While most preneoplastic lesions disappear, some of them progress to advanced medulloblastoma . Importantly, the finding of spontaneous p53 mutations is not limited to the ptch1 model, as we also found spontaneous p53 mutations in tumors from olig1-gnas mice, another model of shh medulloblastoma . 1b), this may highlight some possible interactions between p53 and hh signaling in the brain . Shh, a protein secreted by purkinje cells of the cerebellum, is the most potent mitogen for gcps . In contrast to these proliferative effects of shh, p53 activity supresses proliferation and self - renewal of adult neural stem cells of the subventricular zone, and this effect might be mediated by p21 . This may explain why p53 activity is downregulated during neurogenesis to allow cell proliferation and brain formation . Interestingly, gli activity has been shown to downregulate p53 protein levels in cell lines . Specifically, it has been proposed that high levels of hh signaling in cell lines caused by expression of constitutively active smo mutants or overexpression of gli1 and gli2 leads to an mdm2-dependent degradation of p53 . Consistently, low levels of mdm2 in vivo increase p53 levels, lead to cerebellar hypoplasia and reduce medulloblastoma development in ptch1 mice, showing that p53 signaling negatively controls cerebellum growth and implying that p53 signaling is important for medulloblastoma tumor suppression . Similarly, the proto - oncogene ppm1d, a negative regulator of p53, is overexpressed in medulloblastomas and increases medulloblastoma formation in mice when overexpressed together with shh . Together, these findings provide strong evidence that hh signaling leads to a functional inactivation of p53 signaling that allows gcp proliferation and, in some instances, medulloblastoma formation . However, the presence of somatic p53 mutations in ptch1 medulloblastoma demonstrates that the ability of shh signaling to functionally suppress p53 signaling is not always sufficient to inactivate p53 activity in a tumorigenic context . For many years, it has been known that p53 deletion accelerates medulloblastoma formation and increases medulloblastoma incidence in ptch1 mice; however, the mechanism responsible for this was never investigated . The fact that p53 mutations, which are acquired spontaneously during medulloblastoma formation, lead to senescence evasion provides an explanation for the presence of somatic tp53 mutations in human shh medulloblastoma . Recently, we also found that the ligand shh induces dna damage in gcps, an effect that requires the presence of the hh receptor boc and cyclind1 . These results support the hypothesis that gcps are sensitive to replicative stress and that hedgehog signaling likely causes replication stress . It has been demonstrated that oncogene activation leads to oncogene - induced cell senescence (ois), a tumor - suppressive mechanism that prevents transformation of premalignant lesions into tumors . Moreover, oncogene - induced dna damage seems to be required for ois . In light of this, the fact that we found cell senescence and ptch1 loh in medulloblastoma preneoplastic lesions suggests that high levels of hedgehog signaling in preneoplasia likely cause high levels of oncogenic stress and this leads to cell senescence . Therefore, we propose that high levels of hedgehog signaling shape the molecular evolution of medulloblastoma by leading to ois and creating selection pressure to inactivate p53 in order to evade ois . In addition to oncogenes, loss of tumor suppressor genes has been shown to cause ois . For example, in the context of prostate cancer, pten inactivation causes senescence and leads to the acquisition of p53 mutations . Neurofibromin (nf1) loss also leads to ras - mediated induction of cell senescence . Since we observed a strong association between ptch1 loss and cell senescence in medulloblastoma preneoplasia, ptch1 seems to be a new tumor suppressor whose absence may be capable of causing ois . However, the molecular mechanism leading to tp53 mutations has never been investigated in wnt medulloblastoma . Interesting work has shown that, at least in cell lines, overexpression of the downstream wnt effector - catenin leads to p53 stabilization and activation . Wnt activation in colorectal cancer has been associated with decreased levels of proliferation and accumulation of p53 protein in early tumor stages, suggesting that wnt signaling may lead to cell senescence in precancerous lesions and this could be the cause of the acquisition of tp53 mutations at late stages of colorectal cancer . We thus speculate that wnt activation in the brainstem may also lead to changes compatible with cell senescence and this may be an explanation for the presence of tp53 mutations in advanced wnt medulloblastomas . This is supported by the fact that expression of an active - catenin mutant in the lower rhombic lip of mice leads to the formation of hyperplastic lesions that only progress to advanced medulloblastomas when p53 is also deleted . It has been shown that low - grade astrocytoma lesions display a dna damage response and that loss of components of the atm - chk2-p53 pathway accelerates astrocytoma development . Although cell senescence was not interrogated in those reports, another study showed that pilocytic astrocytomas (pa), the most benign type of astrocytomas, display mapk activation and ois; interestingly, pa are indolent tumors that display long periods of growth arrest, rarely become high - grade astrocytomas, and do not display tp53 mutations . This is in agreement with the idea that pa are long - term senescent astrocytic lesions that do not progress . Eglexternal granule - cell layergcpsgranule cell precursorshhhedgehogiglinternal granule - cell layeroisoncogene - induced senescenceshhsonic hedgehog external granule - cell layer granule cell precursors internal granule - cell layer oncogene - induced senescence work done in the charron lab was supported by grants from the canadian institutes of health research (cihr), the fonds de recherche du qubec - sant (frqs), and the canada foundation for innovation (cfi). Sms is recipient of the mcgill james o. and maria meadows fellowship and ircm challenge - gupards et gazelles gourmands scholarship.
A 33-year - old woman, (gravida 3, para 2), at 34 weeks of gestation, presented with episodes of lightheadedness, and dyspnea . She was asymptomatic until 18 weeks of gestation, when she started having episodes of palpitations associated with presyncope . Initially, the episodes were infrequent (less than once per day) but a gradual increase in frequency and severity was noted by the patient . One month prior to presentation, she noted episodes of paroxysmal nocturnal dyspnea, but no dyspnea on exertion, chest pain, diaphoresis, pedal edema, orthopnea, or neurological symptoms . She had no significant past medical history and had not had complications relating to this pregnancy . All prior pregnancies had progressed to full term without similar symptoms or complications, and resulted in spontaneous vaginal deliveries of healthy newborns . Physical examination revealed an afebrile patient with a regular pulse of 80 beats per minute, blood pressure 110/70 mmhg, and an oxygen saturation of 100% on room air . The examination was normal except for mild non - pitting edema and fine bibasilar crackles . Laboratory investigations revealed normal complete blood count, electrolytes, renal function, cardiac markers and tsh . Cardiac monitoring showed multiple episodes of monomorphic, non - sustained ventricular tachycardia (vt) with left bundle branch morphology, consistent with vt originating from the right ventricular outflow tract (fig . Urgent two - dimensional echocardiogram showed normal left ventricular size and wall thickness with no segmental wall motion abnormalities and normal systolic and diastolic function with a left ventricular ejection fraction of 57% . A 12-lead electrocardiogram showed sinus rhythm with average rate of 80 beats / min with a normal axis, pr - interval, and qrs duration with no evidence of chamber enlargement or infarction . The clinical working diagnosis was pregnancy - related vt of right ventricular outflow tract origin . A 24 year woman (gravida 4, para 2) presented to her local emergency department at 24 weeks gestation because of shortness of breath, cough, and chest tightness developing over the previous day . She had a history of mild asthma but had not had asthma - related symptoms for several years and had never had an asthma exacerbation requiring inpatient or emergency department care . Past medical history and family history there had been no similar symptoms or medical complications in her previous three pregnancies, one of which had ended with spontaneous abortion and the other two with term delivery of healthy children . She smoked one - half to one pack of cigarettes per day but did not drink alcohol or use recreational drugs . Physical examination revealed an afebrile patient with a regular pulse of 78 beats per minute, blood pressure 100/60 mmhg, and an oxygen saturation of 96% on room air . On respiratory exam the rest of the examination was normal except for mild non - pitting peripheral edema . Laboratory investigations revealed normal complete blood count, electrolytes, renal function, cardiac markers, and tsh . The patient was diagnosed with an asthma exacerbation and treated with nebulized salbutamol 2.5 mg, which relieved her symptoms . Shortly thereafter, she developed runs of asymptomatic nonsustained vt with a left bundle branch block pattern (fig . Nebulized salbutamol 5 mg was once again administered, whereupon the patient developed sustained runs of vt of similar morphology . Intravenous lidocaine and magnesium sulfate were administered, leading to resolution of the ventricular arrhythmia . No further salbutamol was administered, and two further days of electrocardiographic monitoring showed only occasional pvcs . Twelve lead electrocardiogram showed sinus tachycardia at 102 beats per minute with occasional premature ventricular beats, normal corrected qt interval and no evidence of ischemia or chamber enlargement . Echocardiography showed normal left ventricular size with normal systolic function with an ejection fraction of 57%, and no valvular abnormalities . Given the absence of structural heart disease and risk factors for coronary disease or arrhythmia, the clinical diagnosis was catecholamine - sensitive pregnancy - related vt of right ventricular outflow tract origin . Ventricular tachycardias (vt) may be seen in pregnancy and can manifest as a new onset arrhythmia or be exacerbated by pregnancy and can cause concern for the well - being of both the mother and the fetus . A study of 11 pregnant women who experienced a new onset of vt during pregnancy showed that onset was distributed equally over the three trimesters and disappeared completely during the postpartum period.1 the characteristics and underlying mechanisms of new - onset vt during pregnancy have not been adequately investigated . Although the majority of vts that occur during the pregnancy are benign, in women with structural heart disease, arrhythmias (especially those of ventricular origin) is one of the five independent predictors of having an adverse outcome during pregnancy.2,3 peripartum cardiomyopathy (ppcm) needs to be considered in women presenting with vt in the last month of pregnancy.4,5 the criteria for the diagnosis of ppcm include: development of congestive heart failure secondary to left ventricular systolic dysfunction in the last month of pregnancy or within 5 months after delivery, absence of preexisting cardiac dysfunction, and absence of identifiable cause of heart failure and lv systolic dysfunction demonstrated by classic echocardiographic criteria.6 hence in women presenting with vt in the last month of pregnancy, an urgent echocardiographic study can assist in establishing a diagnosis of ppcm . Women with certain types of congenital heart disease (chd) either repaired or not, are particularly vulnerable to vt . For example, patients with previous surgeries for correction of tetralogy of fallot may experience vt arising from the right ventricular outflow tract as a result of right ventricular volume overload due to decades of severe pulmonary regurgitation as a consequence of patch augmentation of the right ventricular outflow tract at the time of original corrective surgery.7 a study of 28 female patients with repaired chd demonstrated women with chd had a significantly higher incidence of tachyarrhythmia during pregnancy and in the postpartum period compared to controls.8 congenital long qt syndrome (lqts) is a hereditary disorder of cardiac ion channels causing abnormal electrical activation of the heart.9 women with congenital lqts may experience life - threatening polymorphic vt; however recent reports indicates that their incidence appears to be significantly increased during the postpartum period.10,11 patients with congenital lqts are particularly vulnerable to adverse arrhythmic effects of electrolyte depletion (further prolongation of qt interval) a possible complication in some pregnant women with hyperemesis . Cautious use of mediations that may further prolong the qt interval is necessary in these individuals to avoid precipitation of polymorphic vt . In addition to cardiac diseases, several systemic non - cardiac disorders, including severe electrolyte derangements, thyroid function abnormalities, pulmonary embolism, anemia, and drug overdose may present with ventricular arrhythmias . There are few reports of new onset vt during pregnancy in absence of structural heart disease.12,13 increased sympathetic activity, as well as physiological changes associated with normal pregnancy such as increased heart rate, decreased peripheral resistance, increased stroke volume and psychological stresses are thought to be the most common precipitants of vt in pregnant women with a structurally normal hearts.14,15 the majority of outflow tract vt in structurally normal hearts originate from the right ventricular outflow tract (rvot) just below the pulmonary valve . This is also the most common focus of initiation of pregnancy - related idiopathic vt and usually does not deteriorate to an unstable rhythm . It will often manifest as ectopic beats, bigeminal rhythms or short runs of non - sustained vt . Norepinephrine concentration in the myocardial synaptic cleft has been suggested as a potential mechanism for this arrhythmia.16 only 10% of vt originates from the left ventricular outflow tract (lvot).17 to make a diagnosis, a 12-lead ecg should be recorded ideally during the arrhythmia, although this can be difficult . A typical resting ecg of a pregnant woman may show an increased heart rate with decreased pr, qrs and qt intervals, but there is usually no significant change in the amplitudes of p and t - waves and qrs - complex.15 premature atrial and ventricular beats as well as q wave and inverted t waves in the inferior leads can also be seen during pregnancy.18 on ecg, rvot vt appears as wide qrs complex tachycardia with left bundle brunch block (lbbb) morphology and an inferior axis.17,19 two phenotypic forms of rvot vt can occur, non - sustained monomorphic vt and paroxysmal sustained vt.20 in contrast, lvot vt morphology depends on the site of origin with either lbbb and early precordial transition in leads v12, or rbbb pattern in v1 with broad monophasic r in the precordial leads . In addition, the ecg in patients with vt originating in the lvot will show r: s amplitude ratio of 30% or more or an r: qrs duration ratio of 50% in leads v12.21 a routine 24 to 48 hour - holter monitor is helpful in capturing frequently occurring paroxysmal arrhythmias . To elicit symptoms, an exercise ecg can be reasonably carried out during pregnancy in absence of obstetrical contraindications for exercise . No fetal adverse events are reported for adenosine, except for one case of transient fetal bradycardia.22 the first step in acute management of vt in pregnancy to determine the hemodynamic stability of the pregnant woman . If the woman is unstable or there is evidence of significant fetal compromise which is thought to be related to the vt, dc cardioversion with 50100 j should be given immediately . Dc shocks can be repeated at higher levels of energy (100360 j) if indicated . The risk for the fetus is minimal for all stages of pregnancy, because the amount of current reaching the fetus is small.23 in stable vt, an accurate diagnosis of the type of arrhythmia should be made with a twelve - lead ecg prior to any intervention . Invasive electrophysiological studies are rarely required during pregnancy, as the arrhythmias can be effectively managed pharmacologically . Conservative medical treatment of vt arising during pregnancy is indicated in any patient with sustained vt as long as the patient is hemodynamically stable.14 if possible drug therapy should be avoided during the first trimester and drugs with the longest records of safety should be used as the first - line therapies.24 the literature safety data has examined digoxin, adenosine, flecainide, procainamide, propranolol, propafenone, quinidine, sotalol and verapamil, however; the experience is limited to single or small case series . In stable patients with sustained vt acute therapy can be initiated with either intravenous procainamide, which is safe, well - tolerated and is not associated with teratogenicity . Alternatively lidocaine can also be used to treat stable vt.14 idiopathic vt originating from the rvot and with a lbbb morphology responds well to beta - blockers, while idiopathic lvot vt with rbbb morphology generally responds well to verapamil . Given the sensitivity of majority of vts in pregnancy to catecholamines, a cardioselective beta - blocker, in particular metoprolol, in the absence of contraindications is considered first line therapy.12 in our cases, further investigations did not reveal a secondary cause of the patients arrhythmias . Based on the ecgs (qrs morphology of a left bundle branch block pattern with an inferior axis, and clinical findings) both patients were diagnosed with idiopathic vt originating from the rvot . The lower dose for the second patient was chosen because of suspected asthma exacerbation; however the dose was slowly increased to 50 mg po every 12 hours . Both patients tolerated treatment well and had no further documented episodes of vt on long - term cardiac monitoring . Patient 1 was scheduled for elective c - section at a high risk obstetric center and was discharged home . On the follow - up patient 2 had a spontaneous vaginal delivery at 36 weeks and continued to be on metoprolol after discharge . Pregnant patients may present with ventricular tachycardia during any trimester careful and timely clinical, electrocardiographic, as well as echocardiographic assessment help identify those individuals with structural cardiac abnormalities such as congenital heart disease or peripartum cardiomyopathy, as well as those with long qt syndrome who will need specific management beyond therapy for the arrhythmia . In those stable patients with structurally normal hearts, identification of the location of origin of tachycardia will help in choice of appropriate medical therapy.
Common sources include backyard burning, incineration of plastics, and chlorine bleaching of pulp in paper mills . Documented health effects include acute and chronic effects, including chloracne, various types of cancers, reproductive diseases, circulatory and respiratory diseases, and diabetes . The traditional approach to environmental remediation includes a host of physical and chemical methods, depending on the characteristics of the polluted site and the extent of contamination present . Bioremediation, which is the use of biogenic materials and organisms for environmental cleanup, has also been proposed, including phytoremediation using plants and microbial degradation using primarily bacteria and fungi . Bioremediation is an attractive strategy, as it can destroy the pollutant rather than transferring it from one environmental compartment to another . Common bioremediation strategies include the addition of nutrients, degradative microorganisms, or both . Sphingomonas wittichii rw1 is a microorganism of great interest to the bioremediation community for its ability to biotransform a large number of toxic polychlorinated dioxins and to utilize both nonchlorinated dibenzo - p - dioxin and nonchlorinated dibenzofuran as a growth substrate and sole source of carbon and energy . One of the major challenges in bioaugmentation strategies relying on the addition of nonnative microbes is to ensure their viability and degradative activity toward the target compounds . Monitoring bioremediation is critical to ensure the efficacy of the process and the reduction of contaminant mass to acceptable levels . Traditionally, the most important characteristics investigated for microorganisms used in bioremediation were their ability to transform the substrate, the rate of substrate removal, and the resulting metabolites [69]. To optimize the bacterial degradation of pollutants, it is important to understand how these organisms function during growth on recalcitrant substrates and which factors influence their degradative abilities . This includes analyzing not only the degradative pathways [1012], but also the peripheral processes and mechanisms that are involved in taxis (i.e., directed motion in a chemical gradient), uptake, and transport during exposures to specific substrates . Analysis of dna and rna can shed light on an organism's metabolic potential; however, these measurements poorly correlate to actual protein expression profiles . Therefore, global analyses of protein expression profiles may be a more informative tool for understanding the physiological mechanisms of biodegradation . In addition to identifying important degradative enzymes in a variety of important microbes [1517], proteomic studies have opened the door to a better understanding of system - wide changes in response to differing substrates . The imperative to perform proteomic analyses is particularly true for s. wittichii rw1 because the enzymes in the dioxin degradation pathway are encoded on different loci throughout the genome, certain elements in the pathway are located on a plasmid, and there may be alternative pathways at work . The present study builds on previous work and utilized difference gel eletrophoresis (dige) coupled with mass spectrometry (ms) to exploit recently gathered rw1 genome data . When used together, these tools yield information on the response of cells of s. wittichii rw1 to dioxin exposure and the bacterium's degradative activity toward this recalcitrant compound . The aim of this study was to investigate system - wide changes in protein expression during growth on dibenzofuran, a nontoxic surrogate for dibenzo - p - dioxin, as compared to nonselective growth media . Acetate was selected as the nonselective alternate substrate, as growth on this compound was observed to influence expression of select proteins, including the dioxin dioxygenase . Thus, any changes measured in response to cells grown on dibenzofuran should represent cell - wide effects related specifically to the growth substrate and not to unanticipated extraneous effects . Cultures of s. wittichii strain rw1 (100 ml to 1.0 l) were grown to mid log phase at 30c in m9 phosphate - buffered minimal medium (ph 7.05) supplemented with either dibenzofuran crystals or 50 mm acetate as growth substrates . Cells were grown overnight on dibenzofuran and acetate as sole carbon sources to an optical density of 0.40.6 absorbance units (560 nm). Following biomass processing, protein levels in the samples were on the order of 75200 g / ml . Protein concentrations were normalized prior to analysis by concentration and resuspension in dige sample preparation buffer . Culture purity was confirmed by the streak plate method using luria bertani medium supplemented with 1.5% agar . Harvested biomass was washed, spun again, and the resultant pellet suspended in a small volume of 100 mm ammonium bicarbonate (ph ~7.0). This microbial suspension was then sonicated under cooling with ice, using a microtip sonicator (fisher scientific, pittsburgh, pa, usa) in a sequence of three 10-second bursts delivered in thirty - second intervals . The sonicated cells were then immediately centrifuged at 10,000 xg for 10 minutes at 4c . Briefly, 8 parts of 10% tca in acetone (20c) were added per volume of supernatant and, following mixing on a vortex, the resultant dilution was incubated at 20c overnight . Following centrifugation (10,000 xg, 10 minutes, 4c), harvested biomass was washed in cold acetone for 10 minutes at 20c . Following a subsequent centrifugation, the pellet was resuspended in sample preparation buffer (7 m urea, 2 m thiourea, 2% chaps, 0.2% dtt, 0.02% bromophenol blue) and stored at 20c until analyzed . Protein concentrations were measured using the bicinchoninic acid assay (pierce, rockford, il, usa) following dilution to reduce the concentration of interfering agents . Twenty - five g of crude cell lysates of rw1 biological replicates grown on dibenzofuran (n = 3) and acetate (n = 3) were labeled using cy dyes (ge healthcare) as described elsewhere . Briefly, samples were adjusted to 1 g/l using sample preparation buffer and the ph checked . Subsequently, 0.25 l of 1 pmol/l cy dyes were added to samples for 30 minutes in the dark on ice . To stop the labeling reaction, 0.5 l of 10 mm lysine was added to the samples, which were mixed and incubated on ice for 10 minutes prior to storage at 20c until analysis . Unless stated otherwise, all procedures were carried out in the dark or minimal light to protect the integrity of the fluorescent dyes . Samples were randomized to reduce the effect of dye bias and in - gel variations . A global pool consisting of fractions of each sample was labeled as outlined above using cy 2 and added to each sample as an internal standard . One cy-3- and one cy-5-labeled sample were added to each gel, as defined by the experimental randomizing procedures . To each sample, an additional 175 g of unlabeled sample were added; the volume was increased to a total of 450 l using sample preparation buffer . The reducing agent dtt (dithiothreitol; 1.3 mg per tube) and ipg (immobilized ph gradient) buffer (0.5%) were added, and the samples incubated and mixed in the dark at room temperature for approximately 1 h. samples were then applied to 24 cm ph 47 ipg strips (ge healthcare) and focused for 60 kvh using the following protocol: 12 h rehydration at 30 v; 1 h step and hold at 500 v; 7 hour gradient to 1,000 v; step and hold at 1,000 v for 1 hour; gradient to 8,000 v for 3 h; step and hold at 8,000 v until 60 kvh . Strips were then reduced and equilibrated using 10 mg / ml dtt (15 min) followed by 25 mg / ml iodoacetamide (15 min). The ipg strips were overlaid on 24 26 cm 816% gradient tris - hcl ph 8.8 precast gels (nextgen sciences, ann arbor, approximately 1 ml of agarose was applied to fix the gels and a cy-2-labeled molecular weight marker was applied adjacent to the acidic side of the strip . The gels were then run 1 - 2 w per gel overnight (~2224 hours) at 20c until the marker dye ran off the gel . Gels were then imaged with a typhoon 9400 scanner and processed using decyder v6.5 (ge healthcare) bva batch processor tool . Images were uploaded to decyder (version 6.5) and spurious image objects (water spots, streaks, and mismatches) were identified and excluded from further analysis . Following allocation of changed proteins, individual spots were manually inspected and excluded from analysis if they fell outside acceptable parameters for peak height, area, and slope . Pick lists were generated by selecting proteins whose expression was statistically changed in the two growth conditions (p <0.05) following digital image analysis using decyder, and the corresponding spots were automatically picked using an ettan spot picker (ge healthcare) with ettan spot pick software v.1.1 . Spots were delivered in 100 mm ammonium bicarbonate to a 96-well plate and digested using established protocols . Briefly, gel pieces were sequentially dried using three exchanges of 100% acetonitrile followed by a 10-minute speedvac (savant) drying . Gel pieces were rehydrated in 40 l of 10 ng/l trypsin in 100 mm nh4hco3 on ice for 45 minutes . The supernatant was removed and replaced with 100 mm nh4hco3 and digested at 37c overnight . Peptides were then extracted using 50% acetonitrile/0.1% tfa (trifluoroacetic acid) for 30 minutes at 37c . The peptides were microextracted using omix c18 tips (varian, palo alto, ca, usa) following the manufacturer's instructions and then deposited on a stainless - steel target plate in a matrix consisting of 10 mg / ml 2,5-dihydroxybenzoic acid . Mass spectra were acquired using a voyager de - str matrix - assisted laser desorption / ionization time - of - flight ms (applied biosystems, foster city, ca, usa) in positive reflector mode with delayed extraction using the following parameters: laser energy, 1400 arbitrary units; mass range, 5005,000 da; 120 nsec delay, 100 laser shots per spectrum . External calibration was conducted using a standard peptide mixture (bradykinin, insulin b chain, p14r, and acth), and internal calibration was carried out using trypsin autolysis peaks . Data were processed in data explorer v1.1 (applied biosystems, foster city, ca, usa) using noise reduction (2 standard deviations) and peak deisotoping . Peak masses were searched using the mascot online search engine (http://www.matrixscience.com/) with the following settings: database, ncbi entire database (5.6 million entries); no missed cleavages; monoisotopic peaks; no fixed modifications; variable modification of methionine oxidation; error tolerance of 150 ppm . Database and literature searches were used to further characterize and classify the proteins identified by maldi - tof ms . Where ambiguous names were encountered, blastp searches were used to identify homologous proteins from orthologous species . Image analysis of 24 cm 2d - dige gels loaded with protein of s. wittichii rw1 cells grown on either dibenzofuran or acetate revealed 937 unique spots . Differential in - gel analysis of individual gels determined gel - specific parameters for selection criteria and allowed visual examination of changes between growth on the two substrates (figure 1). Of the 937 identified spots, 595 were matched between all the gels used to statistically compare the quantitative abundance of proteins . Statistical analysis compared triplicate biological observations for each condition, normalized to the internal pooled standard (figure 2). Crude cell lysates from s. wittichii rw1 grown on dibenzofuran showed that, of all proteins observed, 22 proteins were modulated in response to changes in culture conditions . These candidate biomarkers of metabolic activity and phenotype were observed in at least 6 of 9 dige images and were modulated as follows: 16 showed an apparent increase and 6 an apparent decrease (figure 2). These proteins, along with 22 proteins selected due to their high abundance in both growth conditions, were further analyzed and identified using mass spectrometry (figure 3). A mascot search of the entire ncbi database using mass spectral data generated by peptide mass fingerprinting identified 23 of the 44 proteins (52%). All protein identifications corresponded to the genome of s. wittichii strain rw1 . Among the 16 proteins upregulated during growth on dibenzofuran, 7 were successfully identified (table 1). Among the 6 proteins downregulated during growth on dibenzofuran, an additional 13 proteins were identified whose expression level remained unchanged regardless of culture conditions (table 3). Of the 7 identified proteins increased during growth on dibenzofuran, 2 were directly related to the dibenzofuran degradation pathway (figure 4); the others were involved in downstream metabolic processes (catechol 1,2-dioxygenase, adenosylhomocysteinase), cell growth (elongation factor ts), and cell protection (cold shock dna - binding domain protein, alkyl hydroperoxide reductase). The three identified proteins whose expression was decreased (fumarylacetoacetate hydrolase, tonb - dependent receptor, and acyl - coa dehydrogenase) are involved in biosynthesis, catabolism, and transport . The unchanged proteins represented basic cell functions, although biosynthesis, catabolism, and transport proteins dominated the identities . The alpha subunit of the dioxin dioxygenase, the first step in the dioxin degradation pathway, was also identified as unchanged . Dige and 2d electrophoresis are an accepted strategy for mining microbial proteomes for biomarkers related to a number of processes [2830]. The complete protein content of s. wittichii rw1 consists of approximately 5,000 putative proteins from the bacterial chromosome and two megaplasmids . Using simple extraction and purification techniques followed by dige, over 500 protein spots were resolved on a large (24 cm) 2-dimensional gel and matched between the three biological replicates, representing approximately 10% of the entire protein content . Of these 500 proteins, 22 were found to be regulated in response to growth condition changes . Of the genes they identified as being changed in their experiments, only two overlapped with the proteins identified in our study: swit_3144 (tonb - dependent receptor, downregulated) and swit_3376 (chaperonin groel, upregulated). Both of these gene levels responded to short - term perturbation with peg8000 but not sodium chloride in the transcriptomic study and were unchanged in our study . In our study, we also identified a number of proteins that are related to dioxin / dibenzofuran degradation (e.g., dioxin dioxygenase, meta - cleavage product hydrolase, and 2,3-dihydroxybiphenyl 1,2-dioxygenase). Other proteins were identified that showed increases in abundance but whose role was not directly related to the dibenzofuran degradation pathway . The increase in the presence of antioxidants such as alkyl hydroperoxide reductase suggests that there is an increasing stress upon the bacterial cell during growth on dibenzofuran, perhaps due to a change in catabolism resulting in an increase in endogenous peroxide generation . Increases in a cold - shock dna - binding protein may be further evidence of an increased cellular stress . However, proteins of the cold shock family and related ones are also known to have transport and protein processing roles . Among the proteins in the dioxin degradation pathway, the most prominent on the gel was the meta - cleavage product hydrolase . This identification was produced from two adjacent spots, likely representing an artifact due to the protein's extremely high expression or a reflection of the presence of multiple isoforms or a modified enzyme . The one identified in the present study is the product of the swit_3055 locus, a gene also known as dxnb2 . The glyoxalase / bleomycin resistance protein / dioxygenase identified in this study is also annotated as a 2,3-dihydroxybiphenyl-1,2-dioxygenase located on the chromosome at the swit_3046 locus . Again, there are multiple isoforms of this enzyme found both on the chromosome and the megaplasmids . The kegg dioxin degradation pathway identifies swit_4182 as the dihydroxybiphenyl dioxygenase involved in biphenyl metabolism and swit_4902 as the trihydroxybiphenyl dioxygenase in both dioxin and dibenzofuran metabolism . The increased expression in response to dibenzofuran suggests that the swit_3046 dioxygenase plays a more important role in dibenzofuran degradation in vivo . The high degree of redundancy in the dioxin and dibenzofuran degradation pathways, that is, the presence of multiple ring - hydroxylating alpha and beta subunits, glyoxalase / bleomycin resistance protein / dioxygenases, and meta - cleavage product hydrolases, remains to be explained . One possibility is that the various isoforms have different affinities for chlorinated metabolites that would result from chlorinated dioxins and furans . Further experiments are needed to fully distinguish the roles of these enzymes in s. wittichii rw1 degradation pathways . Although not directly implicated in dioxin degradation, the fumarylacetoacetate hydrolase is also of interest because the gene encoding this protein (swit_5089) flanks the ferredoxin fdx1 (swit_5088) that has been identified as part of the electron supply chain supporting dioxin dioxygenase activity . Of the multiple isoforms of this enzyme, the electron supply chain also contains two isofunctional reductases . Neither the ferredoxin itself nor the reductases could be identified . In previous studies, the reductase was present as a much smaller fraction of the soluble cell proteome than either the ferredoxin or the dioxin dioxygenase, so gel - based methods may not be sensitive enough to detect this protein . If transcription of the ferredoxin is linked to the other genes at that locus, as is predicted, the decreased expression in response to dibenzofuran suggests that another ferredoxin is more important in the dioxin degradation pathway in vivo . The detection of the dioxin dioxygenase alpha subunit and related enzymes in both acetate- and dibenzofuran - grown cells is potentially of importance for the field of bioremediation because it suggests an avenue of biostimulation . When utilizing s. wittichii rw1 as a bioremediation agent, it may be possible to induce the expression of the dioxin degradation pathway using acetate . Induction of the dioxin degradation pathway has not been observed when s. wittichii rw1 is grown on glucose or rich medium, and growth in a complex environmental medium (landfill leachate) was correlated with a decrease in copy number of the gene encoding the dioxin dioxygenase alpha subunit . Previous studies using s. wittichii rw1 to transform chlorinated dioxins in soil or fly ash have observed a progressive decrease in degradative activity or viable cells [38, 39], respectively . The addition of acetate may generate sufficient relevant protein biomass to catalyze the successful degradation of dioxin and dioxin - like compounds in environments bioaugmented with s. wittichii rw1 . Proteomic technology has emerged in microbiology more rapidly than in other fields for several reasons . The relatively small genomes code for relatively limited proteomes featuring no or very limited posttranslational modifications compared to higher organisms . Furthermore, microbes are easily controlled and manipulated in the laboratory, both during growth and gene expression . These factors will continue to drive biomarker discovery in microbial proteomes, including phenotypic biomarkers informing on the degradative activity of biomass produced for bioaugmentation of contaminated environments . Furthermore, the field of bioremediation can benefit from methods suitable for monitoring microbial biomarkers in field samples to inform on progress in site bioremediation . This study highlights a number of proteins that were changed in response to dibenzofuran exposure, opens the door to a greater understanding of how s. wittichii rw1 performs and regulates the degradation of dioxins, and suggests ways to enhance the biodegradation of dioxins.
Among dermatological disorders, erythematous dermatitis, or eczema, and psoriasis are the most common.1 dermatitis is defined as a superficial skin inflammation, characterized by poorly delimited erythema, and usually itchy crusts and scales.2 conventional measures to prevent eczema include avoidance of irritants and potential allergens, as well as continued hydration of the skin with thick emollients . Other therapies including phototherapy, antimicrobials, antihistamines, and systemic immunosuppressives are also therapeutic options.1 psoriasis is an autoimmune, chronic, noncontagious, recurrent, inflammatory skin disorder . In western populations, its prevalence is estimated to be as high as 2.8%.3 the typical skin lesions of psoriatic patients are sharply demarcated erythematous plaques covered with silvery or opalescent scales.4 the inflammatory response is supported by the infiltration of polymorphic neutrophils in the skin . Neutrophils are triggered by the chemokines and lymphokines released by keratinocytes and t - lymphocytes, respectively, and, in turn, trigger the activity of lymphocytes and keratinocytes, thus generating a vicious cycle limited to acute lesions but linked in any case to the extended inflammation.5 treatments for psoriasis include immunosuppressive drugs such as methotrexate, cyclosporine, and fumaric acid esters.6 however, advances in mechanistic understanding of signaling pathways involved in the pathogenesis of psoriasis have led to the testing of biological therapies . These include immune suppressive drugs (eg, alefacept) and anticytokine therapies (antitumor necrosis factor [tnf] therapies [adalimumab, etanercept, infliximab, ustekinumab]).7 topical application of anti - inflammatory molecules is a feasible alternative to systemic approaches to treat psoriatic and eczematous symptoms by directly acting on inflammatory processes and generating a marked soothing response in skin.8 a recent study on costs of psoriasis medications has demonstrated that biological treatments represent 67% of total medication costs, and only 5% of patients purchase them . In contrast, topical medications are the most commonly purchased treatments and account for the 18% of total costs.9 furthermore, since almost 10% of patients require at least three topical therapy switches over a 1-year period to gain health benefit, the identification and formulation of novel anti - inflammatory compounds that could be used in topical application are a medical need for psoriasis treatment and a possible approach to reduce costs.9 boswellia serrata gum resin extracts (bses) (or olibanum) are used in traditional ayurvedic medicine to treat inflammatory diseases . The prevalent component of lipophilic fraction of olibanum is represented by the pentacyclic triterpenes boswellic acids (bas), several forms of which have been characterized by analytical techniques, including - and -configured bas (figure 1). Another structural variety is given by the presence of a carbonyl group and an acetyl group in 11-keto bas (kba) and 3-o - acetyl - bas (akba), respectively.10 animal studies and pilot clinical trials support the efficacy of bses for the treatment of a variety of inflammatory diseases such as inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, and asthma.1114 in 2002, the european medicines agency classified bse as an orphan drug for the treatment of peritumoral brain edema.15 the main anti - inflammatory properties of bas are attributed to suppression of leukotriene formation via inhibition of 5-lipoxygenase (5-lo) by kba and akba.15 the mechanistic model has to be fully elucidated, but there is some evidence of an indirect action of bas leading to an irreversible inhibition of 5-lo by a ca2 + -mediated pathway.16 bas also inhibit the human leukocyte elastase, released in inflammation- and hypersensitivity - based human disorders, thus further supporting ba s antiphlogistic properties.17 in activated human monocytes, akba conveys inhibition of nuclear factor (nf)-kb and subsequent downregulation of tnf- expression, via its direct inhibition of i kappa b kinase (ikk).18 noteworthy is that when the ikk inhibition by bas has been investigated in the cd18 hypomorphic (cd18[hypo]) mouse model of psoriasis, the suppressive effects on nf - kb signaling have been confirmed, thus suggesting that targeting nf - kb with bas may be an effective strategy for the treatment of psoriasis.19 since both psoriasis and erythematous dermatitis present a prevalent inflammatory component and bas have a documented inhibitory activity in some pathways with a major role in inflammation, especially in nf - kb pathway, we developed a formulation containing bas with potential efficacy in patients with psoriasis and erythematous dermatitis . In this double - blind study, we compare, for the first time, a novel ba formulation (containing bosexil, inci: lecithin, bse) with a placebo formulation in the treatment of psoriasis or eczema . A third arm of the trial received a formulation of vaccinium myrtillus seed oil, which was previously demonstrated as an efficient local treatment for psoriatic lesions.20 this is a randomized, placebo - controlled, double - blind study performed at the department of dermatology at the university of varese (italy) in collaboration with velleja research and the local health agency 1 (asl1) in the piacenza (italy) area . We separately considered in the study patients who were affected either by psoriasis or by erythematous dermatitis . The diagnosis of erythematous atopic eczema or psoriasis was made by the physician in charge of the department of dermatology involved in the trial . Patients had to be free from any topical or systemic anti - inflammatory treatment for at least 3 months . Patients were randomly assigned, in a 1:1:1 ratio and according to a computer - generated numerical table, to one of the following groups: bosexil, v. myrtillus seed oil, or placebo . Bosexil was formulated according to the phytosome delivery system that is aimed at improving the bioavailability and skin affinity of bioactive molecules.21 all preparations could not be distinguished without a chemical analysis . Patients were asked to apply the cream twice a day to the affected areas in a sufficient quantity to cover the whole skin area involved, for a total period of 30 days . Other concomitant pharmacological treatments were not interrupted or modified, except for those, dermatological or oral, finalized to treat psoriasis . Clinical assessments were carried out at baseline (t0) and at the end of the trial (t30), and included the evaluation of scales and erythema in the case of psoriasis and the evaluation of itch and erythema in the case of eczema . Lesions and clinical manifestations were classified as absent (score 0), moderate (score 1), severe (score 2), or very severe (score 3). Efficacy was statistically assessed through a procedure based on the individual s permanence or migration state (primary end point) depending on the entity of the score assigned to a symptom at t30 in comparison with the corresponding baseline score (t0). Thus, the change of condition generated the following categories of patients: in remission: when individuals with scores> 0 at t0 have score = 0 at t30; improved: when individuals have lower scores at t30 than at t0; unchanged: when individuals have the same scores at t0 and t30; worsened: when individuals have higher scores at t30 than at t0 . We validated this statistical procedure in a previous study on the efficacy of v. myrtillus seed oil.20 as an internal control, we compared the effects of bosexil and v. myrtillus with the same placebo . The psoriasis area severity index (pasi) and the eczema area and severity index (easi) the comparison between groups was performed using pearson s chi - square test; responses were evaluated with a markov - like procedure . The comparison between groups was performed using pearson s chi - square test; responses were evaluated with a markov - like procedure . Each group consisted of ten patients affected from psoriasis and ten patients with erythematous dermatitis (except the bosexil group, in which patients with dermatitis numbered nine). At t0, all groups were similar in terms of severity of the disease . However, three patients in the placebo group, one in the v. myrtillus group, and two in the bosexil group suspended the treatment before t30 and were included with a score of 3 in the statistical analysis . No significant difference in terms of age or sex the results from the change of condition (primary end point) analysis between placebo (group a) and two formulations (bosexil and v. myrtillus seed oil) are summarized in tables 5 and 6 . In patients with psoriasis (table 5), signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo . In particular, patients who received placebo had unchanged scales and erythema, and in 10% of cases worsened . Treatment with bosexil formulation improved scales (70% of cases, p=0.0001) and erythema (60% of cases, p=0.0281) without any case of worsening . Treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases, but 30% of patients were in remission at the end of therapy, therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% (p=0.253) of cases . In terms of scales, v. myrtillus seed oil improved the symptomatology in 80% of patients, and 20% was unchanged (p=0.0003). In patients with erythematous eczema (table 6), the bosexil - containing formulation improved both itch (60% of cases, p=0.0115) and erythema (60% of cases, p=0.0115), without any case of worsening . V. myrtillus seed oil treatment improved itch and erythema in 66.7% (p=0.0020) and 77.8% (p=0.0068) of patients, respectively . The pasi and easi scores observed in the three groups are reported in tables 7 and 8, respectively . Figure 2 shows pictures of some psoriasis patients one treated with bosexil, one treated with v. myrtillus seed oil, and the other assigned to placebo . The results from the change of condition (primary end point) analysis between placebo (group a) and two formulations (bosexil and v. myrtillus seed oil) are summarized in tables 5 and 6 . In patients with psoriasis (table 5), signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo . In particular, patients who received placebo had unchanged scales and erythema, and in 10% of cases worsened . Treatment with bosexil formulation improved scales (70% of cases, p=0.0001) and erythema (60% of cases, treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases, but 30% of patients were in remission at the end of therapy, therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% (p=0.253) of cases . In terms of scales, v. myrtillus seed oil improved the symptomatology in 80% of patients, and 20% was unchanged (p=0.0003). In patients with erythematous eczema (table 6), the bosexil - containing formulation improved both itch (60% of cases, p=0.0115) and erythema (60% of cases, p=0.0115), without any case of worsening . V. myrtillus seed oil treatment improved itch and erythema in 66.7% (p=0.0020) and in patients with psoriasis (table 5), signs of scales and erythema improved both with bosexil and with the v. myrtillus seed oil treatment in comparison with placebo . In particular, patients who received placebo had unchanged scales and erythema, and in 10% of cases worsened . Treatment with bosexil formulation improved scales (70% of cases, p=0.0001) and erythema (60% of cases, p=0.0281) without any case of worsening . Treatment with v. myrtillus seed oil formulation improved erythema in 10% of cases, but 30% of patients were in remission at the end of therapy, therefore indicating that v. myrtillus seed oil treatment had positive effects on psoriatic erythema in 40% (p=0.253) of cases . In terms of scales, v. myrtillus seed oil improved the symptomatology in 80% of patients, and 20% was unchanged (p=0.0003). In patients with erythematous eczema (table 6), the bosexil - containing formulation improved both itch (60% of cases, p=0.0115) and erythema (60% of cases, p=0.0115), without any case of worsening . V. myrtillus seed oil treatment improved itch and erythema in 66.7% (p=0.0020) and 77.8% (p=0.0068) of patients, respectively . The pasi and easi scores observed in the three groups are reported in tables 7 and 8, respectively . Figure 2 shows pictures of some psoriasis patients one treated with bosexil, one treated with v. myrtillus seed oil, and the other assigned to placebo . This study aimed to compare, for the first time to our knowledge, the efficacy of topical preparation of bosexil and v. myrtillus seed oil vs a placebo in individuals with a diagnosis of psoriasis or erythematous dermatitis . Indeed, these formulations are particularly rich in polyunsatured fatty acids (v. myrtillus seed oil) or bas (bosexil) that exert anti - inflammatory effects, thus may provide benefits in skin inflammatory diseases . Bas in bosexil formulation were more active in the treatment of psoriatic scales than erythema, and were similarly efficient to treat itch and erythema in patients affected by eczema . This formulation was designed according to the phytosome delivery system21 in order to improve the bioavailability and skin affinity of bas . Consistently with previous results,20 this study confirmed the efficacy of v. myrtillus seed oil preparation in the treatment of major symptoms in both psoriasis and eczema, such as erythema and scales and erythema and itch, respectively . Noteworthy is that v. myrtillus seed oil was associated with three remissions in the treatment of erythema in psoriatic patients . Bilberry fruits contain small seeds rich in alpha - linolenic acid and linoleic acid, which are considered essential nutrients (the human body, in fact, lacks the necessary enzymes to introduce an unsaturation beyond position omega-9). Linoleic acid and alpha - linolenic acid are involved in the skin barrier function.22 one issue in the management of chronic diseases such as psoriasis is the adherence to treatment . Few data are available in literature, but the most frequently mentioned reasons for nonadherence to topical treatment are low efficacy, time consumption, and poor cosmetic characteristics of topical agents.23 this study has tested a twice - daily administration which determined an effective improvement of disease . However, the study lasted a short period of time and included an overall limited number of patients; therefore, long - term, larger studies should be recommended to assess the adherence to this protocol in real life . Moreover, earlier assessments (eg, at day 15) could have been performed to collect information on the early efficacy of both bosexil and v. myrtillus seed oil . Last, other end points (eg, patient - related severity score or the dermatology life quality index) have not been evaluated in this study . In addition to the anti - inflammatory / barrier - protecting effects of polyunsatured fatty acids or bas, a cosmeceutical formulation of these creams may provide the adjunctive benefits that can restore and protect the skin barrier function, increase the remission times between flare - ups, and enhance the pharmaceutical effects of active compounds.24 in conclusion, bas contained in bosexil and formulated according to the phytosome delivery system21 may be eligible components for the formulation of preparation with anti - inflammatory properties for the treatment of psoriatic and eczematous symptoms . We speculate that this formulation could also be applied to other inflammatory skin disorders, including contact, atopic, seborrheic, nummular, chronic palmoplantar, and generalized exfoliative or chronic lichen simplex dermatitis.
Cell lines, cultures and transfections: a madin - darby bovine kidney cell line (mdbk) (hsrrb, osaka, japan) was cultured in dulbecco s modified eagle s medium (dmem) with 10% fetal calf serum (fcs). A human cervical carcinoma cell line (hela) (riken cell bank, tsukuba, japan) was cultured as described in previous studies [10, 14]. A cattle xlf gene (nm_001075393.1) with an artificial ecori site at the 5 end and bamhi site at the 3 end was synthesized . The fragment was confirmed by sequencing and ligated to the ecori and bamhi sites of the peyfp - c1 vector to give the in - frame fusion gene . Peyfp - cattle xlf, peyfp - cattle xlf (162299), peyfp - cattle xlf (162287) or peyfp - c1 was transient transfected in cells using fugene hd (promega, madison, wi, u.s.a .) The cells were cultured for 2 days and then monitored under an fv300 confocal laser scanning microscope (olympus, tokyo, japan) as previously described [9, 11, 12]. Immunoblotting: the extraction of total lysates and western blot analysis were conducted based on the previous methods [11, 13]. The membranes were blocked in blocking one (nacalai tesque, kyoto, japan) for 30 min . The following antibodies were used: a rabbit anti - xlf polyclonal antibody (a300 - 730a) (bethyl laboratories, montgomery, tx, u.s.a . ), a rabbit anti - gfp polyclonal antibody (fl) (santa cruz biotechnology, santa cruz, ca, u.s.a . ), a mouse anti - ku70 monoclonal antibody (n3h10) (neomarkers, fremont, ca, u.s.a .) Or a mouse anti--actin monoclonal antibody (sigma, st . Three antibodies (i.e., anti - xlf antibody, anti - ku70 antibody and anti - gfp antibody) were diluted in signal enhancer hikari (nacalai tesque), respectively . The binding to each protein was visualized using a select western blotting detection system (ge healthcare bio - sci . Piscataway, nj, u.s.a . ), in accordance with the manufacturer s instructions . Immunofluorescence staining: immunofluorescence staining was conducted as previously described [9, 11]. Briefly, the fixed cells were blocked for 10 min using a blocking solution and then incubated for 30 min at room temperature with a mouse anti-h2ax monoclonal antibody (jbw301) (upstate biotechnology inc ., charlottesville, va, u.s.a .) Or a rabbit anti - xlf polyclonal antibody (x4754) (sigma). After washing with pbs, detection of each protein was performed using alexa fluor 568-conjugated secondary antibodies (molecular probes, eugene, or, u.s.a . ). Local dna damage induction using laser and cell imaging: local dna damage induction using laser and cell imaging was conducted as described previously [9, 11,12,13]. Briefly, a 530% power scan (for 1 sec) from a 405 nm laser was used to induce local dsbs . Images of living cells or fixed cells expressing eyfp - tagged cattle proteins or eyfp alone were obtained using an fv300 confocal scanning laser microscopy system (olympus). Expression and localization of cattle xlf in cattle cells: we examined the expression and subcellular localization of xlf in cattle cells . First, we examined the expression of xlf and ku70 in the cattle cell line mdbk and the human cell line hela by western blot analysis using the anti - xlf antibody and anti - ku70 antibody . (a) total cell lysates from each cell line (mdbk, 50 g; hela, 10 g) were analyzed by western blotting using an anti - xlf antibody, an anti - ku70 antibody or an anti--actin antibody . (b, c) subcellular localization of xlf in cattle (mdbk) cells during the cell cycle . The images shown are a representative example for interphase cells or mitotic phase cells (c). These results demonstrate that the core nhej factors, xlf and ku70, are expressed in cattle cells . Total cell lysates from each cell line (mdbk, 50 g; hela, 10 g) were analyzed by western blotting using an anti - xlf antibody, an anti - ku70 antibody or an anti--actin antibody . (b, c) subcellular localization of xlf in cattle (mdbk) cells during the cell cycle . The images shown are a representative example for interphase cells or mitotic phase cells (c). To elucidate the localization of xlf in cattle cells, we studied the distribution of xlf by confocal laser microscopy (fig . 1b and 1c). Indirect immunofluorescence staining using the anti - xlf antibody showed that fluorescence was detected in the nucleoplasm of mdbk cells during the interphase . On the other hand, the fluorescence was detected throughout the cytoplasm of mdbk cells during the mitotic phase, but not in the condensed chromosomes of the mitotic cells . These observations indicate that the localization of cattle xlf changes dynamically during the cell cycle . To clarify the localization of xlf in living cattle cells during the interphase, we examined the expression and localization of eyfp - cattle xlf in mdbk cells . The expression vector peyfp - c1 containing cattle xlf (peyfp - cattle xlf) was transfected into mdbk cells (fig . (a) schematics of eyfp - cattle xlf chimeric protein and control protein (eyfp). (b) extracts from cattle (mdbk) cells transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western blotting using the anti - xlf, anti - gfp or anti--actin antibody . Living mdbk cells transiently expressing eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy . Eyfp images for the same cells are shown alone (left panel) or merged (right panel) with differential interference contrast images (dic) (center panel).). 2b, a signal of eyfp - cattle xlf was detected in the transfectants by western blot analysis using the anti - xlf antibody and anti - gfp antibody . By confocal laser microscopy, we clarified that eyfp - cattle xlf was localized in the nuclei of living interphase cells in eyfp - cattle xlf transfectants (fig . 2c). Expectedly, in eyfp transfectants, we confirmed that eyfp was distributed throughout the cell excluding the nucleolus (fig . 2c). (a) schematics of eyfp - cattle xlf chimeric protein and control protein (eyfp). (b) extracts from cattle (mdbk) cells transiently expressing the eyfp - cattle xlf or eyfp prepared and subjected to western blotting using the anti - xlf, anti - gfp or anti--actin antibody . Living mdbk cells transiently expressing eyfp - cattle xlf or eyfp were analyzed by confocal laser microscopy . Eyfp images for the same cells are shown alone (left panel) or merged (right panel) with differential interference contrast images (dic) (center panel). Eyfp - cattle xlf accumulates quickly at dsbs induced by laser microirradiation: we examined whether eyfp - cattle xlf accumulates quickly at the 405 nm laser - induced dsb sites (fig . 3afig . (a) the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser irradiation were examined . (b) imaging of living eyfp - cattle xlf - transfected mdbk cells before (upper panel) and at 1 min after (lower panel) microirradiation . Left panel, eyfp - cattle xlf; right panel, differential interference contrast images (dic). (c) immunostaining of microirradiated eyfp - cattle xlf - transfected cells with anti-h2ax antibody . The cells were fixed and stained with the anti- h2ax antibody at 5 min postirradiation . Left panel, eyfp - cattle xlf; center panel, h2ax image; right panel, merged image . (d) time - dependent eyfp - cattle xlf accumulation in living cells (5 - 120 sec) after irradiation . Upper panel, eyfp - cattle xlf; lower panel, differential interference contrast images (dic).). As shown in fig . 3b, we observed that eyfp - cattle xlf accumulated at the microirradiated sites in the living cattle cells . Next, we investigated whether cattle xlf accumulated at 405 nm laser - induced dsb sites by immunostaining with an antibody that detects h2ax . As shown in fig . 3c, eyfp - cattle xlf colocalized with the dsb marker h2ax at microirradiated sites in mdbk cells . Next, we carried out time - lapse imaging of eyfp - cattle xlf - transfected mdbk cells . 3d, we observed eyfp - cattle xlf accumulation at the microirradiated sites 5 sec after irradiation . In eyfp - cattle xlf - transfected cells, these results reveal that after irradiation, eyfp - cattle xlf quickly accumulates and formes foci at laser - induced dsbs in living cells . (a) the localization and accumulation of eyfp - cattle xlf at dsbs induced by 405 nm laser irradiation were examined . (b) imaging of living eyfp - cattle xlf - transfected mdbk cells before (upper panel) and at 1 min after (lower panel) microirradiation . Left panel, eyfp - cattle xlf; right panel, differential interference contrast images (dic). (c) immunostaining of microirradiated eyfp - cattle xlf - transfected cells with anti-h2ax antibody . The cells were fixed and stained with the anti- h2ax antibody at 5 min postirradiation . Left panel, eyfp - cattle xlf; center panel, h2ax image; right panel, merged image . (d) time - dependent eyfp - cattle xlf accumulation in living cells (5 - 120 sec) after irradiation . Upper panel, eyfp - cattle xlf; lower panel, differential interference contrast images (dic). The c - terminal region (ctr) of cattle xlf is essential for the nuclear localization and recruitment of xlf to dsbs in cattle cells: to determine the region essential for nuclear localization of cattle xlf, we investigated the localization of cattle xlf and its mutant . Firstly, the peyfp - cattle xlf and its mutants were transfected into mdbk cells . As shown in fig . 4.the c - terminal region (ctr) is vital for the nuclear localization and recruitment of cattle xlf to dsbs in vivo . (a) extracts from cattle (mdbk) cells transiently expressing the indicated cattle xlf deletions were prepared and subjected to western blotting using the anti - gfp or anti--actin antibody . (b, c) identification of essential domain of cattle xlf for nuclear localization and for accumulation at dsbs . The localization and accumulation of the chimeric proteins at laser - induced dsbs were investigated via live cell imaging . The results are summarized on the right: cellular localization (n, nucleus; c, cytoplasm) and formation of focus (+, accumulated at microirradiated sites; -, not accumulated at microirradiated sites). (d) immunostaining of microirradiated eyfp - cattle xlf (162287)-transfected cells with anti-h2ax antibody . The cells were fixed and stained with the anti-h2ax antibody at 5 min postirradiation . Left panel, eyfp - cattle xlf (162 - 287); center panel, h2ax image; right panel, merged image . (e) identity between the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf (amino acids 288299). (f) the alignment of the primary sequence among homologous xlf proteins . For comparison, the basic (red) or non - basic residues (black), a signal of each eyfp - cattle xlf mutant was detected in the extracts of each transfectant by western blot analysis using the anti - gfp antibody . By confocal laser microscopy, we confirmed that eyfp - cattle xlf was localized in the nuclei of living interphase cells . We observed that n - terminal deletion mutant eyfp - cattle xlf (162299) localized predominantly in the nuclei, whereas eyfp - cattle xlf (162287) as well as eyfp was distributed throughout the cell excluding the nucleolus in mdbk cells (figs . 2c, 4b and 4c). These results indicate that 12 c - terminal amino acids (amino acids 288299) of cattle xlf are vital for the nuclear localization of xlf in cattle cells (fig . Which region of cattle xlf is essential for its accumulation at dsbs in vivo, we tested whether the xlf mutant proteins could be recruited to dsbs induced by microirradiation . We observed that a n - terminal deletion mutant eyfp - cattle xlf (162299) as well as the wild type eyfp - cattle xlf, accumulated at the dsbs sites in the living mdbk cells (fig . 3c and data not shown). On the other hand, the mutant protein eyfp - cattle xlf (162287) failed to accumulate at the dsbs sites, which was detected by the dsb marker h2ax (fig . 4c and 4d), indicating that deletion of the c - terminal end 12 amino acids abolished the recruitment of cattle xlf to dsbs . The cattle and human xlf proteins are 81.9% identical at the amino acid level, whereas the c - terminal domain (amino acids 220299) of cattle xlf retains only 56.4% identity to human (fig . Interestingly, we confirmed that the basic amino acids in the ctr of xlf are evolutionarily highly conserved among humans and domestic animal species, e.g., cattle, goats, horses and avian, but not in yeast (fig . 4f and data not shown), which strongly suggests the biological significance of the xlf ctr in domestic animals . The c - terminal region (ctr) is vital for the nuclear localization and recruitment of cattle xlf to dsbs in vivo . (a) extracts from cattle (mdbk) cells transiently expressing the indicated cattle xlf deletions were prepared and subjected to western blotting using the anti - gfp or anti--actin antibody . (b, c) identification of essential domain of cattle xlf for nuclear localization and for accumulation at dsbs . The localization and accumulation of the chimeric proteins at laser - induced dsbs were investigated via live cell imaging . The results are summarized on the right: cellular localization (n, nucleus; c, cytoplasm) and formation of focus (+, accumulated at microirradiated sites; -, not accumulated at microirradiated sites). (d) immunostaining of microirradiated eyfp - cattle xlf (162287)-transfected cells with anti-h2ax antibody . The cells were fixed and stained with the anti-h2ax antibody at 5 min postirradiation . Left panel, eyfp - cattle xlf (162 - 287); center panel, h2ax image; right panel, merged image . (e) identity between the cattle xlf and human xlf at the amino acid level and the ctr of cattle xlf (amino acids 288299). (f) the alignment of the primary sequence among homologous xlf proteins . For comparison, the basic (red) or non - basic residues (black) to develop next - generation chemotherapeutics for cancer and other disease is important to clarify the molecular mechanisms of c - nhej . Human xlf is the most recently identified core nhej factor, and it appears to play essential roles in c - nhej . Expectedly, xlf - deficient cells derived from human patients and from knockout mice show ionizing radiation sensitivity [3, 15]. In addition, sirna - mediated downregulation of xlf in human cell lines leads to radiosensitivity and impaired nhej . . Homologues of the xlf gene were predicted in several eukaryotic organisms . On the other hand, the expression, function and regulation mechanism of xlf have not been elucidated in animal species other than mice and humans [1, 3, 11, 16, 21]. Domestic cattle are important domestic animals as livestock and draft animals in not only japan, but also many countries . Recently, cattle have been an ideal animal model for assessing chronic radiation exposure [5, 20]. However, the molecular mechanism of c - nhej is still unknown in cattle cells . In this study, we examined the expression and subcellular localization of cattle xlf and its mutants in cattle cell line mdbk . We found that xlf as well as other core nhej protein ku70 is expressed in cattle cells, and the localization of cattle xlf changes dynamically during the cell cycle . In addition, xlf might play a vital role in the repair of dsb immediately after microirradiation of cattle cells . Moreover, our data showed that the ctr of cattle xlf is vital for the nuclear localization of xlf and for the accumulation of xlf at dsbs in vivo . These findings suggest that the mechanisms regulating of the localization and recruitment to dsbs play a key role in the function of cattle xlf . Cattle xlf (nm_001075393.1) as well as goat xlf (xp_005676612.1) and sheep xlf (xp_004004987.1) is a 299-amino acid protein . The cattle and goat xlf genes are 94.6% identical at the amino acid level . In addition, the cattle and sheep xlf genes are 94.3% identical at the amino acid level . On the other hand, human xlf is a 299-amino acid protein, which contains an n - terminal head domain (amino acids 1141), a coiled - coil central domain (amino acids 142230) and a non structured c - terminal domain (amino acids 231299) [1, 2, 17]. Comparison with other eukaryotic homologues shows a high degree of sequence similarity within the 220 n - terminal amino acids, while the c - terminal domain (amino acids 225299) is less conserved [1, 2, 17]. On the basis of experimental findings, there are some reports concerning the role of the c - terminal domain of xlf in humans, but not in animals including cattle . Yano et al . Reported that a 10-amino - acid deletion at the c - terminal end completely abolishes the ku - xlf interaction and the accumulation of xlf at dsbs . On the other hand, malivert et al . Have reported that the c - terminal end (amino acids 231299) of human xlf is dispensable for dna repair in vivo . In this study, our data showed that a 12-amino - acid deletion at the c - terminal end abolishes the accumulation of cattle xlf at dsbs . Altogether, we conclude that the xlf ctr is important for the accumulation of xlf at dsbs in both human and cattle cells, although the role of the c - terminal region of xlf remains controversial in human cells . It was demonstrated that there is a general absence of conservation in the 75 c - terminal amino acids among humans and other species, although the extreme c terminus of xlf contains a small conserved basic cluster, which was proposed as a putative nls (krkk) [1, 2]. Our data revealed experimentally that the ctr of cattle xlf is critical for the nuclear localization of xlf and recruitment to dsbs, whereas the n - terminal domain is not essential . In addition, basic amino acids in the ctr of cattle xlf are evolutionarily conserved among ctr of domestic animals, which suggests the common biological significance of the xlf ctr in domestic animals . We consider that there is only one nls (kvkrkklr) in cattle xlf, and the nls is a classical monopartite nls having a single cluster of basic amino acid residues . We speculate that the c - terminus of 75 amino acids is important for a specific function in each species, whereas the xlf ctr is critical for the regulation of common functions in domestic animals ., we showed that xlf is expressed in cattle cells and the localization of cattle xlf changes dynamically during the cell cycle . In addition, our data showed that the localization and recruitment of cattle xlf to dsb sites at an early stage following irradiation are dependent on the ctr . These basic informations might be useful to develop the molecular - targeting therapeutic drug taking xlf as a target molecule for human and domestic animals . Further studies to elucidate the mechanisms regulating cattle xlf at dsbs will lead to a better understanding of the physiological function of xlf not only in cattle cells, but also in cells of human and other domestic animals . Inherited mutations of core c - nhej factors (e.g., dna - pkcs, dna ligase iv and xlf), have been discovered in humans . On the other hand, inherited mutations of the dna - pkcs, which cause scid, have been identified in not only humans, but also domestic animals, i.e., mice, horses or dogs . Therefore, further comparative studies might provide available information for the development of new clinical medicines and new chemoradiotherapies for humans and domestic animals including cattle.
Folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis . Our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells . This modification can be used for successful targeting of cancer cells expressing the folate receptor . Many conventional cancer chemotherapies are ineffective because of an inability to reach the tumor site in effective concentrations.1 there is little doubt that nanoparticles offer new opportunities in many fields.2 nanotechnology is expected to revolutionize medicine . Nanostructures can play a major role in medicine, especially in cancer diagnosis and therapy.3 magnetic nanoparticles have been investigated for various biomedical applications, nanoparticles, and prospected in diagnostic research for magnetic resonance eg, fe3o4 imaging and application of nanotechnologies in medicine.4 magnetic nanoparticles could enhance therapeutic effects and reduce side effects of drugs when used in combination with conventional cancer treatment.5 the combination of fe3o4 magnetic nanoparticles with different chemotherapeutics may provide new strategies in the treatment of specific cancer cells.6 moreover, fe3o4 nanoparticles are the only magnetic nanomaterials approved for clinical use by the us food and drug administration, and the preparation method is relatively simple.7 we aimed to determine whether the anticancer effects of methacrylamido - folic acid (ma - fol) would have improved anticancer activity if incorporated into magnetic nanoparticles . We demonstrated that magnetic fe3o4 nanoparticles coupled with folic acid can inhibit tumor proliferation and induce apoptosis of cancer cells in a dose- and time - dependent manner . The folate receptor is significantly overexpressed on the surface of human cancer cells.8,9 folate receptor - mediated drug delivery is based on conjugation with folic acid, which is internalized by folate receptor - mediated endocytosis . Folic acid has been immobilized on superparamagnetic particles,10 polymer nanoparticles,11 and incorporated into dendrimer - based therapeutic nanodevices12 for selective targeting of tumor cells . Folate receptors exhibit limited expression on healthy cells, but are often present in large numbers on cancer cells.13 folic acid receptors are overexpressed by epithelial cancers in the ovary, mammary gland, colon, lung, prostate, nose, throat, and brain,14 so represent an important target for tumor - specific delivery of anticancer drugs . Apoptosis, or programmed cell death, is an active process characterized by cytoplasmic shrinkage, chromatin condensation, nuclear fragmentation, and activation of caspases.13 in addition, phosphatidylserine is exposed on the external surface of the cell in the early phase of apoptosis, and this exposure precedes membrane damage and dna fragmentation.15 on the other hand, necrosis is passive, and is characterized by cell swelling, rupture of the plasma membrane, and cell lysis, with leakage of cytoplasmic components, such as lactate dehydrogenase.13 in the present study, folic acid was coupled on the surface of fe3o4 for selective binding to cancer cells and immobilized on the surfaces of magnetic nanoparticles, to disperse particles and improve their cell internalization and target cancer cells, respectively . Further, the apoptotic effects of ma - fol - modified fe3o4 nanoparticles were determined in a 5rp7 (h - ras - transformed rat embryonic fibroblasts) and in a nih/3t3 control cell line (normal mouse embryonic fibroblasts) by flow cytometry and transmission electron microscopy (tem). Nanoparticles are generally internalized into cells via fluid - phase endocytosis,16,17 receptor - mediated endocytosis, or phagocytosis . One strategy to realize efficient and specific cellular uptake of nanoparticles is to modify the nanoparticle surface with a ligand that is efficiently taken up by target cells via receptor - mediated endocytosis.18 the objective of this research was to assess the potential effects of fe3o4 magnetic nanoparticles modified with ma - fol on 5rp7 cancer cells and nih/3t3 cells . Ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution . A solution of methacryloyl benzotriazole19,20 in dioxane (15 ml) was added to the folate solution . Completion of the reaction was monitored by thin layer chromatography, which showed free 1h - benzotriazole spot . Reaction mixture was extracted with ethyl acetate (3 20 ml) to remove 1h - benzotriazole . Water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield . H nuclear magnetic resonance (nmr, 500 mhz, dmso - d6) was as follows: 8.56 (s, 1 h), 7.59 (d, jhh = 8.80 hz, 1 h), 6.65 (d, jhh = 8.80 hz, 1 h), 5.565.53 (m, 1 h), 5.035.00 (m, 1 h), 4.45 (s, 2 h), 4.05 (dd, 3 jhh = 5.05, 7.0 hz, 1 h), 2.252.15 (m, 2 h), 2.101.90 (m, 2 h), 1.96 (s, 3 h) ppm . C nmr (125 mhz, dmso - d6): 174.0, 173.9, 167.2, 167, 164.9, 164.2, 153.1, 151.9, 151.4, 149.4, 141.4, 129.5, 126.7, 118.7, 112.8, 110.6, 53.9, 45.7, 30.1, 27.9, 18.7 ppm . Superparamagnetic iron oxides (size 2050 nm), were obtained from sigma - aldrich chemical co (st . Louis, mo). The surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene, adding 0.5 ml trimethoxysilyl propyl methacrylate . These silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide (sigma - aldrich chemical co). 5rp7 (h - ras - transformed rat embryonic fibroblasts) and nih/3t3 (a control cell line of normal mouse embryonic fibroblasts) were used in these experiments . Cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium, supplemented with 10% fetal bovine serum, 2 mm l - glutamine, 50 iu / ml penicillin, and 50 mg / ml streptomycin ., the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution (0.25% trypsin, 1 mm ethylenediamine tetra - acetic acid) for 2 minutes at 37c to detach the cells, and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin . The cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks . Cell viability was determined through staining with trypan blue, and cells were counted using a hemocytometer . To study the cellular uptake of the nanoparticles by flow cytometry and tem, ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml, 4.5 g / ml, and 9 g / ml . The cells were cultured and then reseeded with the nano - particle - dispersed culture media . After 24 and 48 hours of incubation at 37c, the cells were washed twice with phosphate - buffered saline, detached using trypsin - ethyl - enediamine tetra - acetic acid solution, and resuspended in culture media . In control cultures, the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density . Annexin v - fluorescein isothiocyanate (fitc) is a sensitive probe for identifying apoptotic cells . It binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine, a membrane phospholipid, than for most other phospholipids.21 in apoptotic cells, phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria (bd cor poration, bedford, ma) flow cytometer was used for analysis of the cells . A recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit (bd corporation) as described by the manufacturer . Cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1 10 cells / ml, after which 100 l of solution (1 10 cells) was transferred to a 5 ml culture tube . Annexin v - fitc 5 l and propidium iodide 5 l were added, and the cells were then incubated for 15 minutes at room temperature in the dark, after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria . Uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells, as well as nanoparticle size and morphology, were determined using a tem (fei tecnai biotwin). Cells were deposited on formvar - coated 200300 mesh copper grids and dried, fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer (ph 7.4), and left in phosphate - buffered saline overnight at 4c . After being embedded in agar and after fixation in 2% osmium tetroxide, the cells were dehydrated in graded ethanol, ie, 70, 90, 96, and 100% . They were thin - sectioned using a diamond knife to a maximum thickness of 100 nm . Ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution . A solution of methacryloyl benzotriazole19,20 in dioxane (15 ml) was added to the folate solution . Completion of the reaction was monitored by thin layer chromatography, which showed free 1h - benzotriazole spot . Reaction mixture was extracted with ethyl acetate (3 20 ml) to remove 1h - benzotriazole . Water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield . H nuclear magnetic resonance (nmr, 500 mhz, dmso - d6) was as follows: 8.56 (s, 1 h), 7.59 (d, jhh = 8.80 hz, 1 h), 6.65 (d, jhh = 8.80 hz, 1 h), 5.565.53 (m, 1 h), 5.035.00 (m, 1 h), 4.45 (s, 2 h), 4.05 (dd, 3 jhh = 5.05, 7.0 hz, 1 h), 2.252.15 (m, 2 h), 2.101.90 (m, 2 h), 1.96 (s, 3 h) ppm . C nmr (125 mhz, dmso - d6): 174.0, 173.9, 167.2, 167, 164.9, 164.2, 153.1, 151.9, 151.4, 149.4, 141.4, 129.5, 126.7, 118.7, 112.8, 110.6, 53.9, 45.7, 30.1, 27.9, 18.7 ppm . Superparamagnetic iron oxides (size 2050 nm), were obtained from sigma - aldrich chemical co (st . Louis, mo). The surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene, adding 0.5 ml trimethoxysilyl propyl methacrylate . These silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide (sigma - aldrich chemical co). 5rp7 (h - ras - transformed rat embryonic fibroblasts) and nih/3t3 (a control cell line of normal mouse embryonic fibroblasts) were used in these experiments . Cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium, supplemented with 10% fetal bovine serum, 2 mm l - glutamine, 50 iu / ml penicillin, and 50 mg / ml streptomycin ., the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution (0.25% trypsin, 1 mm ethylenediamine tetra - acetic acid) for 2 minutes at 37c to detach the cells, and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin . The cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks . Cell viability was determined through staining with trypan blue, and cells were counted using a hemocytometer . To study the cellular uptake of the nanoparticles by flow cytometry and tem, ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml, 4.5 g / ml, and 9 g / ml . The cells were cultured and then reseeded with the nano - particle - dispersed culture media . After 24 and 48 hours of incubation at 37c, the cells were washed twice with phosphate - buffered saline, detached using trypsin - ethyl - enediamine tetra - acetic acid solution, and resuspended in culture media . In control cultures, the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density . Annexin v - fluorescein isothiocyanate (fitc) is a sensitive probe for identifying apoptotic cells . It binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine, a membrane phospholipid, than for most other phospholipids.21 in apoptotic cells, phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria (bd cor poration, bedford, ma) flow cytometer was used for analysis of the cells . A recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit (bd corporation) as described by the manufacturer . Cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1 10 cells / ml, after which 100 l of solution (1 10 cells) was transferred to a 5 ml culture tube . Annexin v - fitc 5 l and propidium iodide 5 l were added, and the cells were then incubated for 15 minutes at room temperature in the dark, after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria . Uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells, as well as nanoparticle size and morphology, were determined using a tem (fei tecnai biotwin). Cells were deposited on formvar - coated 200300 mesh copper grids and dried, fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer (ph 7.4), and left in phosphate - buffered saline overnight at 4c . After being embedded in agar and after fixation in 2% osmium tetroxide, the cells were dehydrated in graded ethanol, ie, 70, 90, 96, and 100% . They were thin - sectioned using a diamond knife to a maximum thickness of 100 nm . A logical method to promote internalization of nanoparticles is to modify their surface with a ligand such as folic acid which can be efficiently taken up by cells through receptor - mediated endocytosis.23 folic acid binds to the folate receptors at cell surfaces with very high affinity and is internalized by receptor - mediated endocytosis.24,26 more importantly, folic acid is stable, poorly immunogenic, and has the ability to target cancer cells preferentially because the folate receptor is frequently overexpressed on the surface of cancer cells . Flow cytometry is a process whereby physical or biochemical parameters of single biological cells or particles are measured as the cells move through a fluidic channel.27 the annexin v binding assay provides a very specific, rapid, and reliable technique to detect apoptosis by flow cytometry or by fluorescence microscopy.28,29 fitc is a very useful fluorescent moiety that can be used to label essentially any protein . Fitc - conjugated annexin v is used to detect apoptotic cells in a diverse range of cell types and in response to many different proapoptotic stimuli.28,29 iron nanoparticles were successfully modified with ma - fol as described in the methods section . Ma - fol was used to target preferentially cancer cells with folate receptors expressed on their surfaces, and to facilitate the nanoparticles to transit across the cell membrane . Annexin v - fitc allows detection of cell surface changes that occur early during apoptosis, because annexin v binds to phosphatidylserine which becomes exposed on the outer surface of the plasma membranes by flipping from the inner side of the membrane.22,30,31 exposure of phosphatidylserine during apoptosis precedes nuclear changes . This change in membrane surface is important for macrophages to recognize cells undergoing apoptosis.30,32 annexin v, an anticoagulant protein, binds to phosphatidylserine with high affinity . Thus, by staining cells with annexin v - fitc and additionally with dna - specific fluorochrome, eg, propidium iodide, it is possible to identify live cells, early apoptotic cells, as well as late apoptotic and necrotic cells, by flow cytometry.22,33 subsequently, annexin v - fitc binds to cells expressing phosphatidylserine, an early marker of apoptosis on the cell surface . Target cells are gated upon as propidium iodide - negative and quantified with respect to their annexin v positivity . The shift from annexin v to annexin v is a discrete event, such that all target cells fall within discernible populations with respect to annexin v. dead or dying cells are then stained with annexin v - fitc.34 annexin v has been used to detect apoptotic cells in a wide variety of cell types . Externalization of phosphatidylserine has been demonstrated in plasma membrane permeability changes, as measured by propidium iodide uptake and annexin v binding, and precedes the morphological features of apoptosis as assessed by flow cytometric analysis of cell shrinkage.29,34 the sensitivity and early kinetics of annexin v binding make it an ideal marker for cell death in a flow cytometric assay.34 analysis of annexin v / propidium iodide - stained cells by flow cytometry allows quantitation of the fraction of cells that are annexin v - negative and propidium iodide - negative (double negative), annexin v - positive and propidium iodide - negative (single positive), or annexin v - positive and propidium iodide - positive (double positive).28 a number of detectors are aimed at the point where the stream passes through the light beam, ie, one in line with the light beam (forward scatter) and several perpendicular to it (side scatter) and one or more fluorescent detectors . Forward scatter correlates with the cell volume, and side scatter depends on the inner complexity of the particle (ie, shape of the nucleus, amount and type of cytoplasmic granules, or membrane roughness).35 data analysis was performed with diva software (bd facs diva software 6.0; bd corporation, bedford, ma). Apoptosis was quantitatively confirmed by analyzing the percentage of early apoptotic cells using annexin v - fitc / propidium iodide double staining . A marker of early apoptosis, measured by annexin - v, is phosphatidylserine, which is released as a result of redistribution of the plasma membrane of the cells.22 three main populations of cells were distributed in dot - plots for viable cells q3 (annexin v - negative / propidium iodide - negative), early apoptotic cells q4 (annexin v - positive / propidium iodide - negative), and late apoptotic and necrotic cells q2 the cytotoxicity of folic acid - modified nanoparticles was determined by exposing normal cells and cancer cells to various concentrations in dulbecco s modified eagle s medium for 24 and 48 hours . The results and respective percentages of cells in apoptotic regions for both nih/3t3 and 5rp7 cells are given in figures 1 and 2 . The results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol - modified nanoparticles was 4.5 g / ml or below in cancer cells . As the concentration increased to 9 g / ml, the rate of viability was close to that of normal cells . These results confirm that the cells are saturated with ma - fol - modified nanoparticles at a concentration of 4.5 g / ml . Up to this concentration figure 2 shows cell death due to the ma - fol - modified magnetic nanoparticles at 4.5 g / ml for 24 hours in cancer cells . When cells were exposed to a nanoparticle concentration of 4.5 g / ml for 24 hours, cell survival decreased to 83% . When incubation time was extended to 48 hours as shown in figure 2, folic acid - modified nanoparticles caused worse damage to cancer cells than to normal cells . The population of annexin v - positive live cells increased in a time - dependent manner, and most (12.9%) of the cells became annexin v - positive at 48 hours (figure 2). When cancer cells were incubated with modified magnetic ma - fol nanoparticles for 48 hours (9 g / ml), the population of annexin v - positive live cells was found to be 7.3% (figures 2a and 2b). Cells in the late stages of apoptosis are located in the bottom right quadrant of the dot - plot as double positive annexin v - fitc- and propidium iodide - binding cells because, at this stage, the cell membranes are damaged and propidium iodide has penetrated into the cell . In the later stages of apoptosis, propidium iodide enters the cell, leading to a double positive population.28 cells undergoing necrotic cell death also stain as double positive for annexin v and propidium iodide, as discussed earlier . Flow cytometry by this double staining method enables clear detection of three populations of cells (viable, apoptotic, and necrotic). Thus, the early apoptotic cells bind only to annexin - v fitc, and late apoptotic cells to both annexin v - fitc and propidium iodide, and viable cells do not take up any of the dye . The annexin v - fitc / propidium iodide population was considered to reflect normal healthy cells, whereas annexin v - fitc - positive / propidium iodide - negative cells were taken to show early apoptosis . Annexin v - fitc -positive / propidium iodide - negative cells were in late apoptosis or necrosis.35 our flow cytometric analysis has shown that48 hours of treatment with ma - fol - modified magnetic nanoparticles caused apoptosis in a 5rp7 cell line in a concentration - dependent manner . Ma - fol - modified magnetic nanoparticles at a dose of 4.5 g / ml showed 12.9% cells in the apoptotic zone in the case of nih/3t3 cells, and the same concentration produced apoptosis in 4.7% of cells in dot - plot analysis . Late apoptosis and necrosis increased steadily following the increase at a concentration of 4.5 g / ml . Flow cytometric determination of cell cycle phase distribution has further confirmed that folic acid - modified magnetic nanoparticles cause cell cycle deformation in a cancer cell line . The results of this study confirm that cancer cells have more folate receptors, and that a 4.5 g / ml concentration of folic acid - modified magnetic nanoparticles causes apoptosis in 5rp7 cells . The uptake of folic acid - modified nanoparticles by cancer cells was also much higher than that of normal cells . This indicates that folic acid modification not only facilitates the nanoparticles to target specific cells, but also increases the yield of cell internalization . The interaction between folic acid and folate receptors expressed on the surface of cancer cells might have contributed to the improvement of nanoparticle uptake, based on receptor - mediated endocytosis.37 the schemes for the modification of nanoparticles display tem images (figure 3) of ma - fol - modified with fe3o4 nanoparticles for this purpose, firstly, magnetic nanoparticles were imaged . Figure 4 shows the tem images of nih/3t3 cells not treated with ma - fol - modified magnetic nanoparticles . Figure 5 shows the uptake of folic acid - modified nanoparticles into normal cells at 24 hours and 48 hours . The viability of normal cells after each incubation time with the nanoparticles was close to that of control cells, and was in the 95%96% range in flow cytometric analysis . The uptake of magnetic nanoparticles by cancerous rat 5rp7 fibroblasts is shown in figure 7 . After 48 hours of culture in media containing folic acid - modified nanoparticles, the morphology and viability of normal fibroblast cells containing the modified nanoparticles were close to that of the control cells, suggesting biocompatibility of the nanoparticles . The tem and flow cytometry results showed that folic acid - modified nanoparticles were internalized into both normal cells and cancer cells . The immobilization of folic acid on the nanoparticles was demonstrated by increasing the amount of nanoparticle uptake into cancer cells in comparison with normal cells . This suggests that the modification of magnetic nanoparticles with ma - fol could be used to resist nonspecific uptake and thus avoid their recognition by macrophage cells, and simultaneously facilitate nanoparticle uptake to specific cancer cells for cancer therapy . In flow cytometry results, the population of annexin v - positive live cells in the cancer cell line increased in a time - dependent manner, and most (12.9%) of the cells became annexin v - positive at 48 hours at a concentration of 4.5 g / ml . The results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol modified nanoparticles is 4.5 g / ml or below in cancer cells . As the concentration increased to 9 g / ml, the rate of viability was close to that of normal cells . These results confirm that cells saturated with folic acid - modified nanoparticles are detected in the 4.5 g / ml concentration . Up to this concentration, magnetic nanoparticles were aggregated and had difficulty penetrating the cells . In tem analysis, the concentration of 4.5 g / ml was used to compare the cancer cells and normal cells at 24 and 48 hours . In normal cells, organelles and cell structures were not affected by the presence of ma - fol - modified nanoparticles inside the cells (figure 5). However, cancer cells treated with the 4.5 g / ml concentration of ma - fol - modified nanoparticles underwent apoptosis . A representative picture of intracellular nanoconjugate uptake by nih/3t3 cells during a 24-hour and 48-hour treatment is shown in figures 5 and 6 . The amount of intracellular uptake of the nanoparticles can be seen in normal cells, whereas in the 5rp7 cell line the amount of the nanoconjugate was increased and cell modifications were damaged, figure 7 clearly illustrates the internalization of ma - fol - modified nanoparticles and their localization near the nucleus and a great number of apoptotic particles . This picture shows clearly that nanoconjugate uptake was maximum, with 5rp7 cells having maximum receptor expression, and hence the most intense color of the pellet, whereas the uptake gradually decreased with reduction in folate receptor expression and, accordingly, the intensity of the color of the pellet in the case of nih/3t3 . The picture confirms our hypothesis about the targeting efficacy of nanoconjugates containing folic acid, ie, that a positive correlation exists between maximum folate receptor expression and maximum uptake of nanoconjugates . The 5rp7 cells with 90% receptor expression showed maximum uptake, and the nih/3t3 cells showed the least . This is further substantiated by tem and flow cytometry analysis, confirming that folic acid - modified magnetic nano - particles can be used as a targeting agent . Ma - fol - modified magnetic nanoparticles represent a potential novel delivery system for compounds with anticancer activity because the folate receptor is frequently overexpressed in cancer cells . Moreover, it may increase our ability to target drugs to tumor cells, protect the drug from in vivo degradation, and reduce drug toxicity . This modification containing folic acid can be used for successful targeting of tumor cells expressing the folate receptor . Future studies will focus on determining the stability and targeting efficacy of this modification in vivo . This study has important implications in cancer cell imaging, tumor ablation, and drug delivery because of its targeting efficacy . Furthermore, it needs to be investigated whether nanoparticles could cause long - term changes in systemic activity . However, the effect and usage of such combinations need to be investigated further in in vivo experiments.
This is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax . Data on age, weight, height, and smoking habits are collected routinely when students join the institute, and then the sport in which the student is injured in the low back is registered in the institute health service files . Only students injured in the first or second year were taken into account, because the following years involve only athletics and gymnastics . Body mass index (bmi; kg / m) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . In this study we used the standard bmi with categories of underweight (bmi of <18.5), normal weight (bmi of 18.5<25), overweight (bmi of 25<30), and obesity (bmi of 30). Smoking was categorized as non - smoker, <20 cigarettes per day, and 20 cigarettes per day . Bmi, smoking habits, and history of any pain or injuries were taken for all the students in the beginning of the first grade . On the basis of the collected data, participants were dichotomized into those with or without lbp, while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . In 1 week, all students practice for a total of 16.5 h, spending 1.5 h in each of the following sports: soccer, handball, basketball, volleyball, judo, weightlifting, swimming, athletics, and gymnastics . Lbp was defined as pain or discomfort in the low - back region, from the lower rib curvature to the lower part of the seat region . The question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past . Furthermore, every registered injury causing lbp was recorded with its severity, but the most important detail for us was the sport in which the student was injured . Students who were injured more than one time were taken into account as subjects with lbp only for their first injury . The scientific council of the university of sfax in tunisia gave written permission for the study protocol . Statistical analyses were done using the spss statistical package, version 11.5 (spss, inc ., chicago, il, usa). The chi - square test was used to test associations between lbp and other categorical variables (sex, obesity, and smoking habit), and to compare the prevalence of lbp between males and female . Statistical significance this is a retrospective cross - sectional survey involving students at the higher institute of sport and physical education in the city of sfax . Data on age, weight, height, and smoking habits are collected routinely when students join the institute, and then the sport in which the student is injured in the low back is registered in the institute health service files . Only students injured in the first or second year were taken into account, because the following years involve only athletics and gymnastics . Body mass index (bmi; kg / m) was determined for each participant by dividing the weight in kilogram by the square of the height in meter . In this study we used the standard bmi with categories of underweight (bmi of <18.5), normal weight (bmi of 18.5<25), overweight (bmi of 25<30), and obesity (bmi of 30). Smoking was categorized as non - smoker, <20 cigarettes per day, and 20 cigarettes per day . Bmi, smoking habits, and history of any pain or injuries were taken for all the students in the beginning of the first grade . On the basis of the collected data, participants were dichotomized into those with or without lbp, while indicating the sports discipline in which they were injured or that they were injured after cumulative fatigue due to the large number of hours spent in practicing sports and physical activities . In 1 week, all students practice for a total of 16.5 h, spending 1.5 h in each of the following sports: soccer, handball, basketball, volleyball, judo, weightlifting, swimming, athletics, and gymnastics . Lbp was defined as pain or discomfort in the low - back region, from the lower rib curvature to the lower part of the seat region . The question used systematically by the doctor of the institute health service to identify and estimate lbp cases was about the present injury and if and when they had lbp in the past . Furthermore, every registered injury causing lbp was recorded with its severity, but the most important detail for us was the sport in which the student was injured . Students who were injured more than one time were taken into account as subjects with lbp only for their first injury . The scientific council of the university of sfax in tunisia gave written permission for the study protocol . Statistical analyses were done using the spss statistical package, version 11.5 (spss, inc ., chicago, il, usa). The chi - square test was used to test associations between lbp and other categorical variables (sex, obesity, and smoking habit), and to compare the prevalence of lbp between males and female . Statistical significance table 2 shows that the prevalence of lbp was significantly higher (p<0.001) among females (17.6%) than among males (12.5%). Lbp occurred in 17.1% of those classified by bmi as lean, 14.8% of those in the normal range, 14.3% among those who were overweight, and 12.7% of those who were obese . In addition, 13.9% of students smoking <20 cigarettes and 13.3% of students smoking 20 cigarettes had lbp . Prevalence of lower back pain (lbp) by gender, obesity and smoking lbp: low back pain; standard body mass index (bmi) categories: underweight (bmi of<18.5), normal weight (bmi of 18.5<25), overweight (bmi of 25<30), and obese (bmi of 30); smoking: cigarettes per day; no smoking: not currently smoking . In the analysis of risk factors for lbp in sports training, we found that in both sexes combined, lbp was positively associated with fatigue (caused by the long time spent in training), gymnastics, judo, handball, and volleyball . The increased prevalence of lbp is less in basketball and athletics and even lower in soccer, weightlifting, and swimming (fig . Percent prevalence of lower back pain related to fatigue and type of sports in males and females combined . Figure 2 shows that there is no significant difference (p>0.05) in the prevalence of lbp among males and females separately if they are compared in each practiced sport at the institute . However, if we analyze the prevalence of lbp in terms of sex in each sport, we find that the prevalence of lbp was significantly higher among males than females in handball (p<0.05) and significantly lower in athletics (p<0.05). Percent prevalence of lower back pain related to fatigue and type of sports in males and females . Its incidence and prevalence are so high that it should be studied as an epidemic and social disorder (35). The first salient feature that emerges from this study is that lbp is widespread among students in high schools for athletes (14.8%). The students were injured mainly in gymnastics, judo, handball, and volleyball, in addition to lbp caused by fatigue due to the long time spent in training . These findings are generally in accordance with the literature . In a study focused on identifying the types of physical activity associated with increased occurrence of lbp in schoolchildren, skoffer and foldspang (6) noted that lbp was increased by the number of hours jogging, playing handball, and doing gymnastics . In addition, sato et al . (33) found that the odds ratio for the risk of experiencing lbp was significantly higher in most sports items compared with the no - sports group . The most frequently implicated sports were volleyball, athletics, judo, gymnastics, golf, and rugby, where the odds ratio exceeded 2 . Kirialanis et al . Reported that injuries among gymnasts are anatomically widely distributed (36). As in our study, much research (6, 33, 37) noted that gymnastics was associated with an increased prevalence of lbp . Judo is a grappling style martial art with emphasis on dynamic throws and submissions, which can lead to substantial injuries due to the high velocity of the maneuvers (38). We registered a high prevalence of lbp in judo, as noted in the study of sato et al . Handball, which also includes repeated sudden and often rather violent movements, has been shown to be associated with lbp in our population and in other studies (6, 33). We observed a high prevalence of lbp associated with the fatigue caused by the long time spent in physical activity (16.5 h per week). . However, this point of view is attractive in terms of prevention as physical activity is amenable to behavioral intervention, and physical training programs at school and leisure activities may be adjusted to aim at strengthening the back (6). Moreover, both physical inactivity and intensive sports activity have been associated with lbp in some studies (28, 32, 3941). Our results show that a greater amount of physical activity increased the risk of lbp mainly in gymnastics, judo, handball, and volleyball . The time spent in athletics and basketball was associated with lbp to a lesser extent . Moreover, it appears that soccer, weightlifting, and swimming are correlated with relatively low prevalence of lbp . Skoffer and foldspang (6) reported that of several sports activities, only swimming was associated with a decreased prevalence of lbp . Swimming activates the muscles of the trunk, for example, the erector muscles of the spine, so swimming might be a means to prevent lbp . However, our results on soccer are at odds with those of bejia et al . (42), who reported that soccer was positively associated with chronic lbp in tunisian schoolchildren and adolescents . Similarly, our findings on weightlifting contrast with those of calhoon and fry (43), who reported that lbp was one of the most frequent injuries in this sport . The relatively low prevalence of lbp in our study in soccer and weightlifting may be due to the system of this sports and physical education institute which is focused mainly on knowing how to teach these sports (gestural technique and pedagogy) and not on researching performance . Our study shows that the percentage of females (17.6%) with lbp was significantly higher than that of males (12.5%), as frequently reported in the literature (1217). However, the prevalence rates of lbp do not differ significantly between males and females (p>0.05) if they are compared in each practiced sport separately . There was no significant difference in lbp by bmi (p>0.05), in males and in females combined as reported in other studies (44, 45). In addition, many other studies have reported that active smoking is a risk factor for back pain (47, 48). However, we did not find a significant relationship between smoking and lbp (p>0.05). (49) was that parental smoking seemed to increase the risk of spinal pain for both genders . We believe that students should perform exercises to strengthen the muscles of the arms and back, and increase coordination of the upper extremities and the lumbar region with regular activities that have been proven to be effective for the prevention of lbp . Swimming or gymnastics are the nearest sports to reach this aim as they seem to reduce lbp significantly (50). This study was a survey based on data from the institute health service files of 8 years and therefore had some limitations . To closely evaluate the relationship between lbp and sports activities, more information on the students is needed, including basic information such as their physical activity outside the institute . In this study, the diagnostic of lbp was performed by the doctor of the institute health service and mostly without medical imaging, which could have shown the severity of lbp . Imaging is used to show the severity of the injury rather than the severity of the pain associated with it . Lbp was strongly associated with the amount of time reserved to practice the different sports planned in this sports and physical education institute . The sports identified as posing the greatest risk for lbp were gymnastics, judo, handball, and volleyball, followed by basketball and athletics . Imed ajili) and the administration of the sports and physical education institute of sfax (tunisia) for their assistance.
In modern society, health is a n important component, which requires sustained development (1). In recent decades, a person s health has become one of the most important human rights among social groups throughout the world (2). The meaning of health has been extended beyond borders, and different definitions have emerged, owing to diverse geographic regions and cultural views in societies (3). The definition of health may vary by cultural background, and may influence health practices and compliance to medical treatment (4). Individuals perceive health on the basis of their experiences of ill health (5). So, understanding health as a subjective experience can enable health care providers to manage the care giving procedure based on their clients needs . Furthermore, one of the most important aspects with regard to health perception, manifests when patients suffer from a chronic disease, through which their perception of health is affected (5, 6). Diabetes is incurable, and has the third highest mortality rate on the planet (8). The probability of the incidence and prevalence of this disease has increased recently (9). In 2004, it was estimated that by 2030, this number will have grown to 366 million (10) and in 2010, the number of people afflicted by diabetes was estimated to increase up to 439 million by 2030 (8). Current estimates indicate that the number of diabetic patients in iran is around 3.5 million out of a total population of just over 68 million (11). Previous studies have demonstrated that chronic illnesses such as diabetes can influence a person s perception of health (12). In addition, the attitudes and beliefs regarding health undoubtedly play an important role in promoting healthy behaviour (13). Therefore, the understanding of diabetic patients perceptions can help the development of precautionary programmes for diabetics (14). Although the concept of health is gaining more importance and it has constantly been improved and changed by the passage of time (15, 16), few studies are reported to have focused on health and illness, particularly with regard to diabetic patients belonging to different ethnic groups (17). Health is a relative concept; nonetheless, several criteria have been proposed for exploring it through various ethnic and cultural groups (2, 18). Prior studies have shown lack of awareness about the concept of health among iranian kurdish diabetic patients . Kurds are an ethnic group living in the middle east, particularly in iran, who are estimated to be the third largest ethnic group, constituting 9 percent of the total population (19). Consequently, investigating the perception of health - related meaning among this ethnic group can increase the knowledge about the nature of health and its effects . Therefore, this study explored the meaning of health as perceived by iranian kurdish diabetic patients in order to develop an appropriate multidisciplinary outlook toward this ethnicity of patients . Considering the aim of this research, a qualitative approach with a conventional content analysis approach was used . Content analysis is a unique qualitative method, which encompasses a host of analytic approaches that offer flexibility to a researcher, and are of theoretical and substantive interest with regards to the case being studied (20, 21). The research was conducted over a ten - month period during 2014, in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj, in the center of the kurdistan province in western - iran . A purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre . The inclusion criteria for the participants were: having kurdish ethnicity irrespective of gender, having diabetes type 2, while at the same time having a lapse of at least five years since the diagnosis of diabetes, the absence of psychiatric disorders, and finally, having the ability to communicate . The sample size was determined only when the interviewing stage reached saturation (22). The sample was restricted to 20 because no new data emerged after the twentieth interview . Therefore, the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon . In - depth, face - to - face, semi - structured interviews were used for data collection . The participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . Moreover, an interview guideline was defined to direct the interviews toward the aims of the study . The interviews began with a general question about health and its perception, and proceeded to seek a deep understanding of participants perception . Examples of questions asked during the interview were as follows: when do you feel healthy? Further, the patients were asked to describe all the factors that could affect their health . Twenty interviews were conducted in kurdish language; each interview was tape - recorded with the participants permission . Each interview spanned 4090 minutes (average duration: 60 minutes). Each interview was transcribed and then used as a guide for the other interviews . Two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews . The kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english . The researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish, while making them grammatically correct for english readers . Through these inquiries, the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results, including: the allocation of a proper place and sufficient time for data collection, due communication with participants, using the supplementary views of colleagues, reviewing the participants hand writing and examining the data by all engaged researchers . This study was approved by the research committee of the kurdistan university of medical sciences, no.572.14 . This study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences, no: muk.rec.1387.572.14 . All participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned . The participants were assured that they could leave the study at any time even after they had signed the consent form . They were assured that their care would not be affected if they chose not to participate in the study . They were also assured of the data confidentiality; this meant that their names and other significant details, that might reveal their identity, would not be published in the study report . All the participants names were changed into codes during the transcription of the interviews, data was locked in separate locations and the coded information was used for data analysis and discussions . In this study, the conventional content analysis was used for data analysis; this method is generally adopted with a study design which aims to describe a particular phenomenon . This type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited (20). The researchers defined the categories on the basis of the type of data and avoided using predetermined categories . This method is also described as inductive category development (23), where researchers do not use predetermined data groups . Researchers analysed all the data in order to gain a new insight and expand their knowledge about it . By analyzing the literature that they had collected on the participants, and reading the transcriptions of all the participants interviews word by word, one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives (20, 24). Considering the aim of this research, a qualitative approach with a conventional content analysis approach was used . Content analysis is a unique qualitative method, which encompasses a host of analytic approaches that offer flexibility to a researcher, and are of theoretical and substantive interest with regards to the case being studied (20, 21). The research was conducted over a ten - month period during 2014, in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj, in the center of the kurdistan province in western - iran . A purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre . The inclusion criteria for the participants were: having kurdish ethnicity irrespective of gender, having diabetes type 2, while at the same time having a lapse of at least five years since the diagnosis of diabetes, the absence of psychiatric disorders, and finally, having the ability to communicate . The sample size was determined only when the interviewing stage reached saturation (22). The sample was restricted to 20 because no new data emerged after the twentieth interview . Therefore, the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon . In - depth, face - to - face, semi - structured interviews were used for data collection . The participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . Moreover, an interview guideline was defined to direct the interviews toward the aims of the study . The interviews began with a general question about health and its perception, and proceeded to seek a deep understanding of participants perception . Examples of questions asked during the interview were as follows: when do you feel healthy? Further, the patients were asked to describe all the factors that could affect their health . Twenty interviews were conducted in kurdish language; each interview was tape - recorded with the participants permission . Two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews . The kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english . The researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish, while making them grammatically correct for english readers . Through these inquiries, the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results, including: the allocation of a proper place and sufficient time for data collection, due communication with participants, using the supplementary views of colleagues, reviewing the participants hand writing and examining the data by all engaged researchers . This study was approved by the research committee of the kurdistan university of medical sciences, no.572.14 . This study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences, no: muk.rec.1387.572.14 . All participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned . The participants were assured that they could leave the study at any time even after they had signed the consent form . They were assured that their care would not be affected if they chose not to participate in the study . They were also assured of the data confidentiality; this meant that their names and other significant details, that might reveal their identity, would not be published in the study report . All the participants names were changed into codes during the transcription of the interviews, data was locked in separate locations and the coded information was used for data analysis and discussions . In this study, the conventional content analysis was used for data analysis; this method is generally adopted with a study design which aims to describe a particular phenomenon . This type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited (20). The researchers defined the categories on the basis of the type of data and avoided using predetermined categories . This method is also described as inductive category development (23), where researchers do not use predetermined data groups . Researchers analysed all the data in order to gain a new insight and expand their knowledge about it . By analyzing the literature that they had collected on the participants, and reading the transcriptions of all the participants interviews word by word, one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives (20, 24). Twenty diabetic patients participated in this study; the participants included seven men with an age range from 53 to 80 years (average age: 68 years) and thirteen women with an age range from 32 to 55 (average age: 40 years). Meanwhile, the average number of years which had lapsed since diagnosis of diabetes was seven years with a range of 517 years . Based on the thoughts and feelings of the participants, the following three main themes emerged: the syndrome of the healthy body and the happy heart life without compulsory limitations the syndrome of the healthy body and the happy heart is an important theme that emerged in our study . This theme is a kurdish idiom concerning health . According to this theme (bivy bi), health marks the presence of particular characteristics, which is an established symbol in the history of kurdish literature . It refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity, satisfaction, and calmness of the mind . The syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person . Similarly, the participants wished good health to each other with the idea that health implies a healthy body and a contented heart . One of the participants in the study said, a person with a healthy and contented heart is well - behaved and kind to others (p1). The participants always used local terminology to describe their ideas of health, and their descriptions and perception of health indicated that health is tantamount to worshipping god, for them . Another participant said, health reflects a healthy and happy heart; physical well - being holds less importance as compared to the human spirit, and, both, the heart and the spirit need to be healthy the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries; sadness, or any family crises . Health means that you have achieved peace in your family and home, and as a member of your family, you have exhibited good behaviour . Health means that the child and his or her parents share a cordial relationship, and life is a gift from god that ensures calmness and safety (p 11). Another participant said, health means that we do not have any problems or complaints of illness (p6), and, health brings calmness, and it is like praying to god without having to think about stressful matters (p2), and, when you are healthy, you do nt have any complaints and anxiety, and so the few problems that people have to face seem petty when they have the support of their families . So, the complaints gradually go away (p 18) all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others . One of the women (43 years old) said, when i eat rice, i need more water, and then i know that i am not healthy but fatigued, and i feel like travelling to the mountains and running away for a longer time, and then i suffer from sleeplessness and my body feels weak (sometimes, i want to eat some comestibles that our family bought between us, but i cannot do that, and this is so hard for me when you can participate in a ceremony or accompany someone, do anything that you want, or eat anything that you like, you feel a sense of freedom from others and you can go anywhere without worrying about your medicines, this freedom is very fantastic, spirituality is subjective; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health . Participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them . Most of the participants primarily belonged to the category that perceived health as exalted spirituality, and believed in satisfying self and others and trusting and honoring god . A woman (52 years old) said, some of the people whose heart is near to god have good health, because honoring god helps to heal them, and when the spirit is healthy, the body is healthy . And, a healthy spirit in a healthy body, which believes in prayer and spirituality, finds calmness and peace (p 18), and, healthy patients who have the virtue to trust in god and are honest, do nt lose their spirituality, religion or prophet (p 11) another participant said, when i pray to god, i think about how fortunate i am to be born in this world, and then i am happy (p 6). The syndrome of the healthy body and the happy heart is an important theme that emerged in our study . This theme is a kurdish idiom concerning health . According to this theme (bivy bi), health marks the presence of particular characteristics, which is an established symbol in the history of kurdish literature . It refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity, satisfaction, and calmness of the mind . The syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person . Similarly, the participants wished good health to each other with the idea that health implies a healthy body and a contented heart . One of the participants in the study said, a person with a healthy and contented heart is well - behaved and kind to others (p1). The participants always used local terminology to describe their ideas of health, and their descriptions and perception of health indicated that health is tantamount to worshipping god, for them . Another participant said, health reflects a healthy and happy heart; physical well - being holds less importance as compared to the human spirit, and, both, the heart and the spirit need to be healthy the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries; sadness, or any family crises . Health means that you have achieved peace in your family and home, and as a member of your family, you have exhibited good behaviour . Health means that the child and his or her parents share a cordial relationship, and life is a gift from god that ensures calmness and safety (p 11). Another participant said, health means that we do not have any problems or complaints of illness (p6), and, health brings calmness, and it is like praying to god without having to think about stressful matters (p2), and, when you are healthy, you do nt have any complaints and anxiety, and so the few problems that people have to face seem petty when they have the support of their families . All the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others . One of the women (43 years old) said, when i eat rice, i need more water, and then i know that i am not healthy but fatigued, and i feel like travelling to the mountains and running away for a longer time, and then i suffer from sleeplessness and my body feels weak (p 13). Sometimes, i want to eat some comestibles that our family bought between us, but i cannot do that, and this is so hard for me for some participants, the limitations had led to the feeling that they were different from others when you can participate in a ceremony or accompany someone, do anything that you want, or eat anything that you like, you feel a sense of freedom from others and you can go anywhere without worrying about your medicines, this freedom is very fantastic in general, spirituality is subjective; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health . Participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them . Most of the participants primarily belonged to the category that perceived health as exalted spirituality, and believed in satisfying self and others and trusting and honoring god . A woman (52 years old) said, some of the people whose heart is near to god have good health, because honoring god helps to heal them, and when the spirit is healthy, the body is healthy . And, a healthy spirit in a healthy body, which believes in prayer and spirituality, finds calmness and peace (p 18), and, healthy patients who have the virtue to trust in god and are honest, do nt lose their spirituality, religion or prophet (p 11) another participant said, when i pray to god, i think about how fortunate i am to be born in this world, and then i am happy (p 6). As the results for this group of diabetic patients indicated, health is composed of the syndrome of the healthy body and the happy heart, life without compulsory limitations, and exalted spirituality . Many studies have shown that health perception is a reflection on, not only health but also health as a culture . For all patients, health perception and points of view are the important factors that affect health behaviour and determine any related behaviour (25, 26). An individual may be ill and yet may feel healthy; therefore, many people can compare their health status during suffering from illness with that in the past (22, 27). The themes that emerged in our study showed these instances briefly . In this study, participants talked about the effects of diabetes on physical and psychological health, which indicated that our results were similar to those of other studies (28, 29). The most important theme of this study was the syndrome of the healthy body and the happy heart . The patients always used local terminology for health description, and these terminologies have extended meanings . In this context, health is considered as a feeling that can affect all components of the human body, including their physical, psychological, social, emotional, and spiritual facets . This view about health is mainly accepted about diabetes because diabetes affects all aspects of a patient s life (11, 28, 29). In this study, participants admitted that, not only physical health but also vivacity, satisfaction, and calmness of mind, good relationship with family and good economic status are all considered as aspects of health . Hence, health care professionals should strongly consider the issue that health implies lack of having any problems, complaints, illnesses, etc . Furthermore, physical illness and its adverse effects are important topics in the health experiences of diabetic patients; however, when these patients were healthy, they said that without peace of mind one could not feel healthy . Moreover, in a study about health in spanish immigrants, ailinger and causey found that some concepts of health are related to the patients psychological point of view and peace of mind, calmness in family, absence of complaints, and so on (30). Assessed the effectiveness of self - efficacy and family inter - relationships among young turkish patients afflicted with diabetes type i (29). Their study showed that calmness of mind and some other related factors such as relationship among family members, conflicts or compatibility in the family, and emotional situations can affect self - efficacy, and accordingly, these factors affect their overall health . Hence, considering the important effect of calmness of mind on diabetic patients health perception and points of view, we, as health care professionals, ought to be obliged to associate a disturbed mind with bad health . Overall, by using these implications as the bases of patients culture, we can improve health programmes for all patients . One of the most plausible themes in our interviews was health as life without compulsory limitations . Miklaucich studied the limitations of life as meaningful health experiences in patients with coronary angina (31). Abazari et al . Also demonstrated that diabetic patients have dietary restrictions as well as limitations with regards to bearing children (18). Thomson & gifford indicated that trying to keep a balance was the preferred meaning of health for diabetic participants (32). It is more important for the professional health care teams to help diabetic patients feel that they are not different from others and in this regard, consultation with dieticians can help to obviate some part of this limitation of life . Studies also show that having a regular activity program and regular follow- up consultation with other health care professionals can help diabetic patients to expel limitations (33, 34). In this article, all participants talked about the importance of the spiritual dimension of health and achieving exalted spirituality . Some other studies showed that spirituality is one of the significant factors that can affect health; hence, spirituality plays a very important role in gaining adaptation to a lifestyle, with regards to chronic diseases, and helps patients to manage their health - related problems (35). This theme was plausible in traditional societies; hence, nursing professionals must pay more attention to this influential factor . Patients can adopt a spiritual approach by becoming a member of a religious group, praying, and meditating . Exalted spirituality, in fact, helps patients to cope with the disease, and feel better about their lives (36, 37). He showed that praying helps patients to not only connect with the higher power, but also to relate to each other . Their act of praying is a response to their anxieties about themselves or their families . Walton showed that women with chronic diseases, used prayer to calm themselves and connect with others to support each other and cope with diseases (39). Therefore, health care professionals should be aware of the effects of spirituality on patients having a chronic disease . The results of our study primarily indicated that the factors that affect health perception include complaining about the illness, side effects and outcomes of the disease, and the extent of the complaints that make the patient feel sick . Patients with chronic diseases encountered some social and emotional problems due to changes in their lifestyle (36). Diabetes affects patients quality of life, and especially their perception and attitude toward health (12). It fully depends on how patients and health professionals define health (40, 41). Due to chronic and acute complaints of diabetic patients, we need to create a programme aiming to reduce these adverse effects and promote self - care for diabetic patients . Especially, a program must be developed based on paying attention to body and mind simultaneously, trying to remove the limitation in life of patients by considering the extent of the limitations and limitations that exist for the patients which compel them to believe that the disease has been brought about just for them . This program should not affect or limit the beliefs and spiritual activity of patients because a person s previous experiences influence the perception of present time, which also impact on their future . Besides, having been brought up in a different society and within a different ethnic group, influences a person s health - related behavior . So, health care providers must manage care situations by enhancing health experience to enable patients lives with the least limitations, and giving them an opportunity to preserve exalted spirituality . Studying health perception among patients with chronic diseases is an important factor that facilitates the development of appropriate programmes, to improve health levels among such patients . In this regard, the professional health - care provider team ought to consider health perception among patients, and then they apply this knowledge for setting up suitable health improvement programs . Nurses need to pay more attention to diabetic patients limitations and facilitate a programme to improve their health in conjunction with other health care teams . Although nursing and other health care professionals have the responsibility to help those patients achieve peace of mind, the patients families should also play an active role in the health care programme and treatment process.
However, proper amount of daily water intake in a healthy individual is controversial . There is a prevalent thought that we all have to drink eight glasses of water daily, we try to clarify this topic from the perspective of avicenna and traditional persian medicine (tpm). The most important traditional persian medical encyclopedia, al - qanun fil - tibb (canon of medicine), was reviewed . Furthermore, medline, embase, scopus, iranmedex, and science iranian database (sid) focusing on the keywords traditional medicine, water intake, fluid, and complementary and alternative medicine were reviewed to find relevant information . Avicenna believed that the demand for daily water is not the same in different individuals . The determinant variables mentioned in his book, the canon of medicine, comprises mizaj (temperament), health status, age, sex, season, place, habits, occupation, etc . He believed that water in extra amount quenches the hararat - e - ghariezi, which is the basal internal heat to convey normal homeostasis and metabolism in the body . Several factors determine the actual need of any person to drink water . Consequently, recommending a specific amount of daily water intake for all is illogical . Moreover, important recommendations of tpm sages on an appropriate amount of water intake should be considered to prevent associated disorders.
A 34year - old male presented for general examination on day 49 of a 50-day self - imposed fast, seeking approval to continue his fasting behavior . During this period, the patient reported only consuming water, tea, coffee (with no milk or sugar), and a daily multivitamin tablet (centrum performance; pfizer). Patient history prior to fasting behavior revealed a family history of obesity and the presence of pathologies related to body mass . Personal history involved an oscillating mass between 85 and 134 kg over a 10-year period . At the start of fasting, the patient reported body mass as 96.8 kg, and 29.2% body fat, as recorded by a personal bioelectrical impedance device . History following the onset of fasting behavior was unremarkable, but included severe abdominal cramps between days 7 and 21 of fasting, development and frequent occurrence of vasovagal syncope on transition from sitting to standing, and the near - cessation of fecal movement . Self - reported physical activity and mobility was reduced, but not prevented; this participant was not bedridden . Substantial water and coffee intake was noted (812 cups of coffee daily and 23 bottles of water daily). As expected, body mass loss during fasting was severe and noticeable (fig . Resting metabolic rate (30 minutes by indirect calorimetry) was 1626 kcal day . During the 30-minute rest period resting ecg appeared morphologically normal, patient was bradycardic (52 bpm) and notably hypertensive (150/78 mmhg). Body composition was measured by whole - body air displacement plethysmography (bod pod) as 19.8% body fat, while height was 180.9 cm and mass was 75.4 kg, with a bmi of 23.5 kg m, all recorded on calibrated laboratory devices . Sum of four skinfolds1 was 30.0 mm, approximately half of that of a reference population of similarly aged males (62 25 mm, aged 3039). It was visually noted that subcutaneous fat in the abdominal region was still present, despite severe losses in the peripheral limbs, protruding ribs, and lack of prominent chest and shoulder musculature . 2). During a six - minute walk test, this individual covered 342 m, which was below expected performance for a healthy male adult.2 a mild scoliosis was observed while walking; however, it is unclear if this is related to fasting . Venous blood was taken for serum markers of metabolism, liver, and renal function . Noteworthy findings include plasma glucose of 3.1 mmol l and alkaline phosphatase of 36 u l. total bilirubin was 19 mol l, direct bilirubin was 7 mol l. uric acid was 620 mol l. urine was straw colored, lacked sediment, and was noticeably sweet in aroma . Rapid dipstick analysis (multistix 2161; siemens) was negative for blood glucose, contained trace proteins, and indicated urine ph of below 5 . One month following initial presentation, the patient returned for follow - up examination during which body composition, resting metabolic rate, urine, and venous blood were recorded . Resumption of feeding initially involved liquids such as vege table soups, with the resumption of solid foods three days following breaking of fast . Days 35 involved one solid meal per day, with 23 solid meals during days 57, and the resumption of three solid food meals per day for 7 days following breaking of fast . In the following 23 days, diet was reported to be of caffeine and alcohol free, low carbohydrate (a piece of bread every 13 days), and high servings of fruit and vegetables . Self - estimation of diet by volume was 20% animal protein (chicken and fish) and 80% fruit or vegetables; validated, quantifiable measures of intake and caloric content are not available . Heart rate remained bradycardic at rest (48 bpm), and blood pressure was notably reduced (132/78). Metabolic rate was reduced (1413 kcal day), and body composition was unchanged (75.0 kg; bmi, 23.4 kg m; 16.1% fat mass). Blood biochemistry returned to within normal range, with the exception of direct bilirubin (table 1). It is noteworthy, therefore, in this case study that moderate physical function appears maintained at day 49, as assessed by general observation and results of the six - minute walk test . Frommel et al.3 suggested that fasting in healthy lean individuals was well tolerated until 18% of body mass was lost; the patient reported here lost 20.7% of body mass without substantial loss of function . One prior report of long - term fasting in obese individuals for 3040 days demonstrated body mass losses between 10.6% and 20.5%, but does not report on function of individuals.4 it seems self - apparent and supported by this limited evidence, such that the starting mass is a key variable in survival of extreme fasting, and not the amount of loss . This patient started fasting while obese and ended his fast when anthropometric variables were within the targeted ranges for the general population . Indeed, among the few (nonpublished) examples of complete fasting and mortality,5 patients were frail between 30 and 50 days, and death was noted to occur between days 43 and 70, which this case study has reached without obvious or significant frailty occurring . Current world medical association guidelines prioritize autonomy, recommending nonintervention in fasting to the point of harm and/or death, if the patient makes a written informed statement of intent.5 lack of research into this field precludes evidence - based decision - making or advice . In the limited light of this case study and historic data of fasting in obesity,4 it is tempting to suggest that guidelines to physicians for advice to the fasting individual reflect body mass, and specifically mass of adiposity at start of fasting behavior . Survival in starvation is ultimately governed by physics, the calories available to maintain metabolic function . Availability of adipose tissue for metabolic usage may thus delay the catastrophic degradation of muscular protein, increasing survival time . That said, small animal models reveal organ - specific effects of starvation, with loss of liver and gut size and function noted after only four to six days.6 functional damage to digestive organs cannot be ruled out, and indeed, the elevated direct bilirubin is suggestive of reduced liver filtration . Of concern, direct bilirubin remained elevated 30 days post breaking of fast . Elevated uric acid is likely due to reduced kidney filtration, as has been reported in caloric restriction previously.7 further, hypertension was an unexpected finding . This finding is counter to one of the few published examinations of cardiology in severe starvation, where bradycardia and hypotension (~20 mmhg below fed state) were noted in juvenile pigs following 29 days of complete starvation.8 the porcine heart contains several structural differences relative to the human, most related to bipedal vs. quadrupedal stance.9 as frequent syncope was also noted in that our case study, it is plausible that dysregulated autonomic regulation of blood pressure underlies the observed hypertension . Blood pressure was measured in a supine position, increasing venous return in the human relative to standing position . Simple frank starling mechanics combined with a failure of autonomic regulation may thus explain elevated blood pressure . Thus, it is tempting to suggest that differences observed may be species dependent; however, this is highly speculative on a single case study . Further, long - lasting cardiovascular deficits were noted in the above porcine model on refeeding, as well as chronically following experimental conclusion.8 as death resulting from starvation often occurs via cardiovascular complications, this elevated blood pressure during fast is key to note; this should be carefully monitered in fasting individuals . Caloric restriction and weight loss in overweight or obese individuals is often associated with subsequent regain of weight.10,11 having access to this case study 30 days following resumption of feeding was of interest due to a chance to examine any occurrence of such a rebound effect . It is of interest to note that this was not seen here; however, future gains in weight cannot be ruled out . Here, we report the maintenance of physical function of an individual after 49 days of complete fast . This report does not indicate support for such behavioral choices, and the projections of metabolic energy provision by starting adiposity assume no other perturbations to homeostasis, such as impaired immune response, liver and renal function, or cardiovascular events.
Tangential tractions on the vitreomacular interface are usually responsible for the development of idiopathic macular holes . The prevalence is 3.3/1000 and it shows a strong female predominance.1 in the past, it was known as a rare, untreatable disorder leading to central loss of vision . In 1991, kelly and wendel2 reported that idiopathic macular holes could be closed by vitrectomy and gas tamponade . With a better understanding of the pathogenesis of macular hole development and innovations in macular surgery, one of the important developments in the surgical approach is peeling of the internal limiting membrane (ilm). Some authors have argued that ilm peeling has beneficial effects on the surgical outcome,3,4 whereas others have stated that this approach does not affect surgical success,5,6 and may lead to complications by increasing the surgery duration.7,8 the ilm is formed by mller cell extensions and functions as a kind of basal membrane between retina and vitreus, acting as a surface for glial cell proliferation . Vitreoretinal interface changes due to cellular proliferation lead to ilm distortion and eventually to the development of epiretinal membrane (erm), macular hole, and recurring macular edema.9 with the removal of the ilm, successful results may be obtained in surgical treatment of such vitreoretinal diseases . The most difficult aspect of surgery is due to the tight attachment of the ilm to the retina and its extreme thinness and transparent nature . To increase the visibility of the ilm indocyanine green (icg) and trypan blue have been used by many researchers in peeling of the erm and ilm . There are different opinions on the amount and concentration of dye required, and exposure duration in the vitreous space . In many studies, because of intraretinal accumulation of icg and its toxic effects on retinal pigment epithelium, visual field defects after surgery and possible optical nerve atrophy have been considered.8,10,11 recently, brilliant blue (bb) has been used in preclinical studies as it has minimal toxic effect and provides effective membrane staining.9,12 in this study, the anatomical and visual outcomes of pars plana vitrectomy (ppv) and ilm peeling with bb in the treatment of idiopathic macular holes has been examined . Fifty eyes of 48 patients who had 23 g ppv and ilm peeling as a treatment for macular holes between july 2007 and december 2009 were retrospectively evaluated in the authors clinic . All patients underwent measurement of best - corrected visual acuity and intraocular pressure, anterior segment and fundus examinations before surgery . The possible merits and risks of the treatment were explained to the patients, and informed consent was obtained from all patients in accordance with the helsinki declaration . Inclusion criteria were: diagnosis using noncontact - lens biomicroscopy and optical coherence tomography (oct) of stages 2, 3, and 4 idiopathic macular hole according to the gass classification . Exclusion criteria were: trauma history; previous macular surgery, rhegmatogenous retinal detachment together with macular hole; myopia higher than 10d; macular hole for more than 2 years; and previous retinal vessel disease . Macular examination was performed before surgery and 1, 3, and 6 months after surgery using spectral oct (optovue inc, fremont, ca). All patients had a 23 g transconjunctival three port pars plana vitrectomy . In four eyes having both macular hole and erm, both ilm and erm peeling were performed . To visualize the ilm, 0.3 ml, 16% c3f8 gas was injected into 28 eyes (56%) and 18% c2f6 gas into 22 eyes (44%). Follow - up time points included 1-day, 1-week, 1-month, 3-month, and 6-month postsurgical evaluations and thereafter visits at 3-month intervals . Best - corrected visual acuity, intraocular pressure, and complications were recorded for each visit . Anatomical and functional outcomes of macular hole patients who underwent 23 g ppv and ilm peeling with bb were evaluated . Cases in different macular hole stages were compared in terms of anatomical and functional results . All operations were performed under local anesthesia . Following the introduction of a 23 g infusion cannula at the inferotemporal site, imaging systems used were the volk miniquad xl (volk optical ltd, mentor, oh) during vitrectomy and volk central retina (volk optical ltd) during ilm peeling . After core vitrectomy, triamcinolone acetonide (kenacort - a; bristol myers squibb, new york, ny) was used to remove the posterior hyaloid . Brilliant blue 0.3 ml (fluoron gmbh, ludwigsfeld, germany) was injected to the posterior pole via a 23 g dualbore cannula . After 60 seconds, peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper (synergetics inc, fort collins, co) and 23 g eckardt end - gripping forceps (dorc, zuidland, the netherlands) for an area of at least 3 disc diameters . After liquid air exchange, air gas exchange was performed (16% c3f8 or 18% c2f6). None of the patients required sutures . All operations were performed under local anesthesia . Following the introduction of a 23 g infusion cannula at the inferotemporal site, imaging systems used were the volk miniquad xl (volk optical ltd, mentor, oh) during vitrectomy and volk central retina (volk optical ltd) during ilm peeling . After core vitrectomy, triamcinolone acetonide (kenacort - a; bristol myers squibb, new york, ny) was used to remove the posterior hyaloid . Brilliant blue 0.3 ml (fluoron gmbh, ludwigsfeld, germany) was injected to the posterior pole via a 23 g dualbore cannula . After 60 seconds, peeling of the ilm was performed using a 23 g diamond - dusted membrane scraper (synergetics inc, fort collins, co) and 23 g eckardt end - gripping forceps (dorc, zuidland, the netherlands) for an area of at least 3 disc diameters . After liquid air exchange, air gas exchange was performed (16% c3f8 or 18% c2f6). The patient group consisted of 33 (66%) female and 17 (34%) male subjects (mean age sd; 63.34 9.6). Of these cases, preoperative oct examination revealed that, according to gass classification, 17 eyes (34%) had stage 2 macular holes, 24 eyes (48%) had stage 3, and 9 eyes (18%) had stage 4 . Anatomic success was assessed by the closure of the hole on oct imaging and the withdrawal of subretinal fluid . The hole was closed in 46 (92%) of the 50 eyes after surgery but not closed in 4 (8%) eyes . When postoperative oct controls revealed that subretinal fluid had disappeared, there was no need for a second surgery . Hole persistence was observed in only one patient 2 months after the surgery and it was closed by reoperation . Figure 3 shows pre- and postoperative third - month spectral oct imaging of a patient with a stage 4 macular hole . The mean best - corrected visual acuity of all patients before surgery was 0.71 0.25 logmar . Postoperative mean best - corrected visual acuity (and comparison with preoperative values) was obtained as: 0.48 0.26 at 3 months (p <0.05), 0.52 0.27 at 6 months (p <0.05), 0.42 0.30 at 9 months (p <0.05), and 0.41 0.31 at 12 months (p <0.05). When assessed together, the final visits (at 12 months) demonstrated that visual acuity had increased in 41 eyes (82%), whereas, in 6 eyes (12%), visual acuity remained unchanged . Because of recurrent macular holes, three cases showed a decrease in visual acuity . Two of these three cases had stage 4 macular holes, and one had a stage 3 macular hole . Best - corrected visual acuity results of patients before and after surgery are given in figure 1 and table 2 . When evaluated according to the stage of macular hole, in stage 2 patients, all measurements of visual acuity after surgery showed that it was significantly higher when compared with preoperative measurements (p <0.05). Increase in visual acuity between 6 and 12 months after surgery was statistically significant due to cataract surgery (p <0.05). In stage 3 patients, preoperative visual acuity compared with postoperative 3-month scores showed an increase, but this was not statistically significant (p = 0.056). During the postoperative period, visual acuity reduced between months 3 and 6 due to cataract development . In patients who underwent phacoemulsification after 6 months, therefore, comparisons of postoperative 912-month values showed significant increases (p <0.05). In contrast, in stage 4 patients, visual acuity scores of all time points after surgery failed to reach statistically significant levels with respect to preoperative values . Therefore, the first month visual acuity measurements were excluded from analysis . In 22 phakic patients, the mean preoperative visual acuity of stage 2 macular hole patients was higher than that of stage 3 or 4 patients . Consequently, the most prominent increase in visual acuity was observed in the stage 2 group after surgery . There was no significant difference between stage 2 and 3 macular hole patients in terms of anatomical and visual outcomes, but the functional successes of these two groups were significantly higher than those of stage 4 patients . Iatrogenic retinal tears developed in four patients during surgery and were successfully treated intraoperatively . In one patient, a nasal retinal tear developed near the optic nerve while removing the posterior hyaloid . One of the phakic cases had preoperative lens opacity and vitrectomy and phacoemulsification was performed together with intracapsular lens implantation . Since the first report of kelly and wendel2 on the treatment of macular holes by pars plana vitrectomy and gas tamponade, anatomical and functional success rates have increased with developments in vitreoretinal surgery . Previous studies reported success rates over 90% after one session of surgery13,14 and anatomical success rates as high as 100% in stage 2 or the initial phase of stage 3 macular holes.15 in histopathological studies,16,17 various surgical approaches have been proposed based on the idea that collagen - containing myofibroblasts and actin - containing cells in the structure of ilm and erm may cause contraction and lead to hole formation or widening of an existing hole . Many studies1821 have reported that ilm peeling increases anatomical and functional success in the treatment of macular hole, and during the late phases it reduces reformation of the hole . Kwok et al18 compared anatomical closure of stage 3 or 4 holes in 40 patients with or without ilm peeling . Success rate was 89% in the ilm peeling group whereas it was 59% in the group without ilm peeling . The differences in anatomical closure success may be due to stage differences of macular holes, inclusion of traumatic cases, high myopic eyes, and recurrent cases . Although there is no consensus on the effects of ilm peeling on anatomical and functional success, publications stating that ilm peeling increases hole closure are in the majority.22,23 there are several opinions regarding which patients require ilm peeling . Previous studies have stated that ilm peeling should be considered especially in patients with a hole wider than 400 microns and that is chronic (6 months or longer), traumatic, or recurring.2125 ilm peeling was reported to increase anatomical success and prevent reopening of the hole by decreasing erm development.25,26 besides reports that suggest that ilm peeling increases anatomical success but not functional success,27 there are also reports that suggest functional outcomes are better in ilm peeling groups.18 in this study, ilm peeling was applied to all patients who underwent macular hole surgery . Anatomic closure was obtained in 46 (92%) of the 50 eyes, but the hole was not closed in four patients . Visual outcomes were significantly better in stage 2 or 3 patients than in stage 4 patients (p <0.05). The authors consider that ilm peeling improved anatomical and functional success rates in stages 2 and 3 patients whereas it had no effect on visual outcomes in stage 4 patients due to photoreceptor loss . Cataract development is the most prevalent complication of macular hole surgery in phakic eyes . In this study group, cataract development was observed in 23 (74%) of 31 patients due to an endotamponade application . Besides publications that report early cataract surgery following macular hole surgery may increase the risk of re - opening of the hole,28 there are also studies that report no such risk.29,30 therefore, in patients developing cataract, phacoemulsification surgery was performed at least 6 months after macular hole surgery to prevent a recurrence . As the thin and semitransparent structure of the ilm complicates visualization during surgery, in patients with ilm peeling, there may be small asymptomatic paracentral scotomas, irregularity in nerve fiber layers, and retinal microhemorrhages due to iatrogenic retinal trauma.3136 chromovitrectomy has been developed as a method to improve ilm visibility, shorten the duration of surgery, and reduce iatrogenic retinal trauma.36 many dyes, such as indocyanine green (icg), infracyanine green (ifcg), trypan blue (tb), bb, and triamcinolone acetonide (ta), are used to dye the ilm . Since icg was first introduced for ilm peeling in macular hole surgery, several potential side effects have been noted.37,38 visual field defects, retinal pigment epithelium, and ganglion cell defects are among the most reported side effects.26,32,33 dose - dependent biochemical damage to the retinal pigment epithelium and ganglion cells, photo - oxidative cell damage due to the phototoxic properties of icg, and retinal pigment epithelium damage due to hypoosmotic solution are considered responsible for icg - mediated ocular toxicity.3941 because of the potential toxic side effects of icg in chromovitrectomy, other vital dyes with minimal toxicity have been tried.32,33,38,42 ifcg, unlike icg, does not contain iodine, and it is widely thought that its toxicity to the retinal pigment epithelium is less than that of icg.31 triamcinolone acetonide was reported as both toxic4345 and nontoxic46 to retinal pigment epithelium in preclinical studies . In the authors opinion, there is an insufficient number of randomized clinical trials to make a conclusion . However, in some studies of ta - assisted ilm peeling, it was found similar to icg.43,47 another vital dye, tb, is recommended for erm staining because of its high affinity for proliferation - dense intraocular tissues.48 several preclinical studies with high - concentration tb reported toxic effects on tissue culture of retinal pigment epithelium.49,50 however, many other researchers claim that if it is used in lower concentrations, there will be no toxic effects.51,52 in this study, bb, a relatively new type of vital dye, was used to stain the ilm . Enaida et al9 studied icg on rats and found that low - dose icg administration resulted in no retinal cell damage but high dose icg led to morphological damage of retinal cells . In a rat study by hisatomi et al and enaida et al,53,54 low dose intravitreal bb administration caused no effects on retinal cells, but in higher doses electron microscopy revealed cyst formation in the inner layers of the retina . The authors of this present study think that biological adaptation to bb is better than to icg . The use of bb is easier than icg and tb because of its granular structure and easy dissolution in intraocular irrigation . When compared with bb, higher concentrations of icg are required to dye the ilm.12 in addition, bb is not a fluorescent dye, so its phototoxicity is lower than icg . The authors of this study have observed intraretinal accumulation in postoperative fundus fluorescein angiography of patients for whom icg was used . Ilm peeling in macular hole surgery provides beneficial effects on anatomical outcome, independent of disease stage . In terms of visual outcome
The study included 340 whites from italy with type 2 diabetes (according to american diabetes association 2003 criteria) and coronary artery disease (cad), as indicated by previous myocardial infarction (mi) or> 50% stenosis of at least one major vessel at coronary angiography, or both . These individuals were cases of the cross - sectional case - control gargano heart study (ghs) (30) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 . Ten patients became untraceable before the first follow - up visit; therefore, data were available for 330 patients . They all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines . Exclusion criteria were the presence of malignancies and a medical record of diabetes, although 22 study participants (15.6%) were found to have subclinical diabetes after an oral glucose tolerance test . Because recruitment is still in progress, only patients who were recruited up to 2007, and as such underwent at least one follow - up visit, were included in this study . Two patients became untraceable before the first follow - up; therefore, data were available for 141 patients . This study comprises 283 whites with end - stage renal disease (esrd), with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis (34). Exclusion criteria were dialysis for less than 6 months, left ventricular ejection fraction <35%, history of circulatory congestion, and hospitalization for intercurrent illness, including major infections . No dropouts were observed . Blood samples for dna extraction were unavailable for 17 subjects; thus, 266 patients were included . Of these, 43 (16.2%) had diabetes, a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy (35). The end point considered in all three samples was a major cardiovascular event, defined as nonfatal stroke, nonfatal mi, or cardiovascular death . Information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . If patients did not report to a scheduled visit, information on the occurrence of cardiovascular events was obtained by telephone interview, from their primary care physicians, or from death certificates . Deaths were ascribed to cvd according to the international classification of diseases codes: 410.0410.9, 415.1, 427.4427.5, 428.0428.9, 433.1, 434.1, 444.2, 444.8 (9th edition) or i21.0i21.9, i46.1, i49.0, i50.1, i62.9, i63.0i63.9, i71.3 (10th edition). A total of 339 white patients with type 2 diabetes who had survived an mi were studied . They are part of two ongoing investigations on the genetics of cad in type 2 diabetes (30,36,37). Of these, casa sollievo della sofferenza in san giovanni rotondo (gargano, center east coast of italy), as cases of the cross - sectional case - control ghs (29). Most of these patients (n = 160) were further studied for incident major cardiovascular events in the prospective ghs (see above). Another 170 were recruited in boston from the beth israel deaconess medical center (bidmc) and the joslin clinic (which serves as the bidmc diabetes clinic) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously (37). All subjects underwent a clinical examination and a standardized interview (at the time of recruitment and at each subsequent time point, if applicable), which was identical in all three prospective samples . A fasting blood sample (collected between 8:00 a.m. and 9:00 a.m.) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy . A random blood sample was obtained from the cross - sectional study participants recruited in boston . Bmi was calculated by dividing the weight (in kilograms) by the square of height (in meters). Presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg, or both, or the presence of antihypertensive treatment . Current and former smokers were considered as one group and compared with those who never smoked . All study protocols were approved by the local institutional review boards and performed according to the helsinki declaration . Genotyping of the enpp1 k121q polymorphism (rs1044498) was performed by taqman allele discrimination (assay c_16190162_10; applied biosystems, foster city, ca) on the ht7900 platform (applied biosystems). Patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables . Comparisons between genotype groups were performed by pearson or mann - whitney u tests for continuous or categoric variables, respectively . Because of the low number of qq individuals, only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype (q121) (i.e., kq heterozygotes + qq homozygotes) to k121 homozygotes (kk). In prospective studies, a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios (hrs) along with their 95% ci . The time to event was defined as the time between enrollment date and the date of the first cardiovascular event . For censored subjects, the time variable was defined as the time between the enrollment date and the date of the last available clinical data . The assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . In cross - sectional studies, the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis, and results are given as regression coefficients . Pooled data analyses were performed in an individual patient data meta - analysis fashion (38) (i.e., adjusting for study sample) after excluding genotype - by - sample interactions . Genotype - by - obesity interaction was tested by adding a cross - product term to the regression model . The discriminatory power of prediction models was assessed by estimating the survival c - index (39) and by measuring the integrated discrimination improvement (idi) (40). All analyses were performed using sas 9.1 software (sas institute, cary, nc). The study included 340 whites from italy with type 2 diabetes (according to american diabetes association 2003 criteria) and coronary artery disease (cad), as indicated by previous myocardial infarction (mi) or> 50% stenosis of at least one major vessel at coronary angiography, or both . These individuals were cases of the cross - sectional case - control gargano heart study (ghs) (30) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 . Ten patients became untraceable before the first follow - up visit; therefore, data were available for 330 patients . They all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines . Exclusion criteria were the presence of malignancies and a medical record of diabetes, although 22 study participants (15.6%) were found to have subclinical diabetes after an oral glucose tolerance test . Because recruitment is still in progress, only patients who were recruited up to 2007, and as such underwent at least one follow - up visit, were included in this study . Two patients became untraceable before the first follow - up; therefore, data were available for 141 patients . This study comprises 283 whites with end - stage renal disease (esrd), with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis (34). Exclusion criteria were dialysis for less than 6 months, left ventricular ejection fraction <35%, history of circulatory congestion, and hospitalization for intercurrent illness, including major infections . No dropouts were observed . Blood samples for dna extraction were unavailable for 17 subjects; thus, 266 patients were included . Of these, 43 (16.2%) had diabetes, a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy (35). The end point considered in all three samples was a major cardiovascular event, defined as nonfatal stroke, nonfatal mi, or cardiovascular death . Information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . If patients did not report to a scheduled visit, information on the occurrence of cardiovascular events was obtained by telephone interview, from their primary care physicians, or from death certificates . Deaths were ascribed to cvd according to the international classification of diseases codes: 410.0410.9, 415.1, 427.4427.5, 428.0428.9, 433.1, 434.1, 444.2, 444.8 (9th edition) or i21.0i21.9, i46.1, i49.0, i50.1, i62.9, i63.0i63.9, i71.3 (10th edition). A total of 339 white patients with type 2 diabetes who had survived an mi were studied . They are part of two ongoing investigations on the genetics of cad in type 2 diabetes (30,36,37). Of these, casa sollievo della sofferenza in san giovanni rotondo (gargano, center east coast of italy), as cases of the cross - sectional case - control ghs (29). Most of these patients (n = 160) were further studied for incident major cardiovascular events in the prospective ghs (see above). Another 170 were recruited in boston from the beth israel deaconess medical center (bidmc) and the joslin clinic (which serves as the bidmc diabetes clinic) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously (37). The study included 340 whites from italy with type 2 diabetes (according to american diabetes association 2003 criteria) and coronary artery disease (cad), as indicated by previous myocardial infarction (mi) or> 50% stenosis of at least one major vessel at coronary angiography, or both . These individuals were cases of the cross - sectional case - control gargano heart study (ghs) (30) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 . Ten patients became untraceable before the first follow - up visit; therefore, data were available for 330 patients . They all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines . Exclusion criteria were the presence of malignancies and a medical record of diabetes, although 22 study participants (15.6%) were found to have subclinical diabetes after an oral glucose tolerance test . Because recruitment is still in progress, only patients who were recruited up to 2007, and as such underwent at least one follow - up visit, were included in this study . Two patients became untraceable before the first follow - up; therefore, data were available for 141 patients . This study comprises 283 whites with end - stage renal disease (esrd), with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis (34). Exclusion criteria were dialysis for less than 6 months, left ventricular ejection fraction <35%, history of circulatory congestion, and hospitalization for intercurrent illness, including major infections . No dropouts were observed . Blood samples for dna extraction were unavailable for 17 subjects; thus, 266 patients were included . Of these, 43 (16.2%) had diabetes, a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy (35). The end point considered in all three samples was a major cardiovascular event, defined as nonfatal stroke, nonfatal mi, or cardiovascular death . Information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . If patients did not report to a scheduled visit, information on the occurrence of cardiovascular events was obtained by telephone interview, from their primary care physicians, or from death certificates . Deaths were ascribed to cvd according to the international classification of diseases codes: 410.0410.9, 415.1, 427.4427.5, 428.0428.9, 433.1, 434.1, 444.2, 444.8 (9th edition) or i21.0i21.9, i46.1, i49.0, i50.1, i62.9, i63.0i63.9, i71.3 (10th edition). A total of 339 white patients with type 2 diabetes who had survived an mi were studied . They are part of two ongoing investigations on the genetics of cad in type 2 diabetes (30,36,37). Of these, casa sollievo della sofferenza in san giovanni rotondo (gargano, center east coast of italy), as cases of the cross - sectional case - control ghs (29). Most of these patients (n = 160) were further studied for incident major cardiovascular events in the prospective ghs (see above). Another 170 were recruited in boston from the beth israel deaconess medical center (bidmc) and the joslin clinic (which serves as the bidmc diabetes clinic) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously (37). All subjects underwent a clinical examination and a standardized interview (at the time of recruitment and at each subsequent time point, if applicable), which was identical in all three prospective samples . A fasting blood sample (collected between 8:00 a.m. and 9:00 a.m.) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy . A random blood sample was obtained from the cross - sectional study participants recruited in boston . Bmi was calculated by dividing the weight (in kilograms) by the square of height (in meters). Presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg, or both, or the presence of antihypertensive treatment . Current and former smokers were considered as one group and compared with those who never smoked . All study protocols were approved by the local institutional review boards and performed according to the helsinki declaration . Written informed consent was obtained from each study participant . Genotyping of the enpp1 k121q polymorphism (rs1044498) was performed by taqman allele discrimination (assay c_16190162_10; applied biosystems, foster city, ca) on the ht7900 platform (applied biosystems). Patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables . Comparisons between genotype groups were performed by pearson or mann - whitney u tests for continuous or categoric variables, respectively . Deviations from hardy - weinberg equilibrium were investigated by exact test . Because of the low number of qq individuals, only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype (q121) (i.e., kq heterozygotes + qq homozygotes) to k121 homozygotes (kk). In prospective studies, a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios (hrs) along with their 95% ci . The time to event was defined as the time between enrollment date and the date of the first cardiovascular event . For censored subjects, the time variable was defined as the time between the enrollment date and the date of the last available clinical data . The assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . In cross - sectional studies, the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis, and results are given as regression coefficients . Pooled data analyses were performed in an individual patient data meta - analysis fashion (38) (i.e., adjusting for study sample) after excluding genotype - by - sample interactions . Genotype - by - obesity interaction was tested by adding a cross - product term to the regression model . The discriminatory power of prediction models was assessed by estimating the survival c - index (39) and by measuring the integrated discrimination improvement (idi) (40). A value of p <0.05 was considered significant . All analyses were performed using sas 9.1 software (sas institute, cary, nc). We studied three cohorts of subjects who were at very high risk of major cardiovascular events: the ghs individuals with type 2 diabetes and previously diagnosed cad; the tor vergata atherosclerosis study (tvas)individuals from the general population who had experienced an mi; and the cardiovascular risk extended evaluation in dialysis (creed)individuals with esrd who required dialysis . No significant differences in baseline characteristics across genotype groups were observed in any of the three studies . Clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers, percentage, or mean (sd). The average mean (sd) follow - up was 37.1 (19.4) months (range 191) in the ghs, 30.6 (11.3) months (range 137) in the tvas, and 36.3 (22.0) months (range, 169) in the creed . During follow - up, 43 major cardiovascular events occurred in the ghs, 39 in the tvas, and 94 in the creed, resulting in respective incidence rates of 4.2, 10.8, and 11.7 per 100 person - years (table 2). In all studies, incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes: 5.4 vs. 3.6 in the ghs, 19.2 vs. 8.1 in the tvas, and 14.1 vs. 10.8 in the creed (table 2). The difference was significant in the tvas (p = 0.025) and in a pooled analysis of the three studies (p = 0.005). No difference in the magnitude of the genetic effect was observed among studies (p = 0.32 for interaction). Incidence of major cardiovascular events in ghs, tvas, and creeds * per 100 person - years . The hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 (95% ci 0.802.70, p = 0.21) in the ghs, 2.31 (95% ci 1.224.34, p = 0.01) in the tvas, and 1.36 (95% ci 0.882.10, p = 0.16) in the creed (fig . 1a, b, and c, respectively; p = 0.32 for gene - by - sample interaction). In a pooled analysis (i.e., individual patient data meta - analysis) of the three studies, which included 737 subjects with 176 events, the hr for q121 carriers versus kk homozygotes was 1.56 (95% ci 1.152.12, p = 0.004; fig . Hr 1.04 [95% ci 1.031.06], p <0.0001), diabetes (2.23 [95% ci 1.503.31], p <0.0001), and smoking status (1.71 [95% ci 1.242.36], p = 0.001) were additional predictors of incident events . Bmi (hr 1.03 [95% ci 0.991.06], p = 0.08), hypertension (1.50 [95% ci 0.992.27], p = 0.054), and sex (1.30 [95% ci 0.951.79], p = 0.10) also tended to be associated with increased rate of events, although these did not reach statistical significance . The increased risk of events associated with the q121 variant remained significant (hr 1.55 [95% ci 1.142.12], p = 0.005) after adjusting for bmi and diabetes, both of which had been reported to be associated with the q121 variant (3840), as well as after further adjustment for age, sex, hypertension, and smoking status (hr 1.45 [95% ci 1.052.00], p = 0.022). Kaplan - meier survival curves are shown for major cardiovascular events in ghs (a), tvas (b), and creed (c). The addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age, sex, bmi, smoking status, hypertension and diabetes, as indicated by the survival c - index, which went from 0.704 to 0.713 (p = 0.94), or by the idi, with 0.42% improvement (p = 0.16). Given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance (2527,29,30,3941), we investigated this hypothesis in our study . In the ghs, which entirely consisted of patients with type 2 diabetes, we indeed observed a significant interaction between the q121 variant and obesity . An association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m (hr 3.56 [95% ci 1.2110.5], p = 0.02), but not among the 171 individuals who had a bmi <30 kg / m (0.91 [95% ci 0.402.06], p = 0.82; p = 0.039 for interaction). No evidence of gene - by - obesity interaction was instead observed in the tvas (p = 0.53) and the creed (p = 0.41). Because approximately 85% of these two cohorts consisted of nondiabetic individuals, we hypothesized that the genotype - by - obesity interaction might be specific to diabetes . Indeed, a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes, even though the small sample size prevented statistical significance (data not shown). Thus, we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . In the diabetic stratum (n = 395), the q121 variant was associated with an increased risk of incident events among the 177 obese (fig . 2b) individuals, with adjusted hrs of 5.94 (95% ci 1.8818.78, p = 0.002) vs. 0.62 (95% ci 0.321.24, p = 0.18). The interaction between the q121 variant and obesity was significant (p = 0.003). By contrast, no evidence of interaction was observed in the nondiabetic stratum (n = 344, p = 0.26), with adjusted hrs of 0.82 (95% ci 0.223.11, p = 0.77) in the 39 obese individuals and 1.85 (95% ci 1.182.90, p = 0.008) in the 305 nonobese subjects . Survival curves for major cardiovascular events in obese (a) and nonobese (b) patients with type 2 diabetes . Curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . Among obese diabetic individuals, the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index (p = 0.81). A much larger effect, approaching statistical significance, was observed when the improvement was assessed by idi with a 4.56% improvement (95% ci 0.27 to 9.42, p = 0.09). To seek replication of the gene - by - obesity interaction observed in patients with diabetes, we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi . One sample was from the gargano area in italy, the other was from boston . Salient clinical features of the study subjects are summarized in table 3 . Because no significant genotype - by - sample interaction was observed in the association with age at mi (p = 0.11), pooled analyses were performed by adjusting for study sample . To make the analysis comparable to that of prospective studies, sex, smoking status, hypertension, and bmi, but not age (due to its collinearity with age at mi and diabetes because all study participants were diabetic) 64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes, at 54.5 (9.6) vs. 57.2 (8.9) years of age (p = 0.035). In contrast, no significant difference in age at mi was observed among nonobese subjects: 59.2 (10.5) in 41 q121 carriers versus 57.2 (10.4) in 110 kk homozygotes (p = 0.16; p = 0.025 value for q121-by - obesity interaction). Clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number, percentage, or mean (sd). Virtually identical results were obtained when duration of diabetes was also added to the multivariate model, with q121 carriers having had an mi at a significantly younger age than kk individuals among obese (p = 0.039) but not among nonobese (p = 0.20) individuals (p = 0.032 for interaction). In addition, when bmi was considered as a continuous trait, it was inversely related to the age at mi among q121 carriers (= 0.44 [95% ci 0.75 to 0.12], p = 0.008; fig . 3a), but not among kk individuals (= 0.17 [95% ci 0.41 to 0.07], p = 0.16; p = 0.069; for q121-by - bmi interaction; fig . 3b). Linear regression between bmi and age at mi in diabetic patients with q121 (a) and kk (b) genotypes . Data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston . We studied three cohorts of subjects who were at very high risk of major cardiovascular events: the ghs individuals with type 2 diabetes and previously diagnosed cad; the tor vergata atherosclerosis study (tvas)individuals from the general population who had experienced an mi; and the cardiovascular risk extended evaluation in dialysis (creed)individuals with esrd who required dialysis . No significant differences in baseline characteristics across genotype groups were observed in any of the three studies . Clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers, percentage, or mean (sd). The average mean (sd) follow - up was 37.1 (19.4) months (range 191) in the ghs, 30.6 (11.3) months (range 137) in the tvas, and 36.3 (22.0) months (range, 169) in the creed . During follow - up, 43 major cardiovascular events occurred in the ghs, 39 in the tvas, and 94 in the creed, resulting in respective incidence rates of 4.2, 10.8, and 11.7 per 100 person - years (table 2). In all studies, incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes: 5.4 vs. 3.6 in the ghs, 19.2 vs. 8.1 in the tvas, and 14.1 vs. 10.8 in the creed (table 2). The difference was significant in the tvas (p = 0.025) and in a pooled analysis of the three studies (p = 0.005). No difference in the magnitude of the genetic effect was observed among studies (p = 0.32 for interaction). Incidence of major cardiovascular events in ghs, tvas, and creeds * per 100 person - years . The hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 (95% ci 0.802.70, p = 0.21) in the ghs, 2.31 (95% ci 1.224.34, p = 0.01) in the tvas, and 1.36 (95% ci 0.882.10, p = 0.16) in the creed (fig . 1a, b, and c, respectively; p = 0.32 for gene - by - sample interaction). In a pooled analysis (i.e., individual patient data meta - analysis) of the three studies, which included 737 subjects with 176 events, the hr for q121 carriers versus kk homozygotes was 1.56 (95% ci 1.152.12, p = 0.004; fig . Age at study entry (hr 1.04 [95% ci 1.031.06], p <0.0001), diabetes (2.23 [95% ci 1.503.31], p <0.0001), and smoking status (1.71 [95% ci 1.242.36], p = 0.001) were additional predictors of incident events . Bmi (hr 1.03 [95% ci 0.991.06], p = 0.08), hypertension (1.50 [95% ci 0.992.27], p = 0.054), and sex (1.30 [95% ci 0.951.79], p = 0.10) also tended to be associated with increased rate of events, although these did not reach statistical significance . The increased risk of events associated with the q121 variant remained significant (hr 1.55 [95% ci 1.142.12], p = 0.005) after adjusting for bmi and diabetes, both of which had been reported to be associated with the q121 variant (3840), as well as after further adjustment for age, sex, hypertension, and smoking status (hr 1.45 [95% ci 1.052.00], p = 0.022). Kaplan - meier survival curves are shown for major cardiovascular events in ghs (a), tvas (b), and creed (c). The addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age, sex, bmi, smoking status, hypertension and diabetes, as indicated by the survival c - index, which went from 0.704 to 0.713 (p = 0.94), or by the idi, with 0.42% improvement (p = 0.16). Given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance (2527,29,30,3941), we investigated this hypothesis in our study . In the ghs, which entirely consisted of patients with type 2 diabetes, we indeed observed a significant interaction between the q121 variant and obesity . An association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m (hr 3.56 [95% ci 1.2110.5], p = 0.02), but not among the 171 individuals who had a bmi <30 kg / m (0.91 [95% ci 0.402.06], p = 0.82; p = 0.039 for interaction). No evidence of gene - by - obesity interaction was instead observed in the tvas (p = 0.53) and the creed (p = 0.41). Because approximately 85% of these two cohorts consisted of nondiabetic individuals, we hypothesized that the genotype - by - obesity interaction might be specific to diabetes . Indeed, a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes, even though the small sample size prevented statistical significance (data not shown). Thus, we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . In the diabetic stratum (n = 395), the q121 variant was associated with an increased risk of incident events among the 177 obese (fig . 2b) individuals, with adjusted hrs of 5.94 (95% ci 1.8818.78, p = 0.002) vs. 0.62 (95% ci 0.321.24, p = 0.18). The interaction between the q121 variant and obesity was significant (p = 0.003). By contrast, no evidence of interaction was observed in the nondiabetic stratum (n = 344, p = 0.26), with adjusted hrs of 0.82 (95% ci 0.223.11, p = 0.77) in the 39 obese individuals and 1.85 (95% ci 1.182.90, p = 0.008) in the 305 nonobese subjects . Survival curves for major cardiovascular events in obese (a) and nonobese (b) patients with type 2 diabetes . Curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . Among obese diabetic individuals, the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index (p = 0.81). A much larger effect, approaching statistical significance, was observed when the improvement was assessed by idi with a 4.56% improvement (95% ci 0.27 to 9.42, p = 0.09). To seek replication of the gene - by - obesity interaction observed in patients with diabetes, we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi . One sample was from the gargano area in italy, the other was from boston . Salient clinical features of the study subjects are summarized in table 3 . Because no significant genotype - by - sample interaction was observed in the association with age at mi (p = 0.11), pooled analyses were performed by adjusting for study sample . To make the analysis comparable to that of prospective studies, sex, smoking status, hypertension, and bmi, but not age (due to its collinearity with age at mi and diabetes because all study participants were diabetic) were included as covariates . Among obese subjects, 64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes, at 54.5 (9.6) vs. 57.2 (8.9) years of age (p = 0.035). In contrast, no significant difference in age at mi was observed among nonobese subjects: 59.2 (10.5) in 41 q121 carriers versus 57.2 (10.4) in 110 kk homozygotes (p = 0.16; p = 0.025 value for q121-by - obesity interaction). Clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number, percentage, or mean (sd). Virtually identical results were obtained when duration of diabetes was also added to the multivariate model, with q121 carriers having had an mi at a significantly younger age than kk individuals among obese (p = 0.039) but not among nonobese (p = 0.20) individuals (p = 0.032 for interaction). In addition, when bmi was considered as a continuous trait, it was inversely related to the age at mi among q121 carriers (= 0.44 [95% ci 0.75 to 0.12], p = 0.008; fig . 3a), but not among kk individuals (= 0.17 [95% ci 0.41 to 0.07], p = 0.16; p = 0.069; for q121-by - bmi interaction; fig . 3b). Linear regression between bmi and age at mi in diabetic patients with q121 (a) and kk (b) genotypes . Data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston . Our results indicate that the enpp1 k121q polymorphism predicts acceleration of major cardiovascular events in very high - risk patients . The increased risk conferred by the q121 variant is independent of that of age, sex, bmi, diabetes, and cigarette smoking . Our findings are in agreement with a previous cross - sectional study of 445 mi survivors from central europe (29). By contrast, case - control genome - wide association studies reported that a single nucleotide polymorphism (snp, rs7767502), which is in perfect linkage disequilibrium with the enpp1 k121q polymorphism, was not associated with cad (610). Several differences between our study and the genome - wide association studies, such as the prospective versus cross - sectional designs, the different end points under investigation, and the different baseline cardiovascular risk, with only the patients enrolled in our study being very high - risk as per selection criteria, might be responsible for this apparent discordance . An additional important result of our study is that the effect of the q121 variant was modulated by obesity in diabetic patients among whom the risk of incident events was five times higher in q121 than in kk genotype carriers . Although not the aim of our study, one can infer that obese individuals (fig . 2a) as a whole tend to have a lower risk of future cardiovascular events than nonobese patients (fig . 2b; adjusted hr 0.68 [95% ci 0.4111.124], p = 0.13). This paradoxic protective effect of obesity resembles that observed in patients with cad (41), esrd (42), heart failure (43), and older age (44), all conditions heavily over - represented in our samples . In this context, the q121 variant seems to eliminate the paradoxic protective effect of obesity . An important finding was that the q121 variant - by - obesity interaction observed in the prospective study was replicated in a cross - sectional study on age at mi in diabetic patients . Information on the k121q genotypes tended to improve risk prediction in these patients when the improvement was measured by the idi, the approach that is currently favored to evaluate predictive ability increase conferred by a new marker when added to a well - performing model (39). Thus, pending further validation in larger studies, one can hypothesize clinical implementation of the q121 variant as a marker of early cardiovascular events among obese diabetic patients . Given the increasing incidence worldwide of both obesity and diabetes (24) and the poor ability to stratify cardiovascular risk among diabetic patients, a large sector of society would be likely to benefit in the future from the availability of such a test . The synergistic effect of the genetic marker and obesity in the modulation of cardiovascular risk resembles results repeatedly reported in the risk modulation of insulin resistance and related traits (2527,30,33,4549). Placed in a broader perspective, this is an excellent example of genetic heterogeneity (i.e., different genetic effects being at play in different population subgroups) and clearly illustrates how accounting for such heterogeneity may be critical to dissect the genetic architecture of multifactorial diseases . Understanding the mechanisms through which the q121 variant is associated with cvd is beyond the scope of this study . However, one can speculate that the q121 variant exacerbates cardiovascular risk by inducing systemic insulin resistance (22,2527) and proatherogenic phenotypes (24,29,30,33). It may also act by way of a direct detrimental effect on insulin - dependent endothelial function, as suggested by the observation that human endothelial cells carrying the q121 variant show impaired insulin receptor signaling and, most importantly, reduced release of nitric oxide (24), a potent vasodilator whose deficiency is an established early step in the pathway development of atherosclerosis (50). One can hypothesize that the interaction between the q121 variant and obesity is sustained by the different sites of action on the insulin - signaling pathway . Although enpp1 acts at the insulin receptor level (21,23), obesity acts by different mechanisms, mostly at a postreceptor level (51). It is, therefore, possible that postreceptor insulin - signaling abnormalities are necessary for the q121 variant to be fully effective in inducing insulin resistance and, eventually, related clinical outcomes . The three cohorts of very high - risk individuals that we studied were quite different from each other: one comprised only patients with type 2 diabetes and cad, another included patients with a previous mi who did not have frank type 2 diabetes, and the third included only patients with esrds . Despite such apparent phenotypic heterogeneity, the effect of the q121 variant was not heterogeneous across the three studies . Not only did this allow us to analyze the three cohorts together, increasing statistical power, but it also suggests that our findings may be generalizable to all high - risk patients, irrespective of their background clinical characteristics . Whether the predictive role of the q121 variant extends to situations characterized by a more moderate cardiovascular risk remains to be determined . We acknowledge that, mainly because of the relatively small size of our samples, the significance level of our findings is still compatible with a false - positive result . However, this seems unlikely given that the association between q121 was not heterogeneous across the three cohorts and, importantly, was further confirmed in cross - sectional studies as far as the interaction with obesity in diabetic patients is concerned . We also acknowledge that due to the relatively small sample size of the studies that we analyzed, we cannot exclude that the gene - by - obesity interaction that we observed among diabetic patients also occurs among nondiabetic individuals, as is the case for the modulation of insulin resistance (2527). Therefore, our findings need further replication in larger samples before they can be considered as established . Finally, because this study was entirely performed in individuals of european ancestry, we do not know whether our findings can be extended to populations of different race . In conclusion, pending confirmation in further larger studies, the q121 variant has the potential to become a clinical tool for identifying those very high - risk patients who are especially prone to major cardiovascular events and need, therefore, to be targeted with specific and even more aggressive preventive strategies.
In the past two decades, molecular biology research has revealed the intimate mechanisms of epidemiologically significant diseases, such as cancer, infections and immunological disorders . As the next step beyond seeking the mechanisms involved, scientists are now increasingly making it possible to regulate human biological reactions . In recent years, there have been breakthroughs in genetic engineering related to the inventory and methods necessary to physically construct and assemble biomolecular parts, such as synthetic rna - based regulatory systems . Synthetic biology relies on the engineering of biological systems that perform human - defined functions and on the synthesis of complex, biologically based systems that show functions that do not exist in nature . Despite the possible advantages for clinical applications, more work remains to be done to elucidate the principles of biological design, and to overcome the scientific and technical challenges in designing and building more effective systems that are harmless to humans and therefore useful for clinical applications . A recent study by chen et al . Has produced a significant advance in solving such issues and therefore potentially bridging the gap between the bench and the bedside for synthetic rna - based regulatory systems . The authors developed a modular device composed of a sensor (an aptamer) and a gene - regulatory component (a hammerhead ribozyme) and tested its ability to affect the expression of cytokines important for the function of t - lymphocytes in mouse and human systems . First, it represents the logical continuation of years of experimental work performed by the same group, coming from a team that understands the way a synthetic rna - based regulatory system works and its immediate practical applications . In fact, in a previous study, also published in proceedings of the national academy of sciences of the united states of america, the authors were the first to develop and set up universal rna - based regulatory platforms, called ribozyme switches, by using engineering design principles . In the present report, the authors expanded the advantages of such biomodular platforms to a broader range of applications . They were able to do so by the reliable de novo construction of modular, portable and scalable control systems that can achieve flexible regulatory properties, such as up- and down - regulation of target expression levels and tuning of regulatory responses to fit application - specific performance requirements . Second, the authors applied the synthetic rna regulatory device to a significant medical issue, the use of adoptive cell transfer (act). The act strategy uses t - cell - based cytotoxic responses to attack malignant cells (or any other types of abnormal cells) that escape the body's natural surveillance by using t cells that have a natural or genetically engineered reactivity to a patient's cancer cells . For this purpose, t cells have first to be naturally or genetically engineered to react against a tumor - specific antigen, then expanded and made more effective in vitro, and finally adoptively transferred into a cancer patient . However, the clinical efficacy of act, so far, has been limited . There are many reasons for this, and insufficient persistence and reactivation of infused t cells are among the main ones . Conventional strategies for enhancing the persistence of transferred t cells include ablation of all white blood cells (myeloablative methods), such as total body irradiation and administration of toxic levels of interleukin (il)-2 . However, myeloablation is associated with considerable morbidity, caused by decreased immune response and increased risk of infection . Report on a synthetic rna regulatory system, which marks a new era in adoptive t - cell therapy because of the increase in the amount and survival of infused t cells found with this system . Their system for the control of mammalian t - cell proliferation is based on a platform of assembled rna devices formed by a modular sensor (aptamer) and a gene - regulatory (hammerhead ribozyme) component . This device converts a small - molecule input to an increased gene expression output, in this particular case cytokine production . In more detail, the authors fused a theophylline ribozyme switch to the 3' untranslated region of a tri - functional transgene (cd19-tk - t2a - il15) encoding il-15 (potent survival / proliferative cytokine of t cells), mutant hsv-1 thymidine kinase (acting as a reporter and as a suicide protein in the presence of ganciclovir) and cd19 (a marker for fluorescence - activated cell sorting and immunomagnetic selection). Using this system, they could strictly measure (by monitoring the expression of cd19) and control (by modulating the levels of the input molecule) the biological response (cell proliferation / viability). In addition to all the in vitro evidence, the authors demonstrated that this system worked in vivo and effectively modulated the t - cell growth rate in mice in response to theophylline administration . The growth rate was increased to 32% in the presence of theophylline over a 14 day study in mice . They further investigated its possible clinical application by transducing primary human central memory t cells with this system . In vitro results showed that the population of live central memory t cells increased by 24% and that apoptotic cell population was decreased by 54% in the theophylline - responsive system . Finally, the presented gene regulatory system showed significant advantages over available gene regulatory techniques (synthetic inducible promoters); in particular, it provides a wide range of flexibility for clinical settings . Firstly, the ribozyme switches can be easily programmed to respond to different drug molecules . Secondly, the system can be stringently controlled and finely tuned by adding additional drug - responsive ribozyme switches (up to four), therefore achieving lower basal gene expression levels . Thirdly, this system shows tight drug - mediated regulation of growth over an extended time period . Taking all these features into consideration, it is feasible that this new synthetic rna - based regulatory system could have straightforward clinical utility . It is likely that combining this new modular device framework with upcoming advances in synthetic biology will strongly support the tailoring of rna - based regulatory systems to diverse applications in various clinical and laboratory environments . Yet applying these rna - based regulatory systems in clinical practice in addition, there are many other factors that limit the use of adoptive t - cell therapy for cancer . For example, the failure of adoptive immunotherapy against cancers lies in the absence of tumor - specific sources of t cells . If such obstacles are not overcome, efficacy of these systems will be significantly limited in clinical practice . Also, recent data support the combined roles of protein - coding genes and non - coding rnas, such as micrornas, in the pathogenesis of frequent diseases (such as cancer, immune and cardiac disorders). One question for the future is whether such devices can be adapted for the regulation of the functions of non - coding rnas and micrornas . The published research is good news, but it would be better to hold our cheers until the clinical trials are successfully completed, which we hope will be in the near future . Gac received his md and phd at carol davila university of medicine in bucharest, romania . After working on cytogenetics as an undergraduate student with dragos stefanescu in bucharest, he completed training in cancer genomics in massimo negrini's laboratory at the university of ferrara, italy . In 2000 he became a postdoctoral fellow at the kimmel cancer center in philadelphia, in carlo croce's laboratory . Since july 2007 he has been an associate professor in experimental therapeutics at the md anderson cancer center and studies the roles of micrornas and other non - coding rnas in cancer initiation and progression, as well as the mechanisms of cancer predisposition, and explores new rna therapeutic options for cancer patients . Skl graduated from yonsei university medical school, seoul, south korea with an md and phd and is an assistant professor in the department of gastroenterology, severance hospital, seoul . The focus of his scientific research is understanding the roles of non - coding rnas in gastrointestinal cancers . In march 2009, he began working in gac's laboratory studying the roles of non - coding rnas, including micrornas in the initiation and development of gastrointestinal cancers, as well as the identification of new non - coding rna biomarkers . Gac is supported as a fellow at the university of texas md anderson research trust, as a fellow of the university of texas system regents research scholar and by the cll global research foundation . Work in gac's laboratory is supported in part by nih, by dod, by developmental research awards in breast cancer, ovarian cancer and leukemia spores, and by a 2009 seena magowitz pancreatic cancer action network aacr pilot grant.
The wilms' tumor suppressor gene (wt1), located at chromosome 11p13, has 10 exons, spans -50 kb, and encodes a zinc - finger - transcription factor presumed to regulate the expression of numerous target genes through dna binding (1). Wt1 was initially identified as a gene inactivated in wilms' tumor, although the estimated percentage of wilms' tumors (wts) with wt1 mutations is only 10 - 15% . Investigations of wt1 have revealed the normal physiologic functions of this gene in embryogenesis, gonadogenesis and nephrogenesis (1). Wt1 has been shown to play a role in kidney induction (2) and during later steps of nephrogenesis (3), and there are suggestions that wt1 may play an important role in the maintenance of normal podocyte function (4). Besides wilms' tumor, a number of human diseases have been shown to be associated with mutations of the wt1 gene (5). Genital abnormalities are noted in three of these disorders, wagr syndrome (wilms' tumor, aniridia, genitourinary malformation, and mental retardation), denys - drash syndrome (dds), and frasier syndrome (fs), and the analysis of wt1 knockout mice also suggested a fundamental role of wt1 in gonad development (6). Wilhelm and englert reported that the wt1 regulates early gonad development by activation of steroidogenesis factor 1, sf1, which encodes an orphan nuclear receptor that regulates the expression of several genes involved in steroidogenesis and gonadal development (7). Dds traditionally encompasses patients with the triad of congenital nephrotic syndrome leading to end - stage renal failure (esrf), xy pseudohermaphroditism and wilms' tumor (8, 9). Subsequent reports described patients with incomplete forms of this syndrome (10 - 12), and the definition of dds has now been widened to include xy individuals with mild to severe genital anomalies as well as xx individuals displaying nephropathy and wt (13). The nephropathies in dds are characterized by the histological finding of diffuse mesangial sclerosis (dms), a finding also described in some cases of congenital nephrotic syndrome (isolated diffuse mesangial sclerosis, idms). Analysis of genotype - phenotype correlation in idms suggests that at least some idms patients have a variant form of dds due to wt1 mutations (14). Attempts to correlate the phenotype of dds and different wt1 mutations did not provide clear - cut results (15), and no correlation could be established between any particular mutation and the occurrence of wilms' tumor . However, analysis of a large number of patients might provide clues to the role of each mutation, and might help in understanding the normal physiology of nephrogenesis and the prediction for tumorigenesis . In this report, we present two cases of idms with different wt1 gene mutations that had previously been reported in association with dds . She was born at 37 weeks' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema, ascites and normal female external genitalia . Laboratory findings showed; hemoglobin, 8.2 g / dl; cholesterol, 147 mg / dl; total serum protein, 3.5 g / dl; serum albumin, 2.1 g / dl; serum creatinine, 1.7 mg / dl; bun, 27 mg / dl; total calcium, 5.6 mg / dl; phosphorus, 11.7 mg / dl; sodium, 116 meq / l; potassium, 6.0 meq / l; chloride 102 meq / l; tco2 5.7 meq / l . A renal biopsy was performed at the age of 111 days, and 30 glomeruli were examined . A wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells . Analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution, converting arg to his (fig . The patient is now 13 months old, and is well and on peritoneal dialysis, expecting renal transplantation . She was born at 38 weeks' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . At a local clinic, hypocalcemia, hyperkalemia and azotemia laboratory findings showed: hemoglobin, 9.8 g / dl; cholesterol, 120 mg / dl; total serum protein, 3.7 g / dl; serum albumin, 1.5 g / dl; bun, 75 mg / dl; serum creatinine, 4.1 mg / dl; total calcium, 5.9 mg / dl; phosphorus, 12.7 mg / dl; sodium, 136 meq / l; potassium, 5.5 meq / l; chloride, 113 meq / l; tco2 7.8 meq / l . Albuminuria and a renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis (fig . Analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution, converting asp to tyr, and a heterozygous 395 ser (tcc)> ser (tca) polymorphism (fig . Peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . Sustained hypertension (120/80 mmhg) responded to an angiotensin - converting enzyme inhibitor . She was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization, and the patient died at the age of six months due to multiple serious infections and failure to thrive . She was born at 37 weeks' gestational age with a birth weight of 2,500 g. placenta weight was 450 g. physical examination on admission revealed generalized edema, ascites and normal female external genitalia . Laboratory findings showed; hemoglobin, 8.2 g / dl; cholesterol, 147 mg / dl; total serum protein, 3.5 g / dl; serum albumin, 2.1 g / dl; serum creatinine, 1.7 mg / dl; bun, 27 mg / dl; total calcium, 5.6 mg / dl; phosphorus, 11.7 mg / dl; sodium, 116 meq / l; potassium, 6.0 meq / l; chloride 102 meq / l; tco2 5.7 meq / l . A renal biopsy was performed at the age of 111 days, and 30 glomeruli were examined . A wt1 mutation was identified by direct sequencing of a wt1 pcr product obtained from genomic dna from white blood cells . Analysis of the wt1 exon 8 sequence revealed the presence of a heterozygous g to a base substitution, converting arg to his (fig . The patient is now 13 months old, and is well and on peritoneal dialysis, expecting renal transplantation . She was born at 38 weeks' gestational age with a birth weight of 3,200 g. she was well until two days before admission when she developed generalized tonic clonic seizure . At a local clinic, hypocalcemia, hyperkalemia and azotemia were detected and she was referred to us . On admission, generalized edema was noted . Laboratory findings showed: hemoglobin, 9.8 g / dl; cholesterol, 120 mg / dl; total serum protein, 3.7 g / dl; serum albumin, 1.5 g / dl; bun, 75 mg / dl; serum creatinine, 4.1 mg / dl; total calcium, 5.9 mg / dl; phosphorus, 12.7 mg / dl; sodium, 136 meq / l; potassium, 5.5 meq / l; chloride, 113 meq / l; tco2 7.8 meq / l . Albuminuria and hematuria were detected . A renal biopsy was performed at the age of 29 days and light microscopy showed small glomeruli with various degrees of mesangial sclerosis (fig . Analysis of the sequence of wt1 exon 9 revealed the presence of a heterozygous g to t base substitution, converting asp to tyr, and a heterozygous 395 ser (tcc)> ser (tca) polymorphism (fig . Peritoneal dialysis was commenced with a temporary shift to hemodiafiltration because of dialysate leakage at the exit site . Sustained hypertension (120/80 mmhg) responded to an angiotensin - converting enzyme inhibitor . She was maintained on total parenteral nutrition due to uncontrolled chylothorax that resulted from internal jugular vein catheterization, and the patient died at the age of six months due to multiple serious infections and failure to thrive . The mutation present in the second patient was previously reported in a dds patient with early onset esrf resulting from dms, ambiguous genitalia, and a 46 xy karyotype . Autopsy showed a streak gonad and nephroblastomatosis, but no wts were revealed (16). As in our second patient, this boy reached esrf within a few weeks . Considering that esrf in dms is reached within a few months or years (17), early onset esrf in patients is noted as a feature of this mutation . The mutation in the first patient was also previously reported in a dds patient (46 xy, female genitalia, streak gonad, dms, gonadoblastoma, without wt) (18). It is well known that wt1 has many roles during gonadogenesis, and since wt1 is expressed in the same cell lineage as sry, the y - located testis determinant, wt1 may be acting upstream to sry during development of the genital ridge; perhaps controlling sry expression, or interacting directly with sry during sex determination, or functioning immediately downstream of sry (19). Wt1-null mutant mice of both sexes fail to develop kidneys and gonads, indicating that wt1 acts upstream of the sex - determining decision (6). Frasier syndrome (fs) is another distinctive disease associated with wt1 mutation, characterized by male pseudohermaphroditism and nephropathy with late onset esrf, and a frequent association with gonadoblastoma (20). Molecular analysis of a familial case of fs has been described, which provided a clue to wt1's effects on gonadogenesis . Two sisters in their late teens showed signs of renal disease and each sister has a donor splice - site mutation that was predicted to result in an imbalance of the kts+/kts- isoforms . One sister was 46 xy, with complete gonadal dysgenesis with normal female phenotype, while the other was 46 xx, with apparently normal ovarian development and function . This suggests that either wt1 has a male - specific function in sex determination or that testis formation is much more sensitive to dosage effects than ovarian formation (21). Our two cases are female with karyotype 46 xx, which might be the reason for the absence of gonadal abnormalities, and we suspect that if a patient is born with karyotype 46 xy, there may be a possibility of dds development . Although male patients have been described (14, 22), idms most often occurs in females; however, the percentage of patients with wt1 mutation - positive idms, the sex ratio and the associated risk of wilms' tumor are still unknown . A recent study of seven japanese idms patients reported a low rate of wt1 mutation (2/7) and a low risk of wilms' tumor (0/7) (23). The mutations observed in these japanese patients were different from those observed in the previous reports of dds, and in the removed kidneys, nephrogenic nests were not found . Therefore, it was concluded that in idms with wt1 mutations, the risk of wilms' tumor might not be very high . Our report is limited to two patients, but both mutations were already associated with dds, and the early onset esrf in patients with the 396 asp (gac)> tyr (tac) mutation is distinctively similar . This supports the concept that, at least in part, idms is a variant form of dds, and we suggest that idms patients require the same attention as dds patients because of the possible association with wilms' tumor and/or gonadoblastoma . Hu et al . (24) recently reported two boys with incomplete dds with wt1 mutations who underwent prophylactic bilateral nephrectomy, and the removed kidneys showed nephroblastomatosis, which has malignant potential . The authors suggested that missense mutations in exons 8 and 9 of wt1 can be regarded as risk factors for development of wilms' tumor and proposed prophylactic bilateral nephrectomy and early renal transplantation . It is still debatable whether prophylactic nephrectomies should be carried out in all children with dms before renal transplantation, and schumacher et al . (15) suggested that regular renal imaging might be necessary to evaluate the need for bilateral nephrectomy . Considering the clinical courses of the patients in the previous reports, our patients also might have had the possibility of malignant transformation, but we chose regular renal and gonadal imaging rather than prophylactic nephrectomy, and three - monthly ultrasonogram until death did not show evidence of malignancy in the second patient . The first patient is well on peritoneal dialysis with no sign of malignancy on three - monthly screening and we are considering of nephrectomy at the time of transplantation . The reason for the heterogeneity of the clinical features of patients with wt1 mutations is not yet clear, and we suggest that a classification based on genetic diagnosis rather than syndromatic diagnosis might be more applicable for the prediction of outcome, and routine genetic analysis to detect wt1 mutation should be applied to all idms patients . If wt1 mutation is documented, karyotyping and serial abdominal ultrasonographies are warranted for the evaluation of tumor development in the gonad and kidney, and the analysis of genotype - phenotype correlations should be continued.
Titanium dioxide (tio2) is a component of many sunscreens, soaps, shampoos, toothpastes, cosmetics, paper products, plastics, ink, paint, and building materials 1 in both its bulk form and its nanoform . It is also used in human food as a colorant and inactive ingredient, where it can also be present in both forms 1, 2 . From 1916 to 2011, an estimated total of 165 050 000 metric tonnes of tio2 pigment were produced worldwide (bulk form and nanoform combined), with a current annual estimated production of more than 6 million tonnes / yr 2 . Reviews of nanotio2 toxicology are available across various evolutionary groups of species 3, 4, 5, 6, 7, 8, often summarizing half maximal effective concentration (ec50), half maximal inhibitory concentration (ic50), and median lethal concentration (lc50) values . Nanotio2 is also photoactive and produces reactive oxygen species (ros) on illumination 9 . Reactive oxygen species can be detrimental to many organisms, causing oxidative damage, cell injury, and ultimately death 10 . Recently, it has been argued that photoactivation of nanotio2 under natural levels of sunlight is sufficient to affect the output of lc50 and ec50 values in standard toxicology tests 11, 12 . The majority of studies investigating nanotio2 toxicity did not take photoactivation into account and performed tests either in dark conditions or under indoor commercial artificial lighting that did not simulate natural solar irradiation . The aim of present study was to derive a phototoxicity ratio between the results of the nanotio2 experiments conducted in the absence of sunlight and conducted in the presence of solar or simulated solar radiation (ssr). To achieve this aim, we searched the literature for studies that included nanotio2 experiments both with and without irradiance under the same experimental setup and otherwise identical conditions . Therefore, the phototoxicity ratio can be used to correct endpoints of the toxicity tests with nanotio2 that were performed in absence of natural sunlight or ssr . Such corrections also may be important for regulators and risk assessors when reviewing previously published data . For example, one of the current challenges for conducting risk assessment of nanoparticles such as tio2 is lack of consistent toxicity data because of the varieties of materials and test conditions . Regulatory thresholds for nanotio2 do not exist currently; however, regulation of nanoparticles discharge and monitoring in aquatic environment is anticipated in the future . It is expected that regulators will use the phototoxicity ratio when deriving thresholds for nanotio2 because the majority of the published literature reported toxicity endpoints for nanotio2 in the absence of natural sunlight or ssr . Of course, the phototoxicity ratio value calculated in present study is not absolutely precise and correct for all environmental conditions and species; it does, however, considerably reduce the possible error of data endpoints obtained in the absence of natural sunlight or ssr . It also mitigates uncertainties in the risk assessment process by taking into account the photoactivation and phototoxicity of nanotio2 . A comprehensive literature review was conducted to collect available toxicity endpoints for nanotio2 . The literature search (september 2014) was performed within 4 databases web of science, scopus, google scholar, and the university of british columbia library database using the following keywords in various combinations: titanium dioxide, tio2, nanoparticles, phototoxicity, photoactivation, ec50, lc50, ic50, and lowestobservedeffect concentration (loec). Abstracts of numerous hits were read, and downloaded papers were checked for useful information . Only papers that reported results under the same environmental conditions for 2 different nanotio2 exposure groups (with and without ssr) in the form of ec50, lc50, ic50, loec, or a ratio were selected . Thus, all included data were based on dose response curves, ensuring the highest possible quality . The phototoxicity ratio (pr) was calculated in the form of a ratio: pr = tio2 lc50, ec50, ic50, loec without sunlight or ssrtio2 lc50, ec50, ic50, loec with sunlight or ssra phototoxicity ratio greater than 1 means nanotio2 is phototoxic . In a few isolated cases, results were not given in the form of a number; but it was possible to derive a number based on figures provided . Because the papers reported only irradiance (power of electromagnetic radiation per unit area) intensity and not actual insolation (total amount of solar radiation or ssr energy received on a given surface area during a given time), insolation value was calculated when possible . Insolation was calculated based on the irradiance (w / m), actual duration of irradiance (h), and total duration of the toxicity test . In cases in which irradiance intensity was reported in units other than w / m, the data were converted for consistency . For the studies in which data existed only in the form of full spectrum insolation, it was important to at least approximate the levels of ultraviolet a (uva) and ultraviolet b (uvb) used in the studies . At sea level, uva spectra is accountable for 5.7% of the total sunlight, whereas uvb is accountable for 0.3% of the total sunlight 14 . Thus, uva and uvb approximations were performed on the studies reporting full spectrum with factors of 0.057 and 0.003 for uva and uvb, respectively . Such conversions allowed us to determine whether uva and uvb levels used in the studies were of environmental relevance by comparing the data with published averaged uva and uvb levels over europe . Because the focus of the present study is environmental relevance, in all cases, data points were excluded from evaluation if testing conditions did not represent environmentally relevant exposure conditions, such as in vitro toxicity tests with cells . Nanotio2 toxicity was reported as greater than the highest exposure concentration with no negative toxic effects . In those cases, greater than value was from the control tio2 group that was not exposed to ssr or sunlight . This might have led to a slight underestimation of the phototoxicity ratio value, which can be seen as a conservative approach . In all cases, the crystal structure of the nanotio2 particles, their primary particle size, and their hydrodynamic diameter were reported, and the data are presented in table 1 . Therefore, collected data are a mixture of both anatase and rutile crystal forms as well as various particle sizes . Ecosystems generally contain a mixture of all sizes and types of crystal structures of anthropogenically introduced nanoparticles with which decision makers have to cope simultaneously; thus, the aim of the phototoxicity ratio is to provide a distinct value within a muddle . The coating of nanotio2 was not taken into account when evaluating phototoxicity of nanotio2, since all of the collected studies have investigated exclusively bare nanotio2 . Review of nanotitanium dioxide (tio2) phototoxicity to various species anatase (a) or rutile (r). Derived data based on the presented data . Calculated as 0.45 m distance; 30% shadow angle; and 250 w light source . Not in total darkness . Ec50 = median effective concentration; ic50 = median inhibitory concentration; lc50 = median lethal concentration; loec = lowestobservedeffect concentration; uva = ultraviolet a; uvb = ultraviolet b; pr = phototoxicity ratio; noec = noobservedeffect concentration; na = data are not available . Data were checked for normality with the kolmogorovsmirnov test and were found not to be of normal distribution . Spearman rank correlation was performed between the phototoxicity ratio value and time duration of the reported toxicity test, time duration of irradiance, irradiance intensity, insolation, and the organism taxa to determine whether any of these variables drove the output value . In the case of organism taxa, for the purpose of analysis, a code of 5 different digits was assigned to bacteria, algae, invertebrates, fish, and amphibians . Kruskalwallis analysis of variance with a post hoc multiple comparison and/or mannwhitney u test were also performed where applicable . Validation of phototoxicity ratios in correction of toxicity tests endpoint values was performed on data obtained in absence of sunlight or ssr . Phototoxicity data summarized in table 1 (obtained in the presence of sunlight or ssr), served as a control group . Results were log 10 transformed and then statistically compared with either log 10 (data), log 10 (data / median phototoxicity ratio), or log 10 (data/75% phototoxicity ratio quartile). These 3 groups of results originated from the same set of analyzed studies but were obtained in the absence of sunlight or ssr . The literature search resulted in 25 usable references, from which 62 pairs of data were generated for calculation of a phototoxicity ratio (table 1). In total, experiments were performed on 20 different species, ranging from bacteria to amphibians . Applied total irradiance was between 0.46 w / m and 231 w / m (mean, 35.63 w / m; median, 17 w / m), and effective total insolation was between 0.013 wh / m and 200 wh / m (mean, 17.44 wh / m; median, 2.83 wh / m). The recalculated and approximated insolation mean and median data are, respectively, 5.64 w / m and 1.7 w / m for uva, and 0.243 w / m and 0.015 w / m for uvb . The majority of the studies have used the same nanotio2 products (p25 degussa), resulting in a fairly similar size span of primary particle diameter . Phototoxicity ratio minimum and maximum values were 0.84 and 16 778, respectively, and mean and median values were 407.5 and 3.7 . The discrepancy between the mean and median was caused primarily by the data associated with the cladocera taxon . When the data were analyzed for susceptibility of bacteria, algae, invertebrates, fish, and amphibians to phototoxicity, the invertebrates were significantly different compared with other groups . Nanotio2 was significantly more toxic to invertebrates after exposure to light compared with other groups, resulting in a greater phototoxicity ratio (kruskalwallis followed by post hoc multiple comparison). On the other hand, the spearman rank correlation test was not statistically significant for phylogeny and phototoxicity ratio (decreased or increased phototoxicity of nanotio2 from species on the lower organism stadium, such as bacteria, toward more complex organisms, such as amphibians). Also, there was no correlation between phototoxicity ratio and irradiation intensity, duration of irradiation, or received insolation . Indeed, when exclusive cladocera data were analyzed against all other taxa (figure 1), the statistical difference was highly significant (mannwhitney u test, p <0.01). Because of the clear need for data segregation, separate descriptive statistics were performed for cladocera and noncladocera phototoxicity ratio values (table 2). On average, nanotio2 was 20 times more toxic to noncladocera and 1867 times more toxic to cladocera (median values, 3.3 and 24.7, respectively) after illumination . Descriptive statistics of phototoxicity ratio (pr) values significant statistical difference was observed between true phototoxicity data and data obtained in the absence of sunlight or ssr (figure 2). Once the data were corrected by dividing data obtained in the absence of sunlight or ssr with a median phototoxicity ratio or a 75% quartile phototoxicity ratio value, there was no longer statistical difference compared with the data obtained in the presence of sunlight or ssr (figure 2). However, we do not claim that values for the median phototoxicity ratio and the 75% phototoxicity ratio quartile are definite, because they will change over time as more data points become available from future studies . Comparison of uncorrected data (top), corrected data with median phototoxicity ratio (middle), and corrected data with 75% phototoxicity ratio quartile (bottom) obtained in the absence of sunlight or simulated solar radiation (ssr) with true data obtained in the presence of sunlight or ssr . Cladocera and noncladocera data were corrected separately with the group corresponding median or 75% quartile values . The fact that the cladocera taxon was more sensitive to nanotio2 phototoxicity cannot be explained by the intensity of irradiation or received insolation during testing, because such correlation was not statistically significant (spearman rank correlation test). In cladocerarelated experiments, the original publications from which data were derived provided no evidence that cladocera were exposed to any specific grade, type, or size of nanotio2 particles to which other taxa were not exposed . Ultraviolet sensitivity of the taxon has to be ruled out as well, because appropriate exposure controls to uv were included and no increase in toxicity was detected . Although uv is toxic and lethal to cladocera at higher exposure doses, numerous protection mechanisms prevent hazardous occurrences at lower doses 15 . Both uv and nanotio2 toxicity are based on ros, and oxidative stress was indicated in cladocera exposed to either uv 15, 16 or nanotio2 17 . However, this does not necessarily mean that uv and nanotio2 have the same toxicity mechanism . Whereas generation of ros and consequently oxidative stress following exposure to uv radiation requires endogenous photosensitizer molecules, generation of ros by nanotio2 under uv radiation is a direct photochemical process, and the substantial ros production can readily damage or kill cells or organisms such as cladocera . Why cladocera are more sensitive to irradiated nanotio2 remains unclear, and more targeted research is needed . However, one possible explanation for the high sensitivity of cladocera to nanotio2 phototoxicity is that photoinduced ros on the surface of cladocera carapace may interfere with the respiratory gas exchange . In fact, surface attachment of nanotio2 to cladocera carapace has been observed in previous studies 18, 19, and the inner wall of the carapace is a major site of respiratory gas exchange for cladocera 20 . Sunlight is composed of visible, uv, and infrared spectra . Some of the analyzed studies reported irradiance values exclusively within the uv spectrum, whereas others reported values for the full spectrum . Thus, insolation data also were presented based on reported irradiance spectrum . When the data were segregated into what appeared to be insolation values for the full spectrum, the mean insolation was 25.9 wh / m, and the median was 5 wh / m . The studies that supposedly only reported values for the uv spectrum had a mean insolation of 8.72 wh / m, with a median of 2.83 wh / m . For the purpose of comparison, a solar constant (irradiance of the sun when positioned at 1 astronomical unit compared with earth at zenith) measured at the outer surface of earth's atmosphere is approximately 1360 w / m 21 . A significant amount of the solar constant is lost by the time sunlight reaches a location on the earth's surface, depending on atmosphere, latitude, and time of day . For example, average insolation of the visible spectrum during a decade of measurements over europe is between 5 wh / m and 302 wh / m in winter and between 285 wh / m and 430 wh / m in summer 22 . Therefore, both the mean and median (25.9 wh / m and 5 wh / m, respectively) insolation used in the studies reporting only values for full spectrum are much less than the insolation values over europe . The most likely culprits for tio2 phototoxicity are uva and uvb spectrum because those photons would have enough quantum energy (uva, 3.103.94 ev per photon; uvb, 3.944.43 ev per photon) 23 versus energy of visible light photon (1.63.4 ev) to overcome the band gap . When uva and uvb level approximations were performed on the studies reporting full spectrum and combined with studies directly reporting uva and uvb, mean and median values were 5.64 w / m and 1.7 w / m, respectively, for uva and 0.243 w / m and 0.015 w / m, respectively, for uvb . The actual uv spectrum insolation over europe is, on average, 0.7 wh / m to 37.7 wh / m in winter and 34 wh / m to 64.2 wh / m in summer for uva; for uvb, the average is 0.001 wh / m to 1.08 wh / m in winter and 0.77 wh / m to 2.05 wh / m in summer 22 . The mean and the median values for uva and uvb used in toxicity studies are well within the range of uva and uvb values over europe 22 . Therefore, the current experimental setups represent realistic and natural conditions, and the obtained results should not be doubted . Thus, levels used in experimental setups are credible for the purpose of risk assessment, since they do not exceed natural conditions . It is important to note, however, that approximation to the uva and uvb values were based on the assumption that all of the irradiation lamps spectra used in the phototoxicity studies fully corresponded to sunlight spectrum . Although the technology has advanced significantly over the years, there is no absolute guarantee that all of the studies had the proper irradiation lamps . Furthermore, a significant amount of irradiation at sea level altitude is lost because of reflection and adsorption in the water column, according to the beerlambert law iz = i0ekzwhere z is depth, e is natural logorithm, k is attenuation coefficient, and i 0 is the energy of the sunlight at the surface of the water . Although attenuation in pure water might not affect energy of uv light, reflectance of the water surface may, thus reducing the actual uv energy to which aquatic organisms are exposed . However, an opposite effect may occur in shallow waters because of strong scattering of light, thus multiplying the uv exposure levels 25 . Therefore, although uv insolation levels currently used in nanotio2 phototoxicity studies are credible at the water surface level, it is still not clear whether they are credible for risk assessment below the water surface . The fact that different studies used different exposure time and different irradiances only suggest that current scientific community does not really have a standardized toxicity test to check for the phototoxicity effects of nanoparticles . Therefore, we strongly recommend that universal agreement on irradiation time and irradiance in a standard nanomaterial phototoxicity test is necessary . A validation test of phototoxicity ratio correction (figure 2) showed that after correction with a median phototoxicity ratio value the corrected data are no longer statistically different from the real data obtained in the presence of sunlight or ssr . Data correction for the 75% quartile of the phototoxicity ratio was still not significantly different from the real data (p = 0.052) but in general generated much lower endpoints (higher toxicity), as expected . However, the value of 75% quartile application is that, compared with the median phototoxicity ratio, it can more successfully prevent false toxicity underestimation . The use of phototoxicity ratio does not mean that the newly corrected data are the true representation of endpoints from toxicity tests, but rather that they are likely as close as possible . The true correction of data can be achieved only by defining a function through regression analysis . However, because many variables such as particle size, hydrodynamic diameter, crystal structure, illumination time, irradiance, insolation, species, and organic matter content in test media will likely influence the phototoxicity of nanotio2 (even if their effect is not statistically significant, they will contribute certain percentage of variability), generating such a function will be difficult . Therefore, the use of a phototoxicity ratio is an oversimplified method that can provide an approximate correction with lots of versatility . One recent study 13 deployed a similar methodology to the present phototoxicity ratio approach to determine which is more toxic in the environment, nanosized or dissolved metals . Toxicity ratio was calculated between median lethal dose, lc50, ec50, and ic50 values of dissolved and nanoparticulated metals to provide corrections for threshold values in existing regulatory standards . Therefore, the ratio metric approach whether toxicity ratio, phototoxicity ratio, or nanoratio is an inexpensive, straightforward method that mitigates uncertainties for the purpose of risk assessment and management, assuming enough literature is available . In conclusion, the present study found that nanotio2 is phototoxic to aquatic species, because the phototoxicity ratio values were substantially greater than 1 for the majority of analyzed studies . Existing literature on the subject is likely credible for the purpose of risk assessment because the insolation levels used in experimental setups did not exceed uv levels under natural conditions at the water surface . A significant difference was observed between the phototoxicity ratios of 2 analyzed groups: aquatic species belonging to order cladocera, and all other aquatic species . The order cladocera is very sensitive and prone to nanotio2 phototoxicity, at least in laboratorybased toxicity tests . A median phototoxicity ratio value and a 75% quartile were chosen as the most practical approach for correcting nanotio2 toxicity endpoints obtained in the absence of sunlight or ssr . Using a median phototoxicity ratio value in correction is a more conservative approach, whereas using the 75% quartile lowers the chance of underestimating toxicity and may be favored by risk assessors when analyzing previously published data . The values for the phototoxicity ratio are not definite and may change as more data become available in the future . All of the data used for calculation and statistical analysis are presented in table 1 of the present study, with corresponding references.
We report 2 patients infected with panton - valentine leukocidin (pvl)positive mrsa t034 . Each patient had a medical history typical of that reported for community - acquired mrsa of other lineages, which in most cases are pvl positive (8). The first patient, a previously healthy 36-year - old male physiotherapist, sought medical care in march 2006 for a small abscess in his axilla . Culture of the abscess grew mrsa . Presence of meca gene was confirmed by pcr (9). During the next 2 months, his youngest child, adopted from china, had been found to be mrsa positive (throat, perineum, and a small wound) a month earlier during routine screening for adopted children . During subsequent screening of the family, the older sister, adopted from south korea, both parents were negative for mrsa at that time, which suggests that the father was newly infected when his abscess developed and that he had not acquired the strain abroad . Also, spa typing indicated that the children carried different strains from that of the father and from each other (t286, t1434) (10). Subsequent screening of family members for mrsa on several occasions found only the father to be repeatedly positive . The second patient, a 43-year - old male clerk, also previously healthy, sought medical attention during the summer of 2007 for a mrsa - infected elbow wound . Follow - up examination determined that he carried mrsa also in the perineum and in a chronic external otitis eczema . The patients lived in geographically distinct areas in the western part of sweden and had no connection to each other . No animal contact (e.g., pets, farming) was reported by the 2 patients, their family members, or other close contacts . Both patient strains carried pvl, confirmed by identification of the luks - lukf genes (11), and were resistant to digestion with restriction endonuclease smai when typing by pfge was attempted . They produced -hemolysin according to phenotypic detection methods that used rabbit blood agar with hot cold analysis, which further indicated their animal origin (12). Their drug - susceptibility profiles differed; 1 was resistant to doxycycline and the other was resistant to ciprofloxacin, erythromycin, and clindamycin . These strains carry pvl, a toxin partly responsible for the increased virulence of several of the mrsa clones in the community (8). Despite several recent publications concerning st398 mrsa, few have reported pvl in this lineage, which is believed to be of animal origin (2,4,6,13). Most previous reports have described asymptomatic carriage in persons exposed to occupational hazards (e.g., veterinary personal and pig farmers) (1,2,7). However, severe clinical infections have been described (46). In our patients these strains caused repeated infections that needed medical attention, even hospitalization . Since neither patient had even a remote connection to animals and we found no common source of infection, these strains may already be more common in our region than we had thought . These case reports suggest that strains of this lineage may impose a threat in the community, even to patients with no obvious animal contact.
The selective melatonin receptor agonism effect of ramelteon is useful for insomnia.1) here we wanted to present a refractory chronic migraine case, who had significant improvements in migraine after using ramelteon . The precipitating factors were the stress, increase in physical activities and sudden cold weather . The moderate or severe pain intensity was aggravated by or causing avoidance of increased physical activity . The residual symptom between the migraine episodes were the head tense feeling, but not achieveing headache severity . The computed tomography revealed no significant findings of other causes for headache, such as tumor or hemorrhage . She ever received many kinds of medications, such as aspirin, nonsteroidal anti - inflammatory drugs, triptans, ergots, anticonvulsants and glucocorticoids . However, the severity of migraine still remained moderate (migraine disability assessment test [midas] scores, 19) and the associated symptoms were moderate headache, dizziness, nausea, and sensitivity to sound . The migraine also exacerbated her insomnia problems, with fragmented sleep and inadequate sleep duration as 12 hours (insomnia severity index [isi] score, 23). She did nt have any comorbid mental disorder except insomnia . Due to the fear of abuse potential related to hypnotics after 2 weeks treatment of ramelteon 8 mg / day, her sleep duration prolonged to 45 hours with less fragmentation . In addition, her migraine severity started to decline (midas scores, 11). After 6 months of ramelteon 8 mg / day treatment, the migraine severity continued to relieve (midas scores, 6) with stable sleeping quality (isi score, 14). In this case, we found significant improvements in migraine severity and insomnia after the use of ramelteon . Since the patient did nt have insomnia before the first onset of migraine . Therefore we could speculate that the ramelteon had significant effects in migraine in such mild prolongation of sleep duration . The melatonin might relieve the headache via the following possible mechanisms, such as anti - inflammatory effect, free radical scavenging, reduction of pro - inflammatory cytokine, membrane stabilization, nitric oxide synthase activity and dopamine release inhibition, gaba and opioid analgesia potentitation, glutamate neurotoxicity protection, neurovascular regulation,2) cytoprotection and antiallodynic action.3) the pineal gland, the primary source of serotonin and melatonin, might also play a significant role in the analgesic effect.4) however, the clinical trial of melatonin administration showed no significant improvements in migraine severity,5) which suggested the modulation of melatonin receptor might be a better option for the analgesic effects . The modulation of serotonin system by the ramelteon would also relieve the pain sensation.2) in addition, the chemical structure of ramelteon was similar with that of indomethacin, which is a kind of non - steroidal anti - inflammatory medicine.2) in the study of animal model, the antinociceptive and antiallodynic actions of melatonin have been observed in different kinds of pathways, such as intrathecally or intracerebroventricular routes . The use of ramelteon, a kind of mt1 and mt2 agonist, could also relieve the pain even lack of free radical scavenger effects.3) the state - of - art treatment of refractory chronic migraine also includes the ramelteon due to selective m1 receptor agonism.6) from the successful experience in this case, we can consider the use of ramelteon for refractory chronic migraine with insomnia.
It was previously suggested that atherothrombotic infarction (ati) and lacunar infarction (li), as two different subtypes of ischemic stroke, might also differ in the set of the relevant risk factors, with ati being more associated to the atherogenic risk factors in contrast to li . . However, decreased insulin sensitivity (is), that is, insulin resistance, was observed both in ati and li, which was frequently accompanied with compensatory hyperinsulinemia in t2d patients as well as in nondiabetics [2, 3]. Simultaneously, it has been shown that impaired balance between products of oxidative stress and the level of antioxidant enzyme activities might be the important mechanism underlying the occurrence of ischemic stroke . Moreover, it was elucidated that the brain has only moderate content of glutathione - dependent enzymes, for example, glutathione peroxidase (gsh - px), glutathione reductase (gr), and superoxide dismutase (sod), together with the fact that the intact antioxidant defense could provide first line of protection from initiation and exacerbation of ischemic cerebral injury . In addition, changes in enzymatic antioxidative defense mechanisms in patients with stroke are still controversial . Previous results implied that the majority of antioxidant enzyme activity was significantly reduced in acute ischemic stroke, possibly as a consequence of increased oxidative stress while the recent finding suggested increased levels of glutathione dependent enzymes as an adaptive mechanisms during acute cerebral ischemia . Finally, due to novel facts, oxidative stress can be an important component for astrocytic cell death following metabolic stress . Until now, experimental studies provided evidence of an association between ischemic stroke and increased oxidative stress [8, 9], but data in humans are still heterogeneous and limited . Therefore, our study was aimed to determine is levels and three different types of antioxidant enzyme activities gsh - px, gr, and sod, in t2d with ati and li . In this study we included a total of 93 patients with t2d, ascribed to the following groups: t2d patients with ati (group a, n = 30), and t2d with li (group b, n = 30), and t2d without ischemic stroke (group c, n = 33). Simultaneously, we involved a total of 93 nondiabetics, matched with the t2d patients regarding gender and age, and also comprising the following groups: nondiabetics with ati (group d, n = 30), nondiabetics with li (group e, n = 30), and nondiabetics without stroke (group f, n = 33). T2d was diagnosed in accordance with the criteria of the world health organization . Diagnosis of ati and li was done by a neurologist due to clinical features and brain imaging methods such as cranial computerized scan and/or magnetic resonance imaging in two consecutive examinations, during the first 7 days from the appearance of ischemic stroke . The patients with ati or li were included in the study provided that they had not shown signs of cardioembolic cerebral infarction, or coronary heart disease based on a history of myocardial infarction with definite elevation of serum cardiac enzymes or coronary angiography . T2d patients were treated with insulin therapy, and/or ingestion of antioxidant supplements and drugs, which might affect free radical and antioxidant activity potential; likewise patients who had other endocrine disease or autoimmune diseases, renal or hepatic failure, current infections, neoplasms, polycythemia, or rheumatic diseases were also excluded as well as the patients with history of trauma or operation within the last 3 months . No patient had uncontrolled hypertension, severe alcohol consumption, acute infection, or an inflammatory disease during the last 4 weeks . All the patients, with or without ati and li, showed the similar level of their physical activity . In addition, they were required not to smoke at least 12 hr before the tests were performed . The patients were fully informed about the study and gave the inform consent to participate . The study was conducted at the clinic for endocrinology, diabetes and metabolic diseases and at the clinic for neurology, clinical centre of serbia, faculty of medicine, university of belgrade, and was approved by the institutional ethics committee . The interview, physical examination, metabolic test, and evaluation of antioxidant enzyme activities were performed in all the patients included in the study, for each patient within the same day . Hypertension was diagnosed according to world health organisation criteria (systolic / diastolic blood pressure 140/90 mm hg) or by the use of antihypertensive agents . The metabolic tests were implemented at least after 6 months from the occurrence of the ischemic stroke . The evaluation of insulin sensitivity was done by frequently sampled intravenous glucose tolerance test (fsigt) with minimal model analysis . Briefly, before testing, each patient was required to be at a 12 hr fasting state . During the fsigt, 0.3 g / kg body weight of glucose was injected and the blood samples for plasma glucose (pg) and plasma insulin (pi) determination were taken immediately before and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 23, 24, 25, 27, 30, 40, 50, 60, 70, 80, 90, 100, 120, 160, and 180 minutes after intravenous glucose stimulation . Insulin was injected as a continuous infusion 4 mu / kg / min between minutes 20 and 25 in order to avoid the effect of the potentially blunted insulin response . The insulin sensitivity index (si) was calculated from the results of pg and pi levels by computerized minimal model analysis, using the minmod program (kindly provided by dr . Determination of the antioxidant enzymes sod, gsh - px, gr was conducted using the commercial assays (produced by randox laboratories ltd ., uk), based on spectrophotometer determination methods as described previously . Pg was determined by glucose oxidase method using a beckman glucose analyser (beckman instruments, fullerton, ca). Pi was tested by radioimmunoassay (inep, zemun, rs, double antibody kits). Total cholesterol, hdl cholesterol, and triglyceride concentrations were determined with the chromatography method using commercial kits (produced by boehringer mannheim). The continuous variables within each subtype of ischemic stroke were analyzed with analysis of variance (anova) with a post hoc bonferroni test . All analyses were performed with the spss statistical package (version 16.0 for windows). The clinical characteristics and biochemistry parameters of t2d patients and nondiabetics with ati or li as different subtypes of ischemic stroke are shown in table 1 . The age, gender, duration of diabetes, and duration from the onset of ischemic stroke were similar in t2d patients and nondiabetics with different subtypes of ischemic stroke, together with hba1c levels, implying satisfactory metabolic control before metabolic investigation was done . However, ldl - c level was significantly higher in t2d patients with ati compared to the t2d patients with li and t2d patients without stroke and also in nondiabetics with ati compared to nondiabetics with li and healthy controls . There is no difference in prevalence of hypertension in patients with t2d and ati or li and t2d without ischemic stroke, while it was significantly higher in nondiabetics with ati or li than in healthy controls . We found that si levels were significantly lower in t2d patients with ati (group a) and li (group b) compared to t2d patients without ischemic stroke (group c) (1.14 0.58 and 1.00 0.26 versus 3.14 0.62 min / mu / l 10, resp . Also, the results showed significantly lower si levels in nondiabetics with ati (group d) and li (group e) compared to healthy controls (group f) (3.38 0.77 and 3.03 0.72 versus 6.03 1.69 min / mu / l 10, resp . Simultaneously, pi levels were higher in t2d patients, ati (group a), and li (group b) than in t2d patients without ischemic stroke (group c) (20.94 4.31 and 20.07 0.88 versus 16.06 0.91 mu / l, respectively, p <0.001) and in nondiabetics with ati (group d) or li (group e) in comparison to healthy controls (group f) (15.57 1.86 and 15.59 1.26 versus 7.54 2.03 mu / l, respectively, p <0.001) (figure 2). When we evaluated antioxidant enzyme activities in t2d patients with ati (group a) and li (group b) and without ischemic stroke (group c) and in nondiabetics with ati (group d) and li (group e) and healthy controls (group d), we detected the levels of the gsh - px and gr activity being significantly lower in group a and b versus c and in group d and e versus f (gshpx: a: 21.96 3.56 versus b: 22.51 1.23 versus c: 25.12 1.67 u / ghb, p <0.001; d: 24.75 3.02 versus e: 25.57 1.92 versus f: 28.56 3.91 u / ghb, p <0.001; gr: a: 44.37 3.58 versus b: 43.50 2.39 versus c: 48.58 3.67 u / ghb, p <0.001; d: 24.75 3.02 versus e: 25.57 1.92 versus f: 28.56 3.91 u / ghb, resp ., p <0.001) (figure 3), while the sod levels did not differ between the investigated groups (a: 769.57 72.36 versus b: 768.97 34.50 versus c: 789.18 60.28, d: 795.23 48.28 versus e: 797.80 69.21 versus f: 813.88 45.80 mu / mghb, resp . This analysis identified that decreased insulin sensitivity si and the decreases of gr were related to both ati and li in t2d patients . Simultaneously, this model identified decreased insulin sensitivity si and the level of gr and gsh - px in nondiabetics with ati, but predominantly decreased insulin sensitivity si in nondiabetics with li (table 2). In this study, we directly measured the is level, together with three different types of antioxidant enzyme activities, gsh - px, sod, and gr, in t2d patients and nondiabetic individuals with two different subtypes of ischemic stroke in type 2 diabetics . Our results have shown decreased is level in t2d patients with two different subtypes of ischemic stroke, ati and li, compared to t2d without stroke, while we could not demonstrate the difference between the subtypes, and the same pattern of is changes was found in the nondiabetics . To our knowledge, there are scarce data regarding the changes in is in t2d with ischemic stroke subtypes . Previous study also suggested that insulin resistance measured using different methodology, the short insulin tolerance test, is independently associated with markers of atherosclerosis detected on carotid arteries in t2d patients . Simultaneously, an association has been documented between insulin resistance and other markers at the large vessels, such as the pulsatility index on cerebral arteries . On the other hand, it has been shown that insulin resistance, evaluated by the homeostasis model assessment (homa) index, was higher in diabetic in contrast to nondiabetic patients with li being a small vessel disease . Additionally, it has been suggested that diabetes, hypertension, and metabolic syndrome which share insulin resistance as a common mechanism, contribute to li occurrence [1820]. However, detailed mechanisms of the possible link between the presence of ischemic stroke subtypes and insulin resistance remain to be clarified . In addition, when we measured the level of insulinemia in these patients, the significantly higher level of insulinemia was detected in both groups of t2d patients, with ati and li . The increases in insulin levels might be primarily consequence of the simultaneous presence of insulin resistance in the relevant groups . However, the increases in insulin might contribute to the appearance of the ischemic stroke subtypes . Insulin, as a growth factor, might interfere with the beneficial effects of nitric oxide (no) on the vasculature . Moreover, insulin infusion during euglycemic insulin clamp was able to suppress endothelium - dependent vasodilation in large arteries, which is reported to be based on increased availability of endothelin-1, leading to downregulation of nad(p)h oxidase and superoxide anion production, and endothelial dysfunction is proposed to play an important role in the pathogenesis of cerebral small vessel disease [23, 24]. Also, our results demonstrated lower values of antioxidant enzymes in t2d patients with ati or li than in type 2 diabetics without ischemic stroke . The patients with ati and li did not differ regarding the level of antioxidant enzymes . These findings could be explained by the fact that in diabetes there is already reduced capacity of antioxidant protection, which is further significantly disturbed in the acute phase of ischemic stroke [4, 5]. In our data, in patients with ati and li, we found the decreases in glutathione - dependent enzymes activity in contrast to other types of antioxidant enzyme activity, for example, sod . These results imply a prolonged and severe depression of gluthatione dependent antioxidative defense mechanisms irrespective of stroke subtypes . Since it has been documented that free radicals are extremely difficult to measure directly, antioxidant enzymes have been proposed to represent indirect markers of oxidative stress . Numerous, but mostly experimental, studies provided evidence of an association between ischemic stroke and decreased antioxidant enzyme activity, although this possible association in humans has been less investigated [9, 25]. Moreover, the analyses of treatment with agents in stroke showing an ability to prevent further depression of antioxidant protection and scavenging reactive free radicals were reported to fail to restore gsh - px and gr activities [26, 27]. Generally, a recently study that aimed to assess total antioxidant capacity and oxidative stress in diabetic and nondiabetic acute stroke patients with 2 different stroke subtypes, large and small vessel disease strokes, concluded that oxidative stress and counterbalancing antioxidant capacity are more pronounced in diabetic acute stroke patients than in nondiabetics . The study provided different data in comparison to the previous investigations showing decreased gsh - px activity in both diabetic and nondiabetic patients with coronary heart disease when compared to controls [17, 29, 30]. However, the results from our study have shown the diminished activity of both gsh - px and gr in t2d patients with ati and li, which is consistent with previous reports of decreased gsh - px levels patients with stroke [6, 8, 31, 32]. The levels of sod are found to exhibit great variations in the previous study in patients with the stroke [6, 8, 26, 29, 3137]. Our results could not detect the differences in the sod levels in different groups of patients, and thus they are in line with data showing no changes in respect to sod level in both diabetics and nondiabetics irrespective of different subtypes of ischemic stroke [33, 34, 37]. The tentative explanation for the inconsistent findings regarding sod might reflect the predominant role of intracellular versus extracellular fraction of sod in free radical scavenging . In our study, the detected antioxidant enzyme activities were not affected by other important factors potentially influencing the enzyme levels, for example, by hyperglycemia, aging, duration of diabetes, and presence of macrovascular complications, due to the fact that in all groups of t2d patients had similar levels of metabolic and satisfactory metabolic control and that patients were matched in respect of age, duration of diabetes, and the prevalence of macrovascular complications . The multiple regression analysis applied to our data has demonstrated that decreased is together with the decreases in gr are related to both ati and li in t2d patients . In nondiabetics, the decreases in si levels and the diminished gr the analysis reveals the potential difference in the mechanisms underlying the onset of the two subtypes of the stroke in t2d patients compared to nondiabetics . The results imply that higher levels of insulin resistance combined with lower levels of gr, detected in t2d patients compared to nondiabetics, were underlying the onset of li together ati in t2d in contrast to the findings in nondiabetics . In this context, our results are consistent with the findings that li represents the most frequent ischemic stroke subtype in t2d . Taken together, our data imply that insulin resistance exerts its pathogenic influence on the level of gluthatione dependent antioxidant enzymes, especially in t2d . In conclusion, our results suggest that the presence of different subtypes of ischemic stroke is associated with insulin resistance and diminished antioxidant enzyme activity in both subtypes of ischemic stroke in t2d . The results also imply that atherogenic influence of decreased is in the different subtypes of stroke in t2d might be exerted through a significantly reduced glutathione dependent antioxidant enzyme activity, while the mechanisms relating the effects of insulin resistance and decreased antioxidant enzyme activity remain to be clarified.
The formation of spores is a survival mechanism of microorganisms when exposed to unfavorable environmental conditions (e.g., heavy metal stress, nutrient limitations) leading to a dormant or resting growth state [1, 2]. A variety of bacteria identified in diverse habitats including soil is able to form endospores . These physiological groups include aerobic heterotrophs (e.g., bacillus, paenibacillus, brevibacillus, geobacillus, thermoactinomyces, and sporolactobacillus), anaerobes (clostridium, anaerobacter, and desulfotomaculum), microaerophiles (sporolactobacillus), halophiles (sporohalobacter), and phototrophs (heliobacterium, heliophilum) [3, 4]. Bacterial spores are characterized by a series of unique chemical features which can facilitate their identification in natural environments . Besides the high content of minerals (particularly calcium), spores contain high amounts of dipicolinic acid, dpa . Dpa is uniquely found in bacterial spores in amounts of up to 25% of the spore dry weight and depends on the bacterial species [6, 7]. In solution, a complex is formed in the presence of terbium which shows a very strong and distinctive fluorescence spectrum . Originally, dpa was used to detect very low concentrations of terbium (iii). On this basis, methods for the detection of bacterial endospores have been developed [1013]: by the addition of terbium, the dpa content was determined . However, terbium - dpa fluorescence might be interfered by a series of compounds, especially when dpa has to be determined in complex samples such as sediments or soils . It has been reported that the presence of phosphorus compounds (especially ortho - phosphate) reduced terbium fluorescence by as much as 98% . The addition of aluminium compounds (especially aluminium chloride, alcl3), however, ameliorated the interference caused by the quenching substances . From a series of organic compounds (benzoate, tryptophan, tyrosine, phenylalanine, glucose, malate, riboflavin, nad, and tryptone) only the latter two (especially tryptone) reduced fluorescence significantly . Carbohydrates (e.g., starch, dextrine) were reported not to interfere with the terbium fluorescence . Inorganic compounds such as calcium carbonate, sodium chloride, potassium chloride, ammonium sulphate, ammonium nitrate, and sodium nitrate did not lead to a reduction of the fluorescence, but only dipotassium phosphate did . The aim of this study was to adopt the fluorescence - based method to determine the spore content in soils sampled from various locations . In particular, we were interested in the differentiation between different types of soil such as grasslands (pasture, meadow), allotment gardens, and forests, as well as fluvial sediments, the relationship of soil parameters (carbon - to - nitrogen ratio) on the occurrence of bacterial spores, and the distribution of spores in relation to sampling depth . Different bacillus species (b. megaterium, b. subtilis) were cultivated in liquid medium containing (in g / l): glucose (3.6), ammonium chloride (2.5), magnesium sulfate (0.2), calcium chloride (0.07), iron sulfate (0.01), edta (0.01), potassium dihydrogen phosphate (0.6), dipotassium hydrogen phosphate (0.9), and yeast extract (1.0). Erlenmeyer flasks (250 ml) containing 100 ml of growth medium were inoculated and incubated for 10 to 15 days (150 rpm, 30c). To initiate and stimulate sporulation, bacteria were subsequently transferred to a sporulation medium (identical composition, but without glucose and less ammonium chloride [only 1 g / l]). After additional 30 days of incubation until vegetative cells were not present anymore after inspection by microscopy spores were harvested by centrifugation, immediately frozen in liquid nitrogen followed by lyophilization . Samples from different locations were collected using a stainless steel soil corer (15 mm in diameter), which was sterilized before each sampling . Cores with a maximum length of 25 cm were obtained, cut in sections of 5 cm, transferred to sterile screw cap falcon tubes (20 ml), and stored on dry ice . After return to the laboratory, samples were immediately lyophylized or stored at 80c until further processing . Sampling sites were located in the surroundings of zurich (switzerland): grassland soil, meadow (municipalities of mnnedorf; uerikon; stfa; and dbendorf), allotment garden (university of zurich, irchel campus), pasture (university of zurich, irchel campus; municipality of wdenswil), forest soil (municipality of stfa), and aquatic sediments (river glatt in dbendorf). Lyophilized aliquots of approximately 1 g were transferred to an eppendorf micro test tube (2 ml) and ground (by adding a 6 mm glass bead) in tissuelyser (retsch, haan, germany) for 5 1 min . Elemental composition (carbon, hydrogen, nitrogen) of soil was performed with a chn-932 elemental analyzer (leco corp ., st . Composition (in% of dry soil) varied between 2.2 and 15.4, 0.2 and 1.4, and 0.2 and 2.0 for total carbon, total hydrogen, and total nitrogen, respectively . Phosphate in aqueous soil extracts (250 mg soil in 5 ml sodium acetate buffer; 0.2 m, ph 5) was determined using commercially available kits (lck 348 and 349; hach lange ag, hegnau, switzerland). 10 mg of dry spore powder was resuspended in 10 ml sodium acetate buffer (0.2 m, ph 5). Soil samples were thawed and 50 mg were suspended in 0.9 ml sodium acetate buffer and 0.1 ml aluminium chloride (alcl3, 0.5 m). Samples were microwaved (berghof microwave digester mws-1, with built - in in situ infrared temperature control) in teflon tfm screw cap digestion vessels . Temperature and power were set to 140c and approximately 680 w (80%), respectively . Alternatively, dpa was released from spores by autoclaving the samples in screw cap glass test tubes for 15 minutes at 121c . The identical dpa extraction protocol was applied for soil samples . However, microscopy was not possible due to the presence of mineral particles interfering the observation . After cooling for 30 minutes, 100 l of the spore suspensions were mixed with 100 l terbium chloride solution (tbcl3, 30 m) in white 96-well microtiter plates (in 8 replicates). Fluorescence was immediately measured using a plate reader (spectramax m2, bucher biotec, basel, switzerland) with the following settings: time - resolved fluorescence (delay 50 s, interval 1200 s) at an excitation wavelength of 272 nm, emission wavelength of 545 nm, and 10 endpoint readings per sample at 30c . The number of spores in the soil samples was determined using standard addition method with spores of b. subtilis . Microwave treatment of spore suspensions and soil samples led to a fast release of dpa (figure 1). Within two minutes, maximum release was obtained . Highest spore numbers up to 4 10 spores per gram dry soil were found in agriculturally used land (meadow, pasture), less in forest soil . The interference of different compounds present in soil (e.g., phosphate) might lead to quenching of the fluorescence signal . This drawback has been overcome by the addition of aluminium chloride as already shown for the determination of bacterial spores in aquatic sediments . Concentration of ortho - phosphate in soil extracts (22.5 m) was reduced by the addition of aluminium chloride to concentrations below the detection limit (<1.2 m). Concomitantly, a de - colorization of the extract was observed suggesting the removal of humic acids which have also the potential to form complexes with terbium and quench the fluorescence signal . The method based on terbium fluorescence for the detection of total numbers of bacterial endospores in soils is fast and easy . A transect (approximately 100 m in length) through a grass field with different land use management (unused meadow, allotment garden, and pasture) gave spore numbers in the range of 5 to 9 10 spores per gram of dry soil (figure 3). Spore counts were not related to the type of land use: in allotment garden soil, counts were not significantly different from soil samples taken from a pasture (p = 0.423; t - test). Regarding the different sampling sites, our results show that grassland soils (meadow, allotment garden, and pasture) contains much more bacterial spores than forest soils and fluvial sediments . Spore content was related to the carbon - to - nitrogen ratio (figure 4). At c / n ratios> 20 only low spore counts (0.5 10 spores per gram of dry soil) were detected as compared to c / n ratios <20 . It has been demonstrated in pure cultures of bacillus thuringiensis in a stirred bioreactor that low carbon - to - nitrogen ratios of 4: 1 resulted in high spore counts . In contrast however, spore formation in streptomyces coelicolor was stimulated under nitrogen - limiting conditions . In particular, c / n ratios between 50 and 100 promoted sporulation, whereas c / n rations <40 did not allow spore formation . Our results showed that in soils with extremely high c / n ratios, spore content was low . The importance of c / n ratio was stressed by gao and coworkers regarding the sporulation of fungi, although fungal spores do not contain dpa . A carbon - to - nitrogen (c / n) ratio of 20 stimulated spore formation by fungi such as penicillium camembertii . The fungus colletotrichum coccodes produced highest spore counts at a c / n ratio of 5 to 10, whereas at a ratio of 40, spore formation was significantly lower . Similarly, in plectosporium tabacinum optimal spore formation was found when c / n rations were between 5 and 10 . The distribution of spores in marine sediments (determined as dpa) showed only a low correlation with the content of total organic carbon and varied with the sediment type . Highest numbers have been found in organic - rich black sediments, lowest number in sandy sediments . It was hypothesized from anthrax outbreaks, that the high numbers of bacillus spores might be related to soils rich in organic matter that is, to a high c / n ratio . These soil environmental conditions are suggested to support the presence and viability of b. anthracis spores . Depth distribution of spores from an area currently used as allotment garden (cultivation of flowers and vegetables) showed highest numbers in a horizon of 5 to 10 cm (figure 5). The two methods evaluated (microwaving, autoclaving) for the mobilization of dpa from bacterial spores gave similar results . However, microwaving was less time - consuming, whereas autoclaving allowed faster throughput of samples . In summary, microwave treatment of soil samples followed by the measurement of fluorescence after addition of terbium proved to be a fast and easy method to assess the content of bacterial spores . Our study might provide a basis for the detection of hot spots of endospores in soil.
Alzheimer's disease (ad) is a common neurodegenerative disease in the central nervous system . The pathological features of ad include senile plaques formed by extracellular amyloid (a) aggregation, neurofibrillary tangles formed by abnormal accumulation of hyperphosphorylated tau protein, synaptic impairments, and neuronal loss in the cerebral cortex and hippocampus . A aggregation in the brain is central to the pathological changes of ad and leads to a series of pathological events, which further promote a aggregation, resulting in cascade amplification . The endoplasmic reticulum (er), a dynamic membranous organelle, is involved in protein synthesis, posttranslational modification, folding, calcium storage, lipid metabolism, and steroid hormone synthesis . Specific stress conditions such as hyperglycemia, hyperlipidemia, hypoxia, chemical toxicants, and genetic mutations can induce the accumulation of unfolded or misfolded proteins in the er and changes in er functions, resulting in endoplasmic reticulum stress (ers). In turn, ers stimulates the unfolded protein response (upr) and restores cellular homeostasis while sustained ers leads to cell apoptosis . Many studies have shown that ers is involved in the development and progression of neurodegenerative diseases such as ad, parkinson's disease, huntington's disease, and amyotrophic lateral sclerosis although the underlying mechanisms still remain blurred . The activation of the upr pathway requires the activation of three sensor proteins: inositol - requiring enzyme 1 (ire-1), activating transcription factor 6 (atf6), and protein kinase rna - like er kinase . These sensors serve to promote the expression of chaperone protein, glucose - regulated protein (grp) 78, to repair misfolded or unfolded proteins, to accelerate er - associated protein degradation (erad), and to aggravate the phosphorylation of eukaryotic translation initiation factor 2a . Sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of the transcriptional activator ccaat / enhancer - binding protein homologous protein (chop) and caspase-12 activation . Activated ire-1 interacts with tumor necrosis factor receptor - associated factor 2 (traf2) and apoptosis signal - regulating kinase 1 (ask1) via the cytosolic enzyme domain to form ire-1-traf2-ask1 complexes, which in turn stimulate the c - jun amino - terminal kinase (jnk) pathway to promote cell apoptosis, thus preventing the damaging impact of misfolded and secreted proteins on tissues and organisms . Genetic mutations in ad lead to a overexpression in the brain and subsequent neurotoxicity, which leads to pathogenesis . The five familial alzheimer's disease (5fad) mice were established by overexpressing the five familial - inherited ad mutant genes (app k670n / m671l [sweden]+i716v [florida] + v717i [london]+ps1 m146l+l286v) under the control of the neuron - specific thy-1 promoter . This mouse model is characterized by many pathological features similar to ad, including amyloid plaque deposition, gliosis, neuronal degeneration, neuronal loss, and cognitive deficits at the age of 45 months . More importantly, this mouse model has been documented to show the earliest signs of intracellular a aggregation in neurons in 1.5-month - old mice, which indicates these mice as a suitable candidate to investigate the early events of ad . In this study, we investigated the time - ordered changes of pro - apoptotic and anti - apoptotic factors to determine the role of the upr signaling pathway in cognitive decline of 5fad mice . The outcome may clarify the role of ers in the pathological progression of ad . The 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes (app k670n / m671l [sweden]+i716v [florida]+v717i [london]+ps1 m146l+l286v) under the control of the neuron - specific thy-1 promoter and wild - type (wt) b6/sjl mice with the identical genetic background were provided by prof . Marry jo ladu (department of anatomy and cell biology, university of illinois at chicago, usa). Experimental animals were housed in a pathogen - free colony (ivc system, tecniplast, italy) and allowed free access to food and water . They were raised, 45 animals / cage, under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . All protocols and procedures used in this study were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health the 5fad transgenic and wt mice at 2, 7, or 12 months of age were used for subsequent experiments with 1014 animals per group (n = 12 in each 2-month - old group, n = 10 in each 7-month - old group, n = 10 in the 12-month - old wt group, and n = 14 in the 12-month - old 5fad group). The swimming trace was monitored by camera and analyzed with smart 2.0 software (panlab, barcelona, spain). The escape latency(s) and number of crossings over a hidden platform in 60 s were recorded . Mice were anesthetized using 10% chloral hydrate by intraperitoneal injection (3 ml / kg). Then, left ventricular perfusion was performed and brain tissues were isolated quickly on ice . Brain tissues were cut along the central sagittal suture, and the left hemisphere was fixed in 4% paraformaldehyde/0.1 mol / l phosphate - buffered saline (ph 7.4) at 4c for 24 h, followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome (cm1850, leica, wetzlar, germany) and stored at 20c . Immunohistochemistry was performed as follows: brain slices were washed with tris - buffered saline (tbs) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity . The brain slices were then blocked with a buffer (containing 5% goat serum [gs], 0.25% bovine serum albumin [bsa], 0.3% triton x-100, tbs) at room temperature for 1 h. primary antibodies 6e10 (1:8000, covance, princeton, nj, usa) and neun (1:4000, abcam, cambridge, uk) were diluted in a buffer (containing 2% gs, 0.25% bsa, 0.3% triton x-100, tbs) and incubated at 4c overnight . Biotin - labeled secondary antibodies anti - mouse igg (1:600) and anti - rabbit igg (1:400) (vector laboratories, burlingame, ca, usa) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed, and slices were air - dried at room temperature overnight . The slices were then hydrated for 5 min, dehydrated using an ethanol gradient, treated with xylene, and finally mounted with neutral balsam . The slices were imaged using a microscopy (leica dm 4000b, germany), and image acquisition was performed with image - pro express 5.1 image analysis software (media cybernetics, rockville, md, usa). For quantitative analysis, 6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured . The prefrontal cortex region was selected as regions of interest, and the identical area within the measuring frame in a 10 objective lens was labeled . The clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected . Cells that were lightly stained or had irregular shapes were excluded from quantification . Brain slices were washed with tbs buffer and blocked with a specific buffer (containing 5% donkey serum [ds], 0.25% bsa, 0.3% triton x-100, tbs) at room temperature for 1 h. the primary antibodies (diluted in 2% ds, 0.25% bsa, 0.3% triton x-100, tbs) used were: 6e10, grp 78 (1:50, santa cruz, ca, usa), chop (1:50, santa cruz), glial fibrillary acidic protein (gfap) (1:4000, millipore, boston, usa), and -iii - tubulin (1:4000, abcam). The 6e10 was co - incubated with grp 78, chop, gfap, and tubulin primary antibodies . Alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg (1:1500, invitrogen, carlsbad, ca, usa) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole (dapi) (diluted by 1:8000 in h2o) was then added and incubated for 5 min . Prolong gold antifade reagent (invitrogen) was used for slice mounting, and slices were imaged using confocal microscopy (leica tcs sp5, leica microsystems wetzlar gmbh, germany). Triton x-100, 50 mmol / l sodium fluoride, 2 mmol / l sodium orthovanadate, 10 mmol / l -sodium glycerophosphate, 10 mmol / l sodium pyrophosphate, 1% protease inhibitor cocktail [p8340, sigma - aldrich, st . Louis, mo, usa] dissolved in tbs buffer, ph 7.4) at a ratio of 1:10 (mg / ml). The mixture was incubated on ice for 30 min, and lysis was performed using the ultrasound method . Supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page). Subsequently, the proteins were transferred to polyvinylidene fluoride (pvdf) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies: grp 78 (1:200), chop (1:500), sapk / jnk (1:1000, cell signaling, boston, usa), p - sapk / jnk (thr183/tyr185) (1:1000, cell signaling), caspase-12 (1:500, cell signaling), cleaved caspase-3 (1:500, cell signaling), syvn1 (1:2000, abcam), -actin (1:2000, abcam), and -iii tubulin (1:100,000). Horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs (1:2000) were subsequently added and incubated at 37c for 1.5 h. imagej software (national institutes of health, bethesda, ma, usa) was used for quantitative analysis . Quantitative data were expressed as the mean standard error (se) and analyzed with the graphpad prism 6.01 software package (graphpad, san diego, ca, usa). Escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance (anova) while other data were evaluated using two - way anova . If the main effects and the interaction were significant, the genotypes were compared by student's t - test, or multiple conditions among the same genotype were evaluated with one - way anova . The 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes (app k670n / m671l [sweden]+i716v [florida]+v717i [london]+ps1 m146l+l286v) under the control of the neuron - specific thy-1 promoter and wild - type (wt) b6/sjl mice with the identical genetic background were provided by prof . Marry jo ladu (department of anatomy and cell biology, university of illinois at chicago, usa). Experimental animals were housed in a pathogen - free colony (ivc system, tecniplast, italy) and allowed free access to food and water . They were raised, 45 animals / cage, under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . All protocols and procedures used in this study were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health the 5fad transgenic and wt mice at 2, 7, or 12 months of age were used for subsequent experiments with 1014 animals per group (n = 12 in each 2-month - old group, n = 10 in each 7-month - old group, n = 10 in the 12-month - old wt group, and n = 14 in the 12-month - old 5fad group). Morris water maze tests were performed as described previously . The swimming behavior of mice was evaluated four times a day for 5 days . The swimming trace was monitored by camera and analyzed with smart 2.0 software (panlab, barcelona, spain). The escape latency(s) and number of crossings over a hidden platform in 60 s were recorded . Mice were anesthetized using 10% chloral hydrate by intraperitoneal injection (3 ml / kg). Then, left ventricular perfusion was performed and brain tissues were isolated quickly on ice . Brain tissues were cut along the central sagittal suture, and the left hemisphere was fixed in 4% paraformaldehyde/0.1 mol / l phosphate - buffered saline (ph 7.4) at 4c for 24 h, followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome (cm1850, leica, wetzlar, germany) and stored at 20c . Immunohistochemistry was performed as follows: brain slices were washed with tris - buffered saline (tbs) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity . The brain slices were then blocked with a buffer (containing 5% goat serum [gs], 0.25% bovine serum albumin [bsa], 0.3% triton x-100, tbs) at room temperature for 1 h. primary antibodies 6e10 (1:8000, covance, princeton, nj, usa) and neun (1:4000, abcam, cambridge, uk) were diluted in a buffer (containing 2% gs, 0.25% bsa, 0.3% triton x-100, tbs) and incubated at 4c overnight . Biotin - labeled secondary antibodies anti - mouse igg (1:600) and anti - rabbit igg (1:400) (vector laboratories, burlingame, ca, usa) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed, and slices were air - dried at room temperature overnight . The slices were then hydrated for 5 min, dehydrated using an ethanol gradient, treated with xylene, and finally mounted with neutral balsam . The slices were imaged using a microscopy (leica dm 4000b, germany), and image acquisition was performed with image - pro express 5.1 image analysis software (media cybernetics, rockville, md, usa). For quantitative analysis, 6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured . The prefrontal cortex region was selected as regions of interest, and the identical area within the measuring frame in a 10 objective lens was labeled . The clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected . Cells that were lightly stained or had irregular shapes were excluded from quantification . Brain slices were washed with tbs buffer and blocked with a specific buffer (containing 5% donkey serum [ds], 0.25% bsa, 0.3% triton x-100, tbs) at room temperature for 1 h. the primary antibodies (diluted in 2% ds, 0.25% bsa, 0.3% triton x-100, tbs) used were: 6e10, grp 78 (1:50, santa cruz, ca, usa), chop (1:50, santa cruz), glial fibrillary acidic protein (gfap) (1:4000, millipore, boston, usa), and -iii - tubulin (1:4000, abcam). The 6e10 was co - incubated with grp 78, chop, gfap, and tubulin primary antibodies . Alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg (1:1500, invitrogen, carlsbad, ca, usa) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole (dapi) (diluted by 1:8000 in h2o) was then added and incubated for 5 min . Prolong gold antifade reagent (invitrogen) was used for slice mounting, and slices were imaged using confocal microscopy (leica tcs sp5, leica microsystems wetzlar gmbh, germany). Isolated mouse cortical tissues were added into tissue lysates (1% triton x-100, 50 mmol / l sodium fluoride, 2 mmol / l sodium orthovanadate, 10 mmol / l -sodium glycerophosphate, 10 mmol / l sodium pyrophosphate, 1% protease inhibitor cocktail [p8340, sigma - aldrich, st . Louis, mo, usa] dissolved in tbs buffer, ph 7.4) at a ratio of 1:10 (mg / ml). The mixture was incubated on ice for 30 min, and lysis was performed using the ultrasound method . Supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds - page). Subsequently, the proteins were transferred to polyvinylidene fluoride (pvdf) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies: grp 78 (1:200), chop (1:500), sapk / jnk (1:1000, cell signaling, boston, usa), p - sapk / jnk (thr183/tyr185) (1:1000, cell signaling), caspase-12 (1:500, cell signaling), cleaved caspase-3 (1:500, cell signaling), syvn1 (1:2000, abcam), -actin (1:2000, abcam), and -iii tubulin (1:100,000). Horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs (1:2000) were subsequently added and incubated at 37c for 1.5 h. imagej software (national institutes of health, bethesda, ma, usa) was used for quantitative analysis . Quantitative data were expressed as the mean standard error (se) and analyzed with the graphpad prism 6.01 software package (graphpad, san diego, ca, usa). Escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance (anova) while other data were evaluated using two - way anova . If the main effects and the interaction were significant, the genotypes were compared by student's t - test, or multiple conditions among the same genotype were evaluated with one - way anova . To investigate cognitive changes, we tested mice's behavioral performance in the morris water maze . We found that compared with age - matched wt mice, 7- and 12-month - old 5fad mice displayed a prolonged escape latency (f = 14.710, p <0.01 for 7-month - old; f = 5.939, p <0.05 for 12-month - old), indicating an obvious decline in learning ability and memory retention . In the probe trial, when the platform was removed, the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice (t = 2.331, p <0.05 for 7-month - old; t = 2.075, p <0.05 for 12-month - old) [figure 1a]. Immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice . A accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice, a large amount of plaque deposition was observed in the brain tissues [figure 1b]. Furthermore, we examined cleaved caspase-3 (the activated form), an executor molecule, in the apoptotic cascade by western blots . Levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner . Cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points . The increase was significant at the age of 12 months (n = 8, t = 2.504, p <0.05) [figure 1c]. The number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice, indicating neuronal loss in 5fad mice (n = 6, t = 2.987, p <0.05) [figure 1d and 1e]. The declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones . Escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice (n = 1014, * p <0.05, p <0.01 vs. age - matched wild - type mice). (b) 6e10 staining in 2-, 7-, and 12-month - old 5fad mice and wild - type mice . Scale bar = 100 m . (c) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age (n = 8, * p <0.05 vs. wild - type mice). (d) the neurons within the fifth layer of the cortex was quantified (n = 6, * p <0.05 vs. wild - type mice). (e) neun staining for neural nuclei in the frontal cortical slices from 2-, 7-, and 12-month - old mice . Scale bar = 100 m; 5fad: transgenic mice with five familiar alzheimer's disease; wt: wild - type mice; a: amyloid; v: the fifth layer of the frontal cortex . Grp 78 is a chaperone specifically localized in the er, whose expression increases in response to ers and serves as an er - specific marker . In wild mice, grp 78 expression increased insignificantly from the age of 27 months . In 5fad mice, grp 78 expression decreased from the age of 2 to 12 months (n = 6, t = 5.629, p <0.01 for 12 vs. 2 months). Interestingly, at the age of 2 months, grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice (n = 6, t = 2.549, p <0.05) [figure 2a]. Grp 78 expression (stained green) in wt mice increased gradually from the age of 212 months . In 2-month - old 5fad mice, grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [figure 2c]. Furthermore, grp 78 staining was localized mainly in the staining of -iii tubulin (red), not in the staining of gfap (red). Meanwhile, the distribution of a and grp 78 staining (red) was localized totally with the 6e10 staining (green), as shown in figure 2d . These data indicate that grp 78 is expressed mainly in neurons, not in astrocytes . Significant upregulation of anti - apoptotic factors, grp 78 and syvn1, in 2-month - old 5fad mice . (a) grp 78 expression of 5fad mice and age - matched wt mice at 2, 7, and 12 months old (n = 6, * p <0.05 vs. 2-month - old wt mice; p <0.01 vs. 2-month - old 5fad mice). (b) syvn1 expression of 5fad mice and age - matched wt mice at 2, 7, and 12 months old (n = 5, * p <0.05 vs. 2-month - old wt mice). (c) confocal images of grp 78 (green), 6e10 (a, red) and nuclei (dapi, blue) in the cortical slices of 2-, 7- and 12-month - old mice by immunofluorescence staining . (d) confocal images of grp 78 (green), gfap (red) or -iii - tubulin (red) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining . The bottom row of (d) indicates the immunofluorescent staining of grp 78 (red) and 6e10 (green, targeting a). 5fad: transgenic mice with five familiar alzheimer's disease; wt: wild - type mice; grp 78: glucose - regulated protein 78; syvn1: ubiquitin ligase synovial apoptosis inhibitor 1; a: amyloid; 6e10: the antibody targeting a; gfap: glial fibrillary acidic protein; dapi: 6-diamidino-2-phenylindole . Syvn1, a component of the erad pathway, is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . At the age of 7 months and 12 months, however, at the age of 2 months, syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice (n = 5, t = 2.000, p <0.05) [figure 2b]. Although chop is rarely expressed in normal neurons, its expression can be increased by ers to promote er - specific apoptosis . In the current study, western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice (n = 7, t = 2.806, p <0.05 for 12-month - old wt mice vs. 2-month - old wt mice). Of note, compared with age - matched wt mice, chop expression increased significantly in 2-month - old 5fad mice (n = 7, t = 2.322, p the chop expression was further detected by immunofluorescence and the chop staining (red) showed the same changing trend as that of the western blots assay [figure 3d]. Punctate distribution of chop in the cytoplasm and nucleus indicated chop activation, which serves as a transcription factor . Furthermore, chop staining (green) was localized mainly in the staining of -iii tubulin (red), not in the staining of gfap (red). Meanwhile, chop staining (red) was localized totally with the 6e10 staining (green), as shown in figure 3e . These data indicate that chop is predominantly expressed in 6e10-positive neurons, not in astrocytes . The cleaved caspase-12 expression was gradually increased with age in both wt mice (n = 6; t = 6.280, p <0.01 and t = 2.625, p <0.05, for 7- and 12-month - old mice vs. 2-month - old ones, respectively) and 5fad mice (n = 6, t = 3.320, p <0.05, t = 3.427, p <0.05 for 7- and 12-month - old mice vs. 2-month - old ones, respectively). Of note, caspase-12 activation was higher in 5fad mice at 2-, 7-, and 12-month - old when compared with the activation status observed in wt mice . This increase was significant at 2 and 7 months of age (n = 6, t = 5.365, p <0.01 for 2-month - old mice; t = 2.869, p <0.05 for 7-month - old mice) [figure 3b]. Significant increase of pro - apoptotic factors, chop and cleaved caspase-12 in 5fad mice . (a) western blots analyses revealed that chop protein increased with age in 5fad mice (n = 7, * p <0.05 vs. 2-month wt mice). (b) western blots analysis showed that caspase-12 activation increased with age in 5fad mice (n = 6, * p <0.05, p <0.01 vs. age - matched wt mice; p <0.05,p <0.01 vs. 2-month wt mice; p <0.05 vs. 2-month 5fad mice). (c) western blots analysis showed the expression of p - jnk / jnk . (d) confocal images of chop (red), 6e10 (a, green) and nuclei (dapi, blue) in the cortical slices of 2-, 7-, and 12-month - old mice by immunofluorescence staining . (e) confocal images of chop (green), gfap (red) or -iii - tubulin (red) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining . The bottom row of (e) indicates immunofluorescent staining of chop (red) and 6e10 (green, targeting a). 5fad: transgenic mice with five familiar alzheimer's disease; wt: wild - type mice; chop: ccaat / enhancer - binding protein homologous protein; jnk: c - jun amino - terminal kinase; a: amyloid; 6e10: the antibody targeting a; gfap: glial fibrillary acidic protein; dapi: 6-diamidino-2-phenylindole . A recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis . However, it remains unknown if jnk signaling is activated to trigger chop . The present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different . These observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [figure 3c]. Taken together, these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice . To investigate cognitive changes, we tested mice's behavioral performance in the morris water maze . We found that compared with age - matched wt mice, 7- and 12-month - old 5fad mice displayed a prolonged escape latency (f = 14.710, p <0.01 for 7-month - old; f = 5.939, p <0.05 for 12-month - old), indicating an obvious decline in learning ability and memory retention . In the probe trial, when the platform was removed, the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice (t = 2.331, p <0.05 for 7-month - old; t = 2.075, p <0.05 for 12-month - old) [figure 1a]. Immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice . A accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice, a large amount of plaque deposition was observed in the brain tissues [figure 1b]. Furthermore, we examined cleaved caspase-3 (the activated form), an executor molecule, in the apoptotic cascade by western blots . Levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner . Cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points . The increase was significant at the age of 12 months (n = 8, t = 2.504, p <0.05) [figure 1c]. The number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice, indicating neuronal loss in 5fad mice (n = 6, t = 2.987, p <0.05) [figure 1d and 1e]. The declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones . Escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice (n = 1014, * p <0.05, p <0.01 vs. age - matched wild - type mice). (b) 6e10 staining in 2-, 7-, and 12-month - old 5fad mice and wild - type mice . Scale bar = 100 m . (c) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age (n = 8, * p <0.05 vs. wild - type mice). (d) the neurons within the fifth layer of the cortex was quantified (n = 6, * p <0.05 vs. wild - type mice). (e) neun staining for neural nuclei in the frontal cortical slices from 2-, 7-, and 12-month - old mice . Scale bar = 100 m; 5fad: transgenic mice with five familiar alzheimer's disease; wt: wild - type mice; a: amyloid; v: the fifth layer of the frontal cortex . Grp 78 is a chaperone specifically localized in the er, whose expression increases in response to ers and serves as an er - specific marker . In wild mice, grp 78 expression increased insignificantly from the age of 27 months . In 5fad mice, grp 78 expression decreased from the age of 2 to 12 months (n = 6, t = 5.629, p <0.01 for 12 vs. 2 months). Interestingly, at the age of 2 months, grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice (n = 6, t = 2.549, p <0.05) [figure 2a]. Grp 78 expression (stained green) in wt mice increased gradually from the age of 212 months . In 2-month - old 5fad mice, grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [figure 2c]. Furthermore, grp 78 staining was localized mainly in the staining of -iii tubulin (red), not in the staining of gfap (red). Meanwhile, the distribution of a and grp 78 staining (red) was localized totally with the 6e10 staining (green), as shown in figure 2d . These data indicate that grp 78 is expressed mainly in neurons, not in astrocytes . Significant upregulation of anti - apoptotic factors, grp 78 and syvn1, in 2-month - old 5fad mice . (a) grp 78 expression of 5fad mice and age - matched wt mice at 2, 7, and 12 months old (n = 6, * p <0.05 vs. 2-month - old wt mice; p <0.01 vs. 2-month - old 5fad mice). (b) syvn1 expression of 5fad mice and age - matched wt mice at 2, 7, and 12 months old (n = 5, * p <0.05 vs. 2-month - old wt mice). (c) confocal images of grp 78 (green), 6e10 (a, red) and nuclei (dapi, blue) in the cortical slices of 2-, 7- and 12-month - old mice by immunofluorescence staining . (d) confocal images of grp 78 (green), gfap (red) or -iii - tubulin (red) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining . The bottom row of (d) indicates the immunofluorescent staining of grp 78 (red) and 6e10 (green, targeting a). 5fad: transgenic mice with five familiar alzheimer's disease; wt: wild - type mice; grp 78: glucose - regulated protein 78; syvn1: ubiquitin ligase synovial apoptosis inhibitor 1; a: amyloid; 6e10: the antibody targeting a; gfap: glial fibrillary acidic protein; dapi: 6-diamidino-2-phenylindole . Syvn1, a component of the erad pathway, is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . At the age of 7 months and 12 months, however, at the age of 2 months, syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice (n = 5, t = 2.000, p <0.05) [figure 2b]. Although chop is rarely expressed in normal neurons, its expression can be increased by ers to promote er - specific apoptosis . In the current study, western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice (n = 7, t = 2.806, p <0.05 for 12-month - old wt mice vs. 2-month - old wt mice). Of note, compared with age - matched wt mice, chop expression increased significantly in 2-month - old 5fad mice (n = 7, t = 2.322, p <0.05) [figure 3a]. The chop expression was further detected by immunofluorescence and the chop staining (red) showed the same changing trend as that of the western blots assay [figure 3d]. Punctate distribution of chop in the cytoplasm and nucleus indicated chop activation, which serves as a transcription factor . Furthermore, chop staining (green) was localized mainly in the staining of -iii tubulin (red), not in the staining of gfap (red). Meanwhile, chop staining (red) was localized totally with the 6e10 staining (green), as shown in figure 3e . These data indicate that chop is predominantly expressed in 6e10-positive neurons, not in astrocytes . The cleaved caspase-12 expression was gradually increased with age in both wt mice (n = 6; t = 6.280, p <0.01 and t = 2.625, p <0.05, for 7- and 12-month - old mice vs. 2-month - old ones, respectively) and 5fad mice (n = 6, t = 3.320, p <0.05, t = 3.427, p <0.05 for 7- and 12-month - old mice vs. 2-month - old ones, respectively). Of note, caspase-12 activation was higher in 5fad mice at 2-, 7-, and 12-month - old when compared with the activation status observed in wt mice . This increase was significant at 2 and 7 months of age (n = 6, t = 5.365, p <0.01 for 2-month - old mice; t = 2.869, p <0.05 for 7-month - old mice) [figure 3b]. Significant increase of pro - apoptotic factors, chop and cleaved caspase-12 in 5fad mice . (a) western blots analyses revealed that chop protein increased with age in 5fad mice (n = 7, * p <0.05 vs. 2-month wt mice). (b) western blots analysis showed that caspase-12 activation increased with age in 5fad mice (n = 6, * p <0.05, p <0.01 vs. age - matched wt mice; p <0.05,p <0.01 vs. 2-month wt mice; p <0.05 vs. 2-month 5fad mice). (c) western blots analysis showed the expression of p - jnk / jnk . (d) confocal images of chop (red), 6e10 (a, green) and nuclei (dapi, blue) in the cortical slices of 2-, 7-, and 12-month - old mice by immunofluorescence staining . (e) confocal images of chop (green), gfap (red) or -iii - tubulin (red) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining . The bottom row of (e) indicates immunofluorescent staining of chop (red) and 6e10 (green, targeting a). 5fad: transgenic mice with five familiar alzheimer's disease; wt: wild - type mice; chop: ccaat / enhancer - binding protein homologous protein; jnk: c - jun amino - terminal kinase; a: amyloid; 6e10: the antibody targeting a; gfap: glial fibrillary acidic protein; dapi: 6-diamidino-2-phenylindole . A recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis . The present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different . These observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [figure 3c]. Taken together, these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice . In 5fad mice, we observed that neuronal loss and cleaved caspase-3 increase at the age of 12 months, cognitive deficit at the age of 7 months and 12 months . Interestingly, at the age of 2-month - old 5fad mice, the related factors involved in the ers - associated upr pathway, including chop, cleaved caspase-12, grp 78, and syvn1, were significantly increased compared with those in age - matched wt mice . These findings suggest that 2-month - old 5fad mice exhibit enhanced ers - associated upr pathway, consistent with intracellular a aggregation in neurons . Oakley et al . Reported, in 5fad brain, accumulated intraneuronal a42 starting at 1.5 months of age, the presence of cerebral amyloid plaques and gliosis at 2 months of age, decreased synaptic markers including synaptophysin and postsynaptic density 95 (psd95) accompanied by cognition deficit at 4 months of age, and pyramidal neuron loss in cortical layer 5 at 9 months of age . Consistently, in our study, the deposit of intra- and extra - neuronal a increased with age in 5fad brain; neuronal loss in cortical layer 5 was observed at 12 months of age; however, before significant neuron loss, impaired memory of 5fad mice at 7 months of age was monitored in the morris water maze . Our previous study has also shown that cognitive impairment present in 5fad mice was accompanied by structural degradation of synapses and decreased expression of synaptophysin and psd95 in the brain at the age of 67 months . Under physiological conditions, approximately one - third of proteins in the er are assembled abnormally and do not form mature proteins . Aberrant proteins are bound by chaperone proteins, such as grp 78, to be repaired or transported into the er - associated ubiquitin - proteasome system for degradation via the erad pathway, which serves as an accurate quality control system . Erad promotes the interaction between ubiquitin and proteins waiting for degradation via e1, e2, and e3 . An impaired proteasome system leads to accumulation of aberrant proteins and increased risk of cell death . Sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of chop and caspase-12 activation . Ers characterized by increased ers - specific apoptosis is obvious in postmortem examinations of brain tissues of ad patients . In vitro experiments have also confirmed that a induces the cellular ers response directly, indicating that abnormal aggregation of a is responsible for ers induction . Other studies have shown that astrocytes and macrophages also induce ers . However, in our study, the co - immunostaining of grp 78 and chop with astrocytic or neuronal markers showed that ers in cortical tissues occurred mainly in neurons . Therefore, the ers - associated proteins examined here represent neuronal ers and reveal the impact of the upr on neuronal functions under a-induced stress conditions . In this study, we selected mice at 2, 7, and 12 months of age to investigate the changes of ers - associated proteins in 5fad mice and wt mice . Our results showed that the expression of grp 78 and syvn1 changed without statistical significance but displayed an early - increase and subsequent - decline tendency from 2 to 12 months in wt mice . Interestingly, at the age of 2 months, the expression of grp 78 and syvn1 in 5fad brains was significantly higher than that in wt mice; however, at the age of 12 months, grp 78 level in 5fad brains significantly declined when compared with that of 2-month - old 5fad mice . Meanwhile, the expression of chop and cleaved caspase-12 increased continuously with age, either in 5fad mice or in wt mice . Of note, the level of chop and cleaved caspase-12 in 5fad mice was obviously higher than that in wt mice . Altogether, we speculate that a in 2-month - old 5fad brains lead to higher ers level than that of wt mice, which produces a cellular protective effect, on the one hand, up - regulating the expression of er chaperones and related degradation proteins mainly via pire1a - xbp1s and atf6a pathways at the early stage of ad to eliminate a; on the other hand, inducing increased expression of downstream signaling pathways molecules, especially pro - apoptotic proteins (cleaved caspase-12 and chop). Interestingly, no significant differences in pjnk / jnk levels were found between 5fad mice and wt mice at different periods, which indicates that the pjnk / jnk pathway is not involved in a-induced ers . Under sustained stress conditions caused by the a-associated toxic effects, the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually . The pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death [figure 4]. Schematic drawing shows that activated upr pathways of ers induced by intraneuronal -amyloid in 5fad mice . A in 2-month - old 5fad brains lead to higher ers level than that of wt mice, which produces a cellular protective effect, on the one hand, up - regulating the expression of er chaperones and related degradation proteins mainly via p - ire-1-xbp1s and atf6 pathways at the early stage of ad to eliminate a; on the other hand, inducing increased expression of downstream signaling pathways molecules, especially pro - apoptotic proteins (cleaved caspase-12 and chop). Under sustained stress conditions caused by the a-associated toxic effects, the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually . The pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death . Upr: unfolded protein response; ers: endoplasmic reticulum stress; a: amyloid; 5fad: transgenic mice with five familiar alzheimer's disease; p - perk: phosphorylated protein kinase rna - like er kinase; p - eif2: phosphorylated eukaryotic translation initiation factor 2; atf4: activating transcription factor 4; chop: ccaat / enhancer - binding protein homologous protein; p - ire-1: phosphorylated inositol - requiring enzyme 1; xbp1s: spliced x box - binding protein 1; atf6: activating transcription factor 6; grp 78: glucose - regulated protein 78; erad: endoplasmic reticulum - associated protein degradation; syvn1: ubiquitin ligase synovial apoptosis inhibitor 1; traf2: tumor necrosis factor - receptor - associated factor 2; ask1: apoptosis signal - regulating kinase 1; jnk: c - jun amino - terminal kinase . In conclusion, current data reveal that intracellular a aggregation induces obvious ers in neurons at the early stage of 5fad brains . Imbalanced regulation of grp 78 or syvn1 and chop or cleaved caspase-12 may be involved in the resistance to the restoration of er homeostasis in a-secreting neurons . If the development of ers can be delayed or reduced appropriately or the protective functions of the upr can be enhanced exogenously, the capacity of the er to tolerate abnormal proteins can be increased to delay cell damage and disease progression . Targeting at ers to control the pathophysiological changes at the early stage of ad may be a better strategy for the prevention and treatment of ad . This work was supported by grants from the national natural science foundation of china (no . Xiao - dong pan) and the national and fujian province's key clinical specialty discipline construction programs . This work was supported by grants from the national natural science foundation of china (no . Xiao - dong pan) and the national and fujian province's key clinical specialty discipline construction programs.
Cvds are the leading causes of death in diabetic patients and the substantial rise in diabetes prevalence will ultimately lead to an increase in the burden of diabetes - related cvd in coming decades . Among the cardiovascular diseases, ischemic heart disease is the most frequent [5, 6] and is associated with the highest morbidity and mortality in patients with type 2 dm . Despite the advances in the treatment of coronary artery disease (cad), morbidity and mortality are still high, especially in patients with type 2 dm . Moreover, despite the evolution in the treatment of hyperglycemia, patients with both cad and diabetes have worse clinical outcomes, irrespective of the treatment applied . Although dm alters the healthy functioning of arteries and blood compounds, some clinical studies suggest that myocardial responses to ischemic insults may be deficient in diabetic patients [11, 12], leading to higher myocardial damage and risk of complications . Thus, recent studies have focused on the understanding of such myocardial responses, aiming at discovering the mechanisms of myocardial protection to achieve less aggression and a better prognosis . A substantial effort has been put forth to investigate any promising cardioprotective strategy to effectively reduce myocardial infarct size . And this matter is of particular importance to diabetic cad patients . By 1986, in a landmark experimental study, murry and colleagues demonstrated that a short antecedent period of ischemia could result in a great reduction in myocardial infarct size . For the first time, a method for reducing myocardial cell death other than reperfusion had been discovered . Since its discovery, this phenomenon, termed ischemic preconditioning (ip) has been extensively studied . Currently, ip is the intrinsic myocardial mechanism that provides the greatest protection regarding the reduction in myocardial ischemic damage . It has demonstrated a 75% reduction in the infarcted area in animal models that have undergone the preconditioning protocol . Ip is assumed as a mechanism of myocardial protection in which brief episodes of sublethal myocardial ischemia followed by reperfusion trigger multiple intracellular pathways that ultimately result in greater myocardial resistance to a subsequent intense ischemic injury . Experimentally, this phenomenon was demonstrated by the reduction in the infarcted area by short episodes of ischemia prior to a pronounced ischemic insult . Although ip was initially thought to result from the opening of collateral vessels and higher coronary flow, some interesting studies have shown that the phenomenon occurs irrespective of coronary flow changes [17, 18]. Such studies also demonstrated that ip can be observed by sequential exercise tests (sets), in which the improvement in ischemic parameters in the second of 2 sets were confirmed by invasive measurements of myocardial oxygen consumption . Moreover, the clinical observation that patients with chronic ischemic heart disease frequently describe attenuation or even cessation of angina symptoms if they rest and restart the exercise is termed warm - up or walk - through angina . This phenomenon has been related to ip and documented in studies using sets [19, 20]. Following the demonstration of ip, the cellular mechanisms underlying the phenomenon began to be investigated . Although some cellular pathways have not been fully elucidated, there is a growing understanding of some phenomena and currently it is assumed that ip may be modulated by external factors like age, diseases [2224], as well as some specific classes of medications [2527]. Currently, the opening of k - atp channels plays a fundamental role in the cellular cascades of ip . It is also assumed that medications that bind to this channel may interfere with the ip mechanism . Thus, some classes of medications like the oral hypoglycemic agents may also block such channels in extrapancreatic sites, such as in the heart . It has been shown by some authors that these medications may cause the blockage of myocardial ip . However, despite the fact that some oral hypoglycemic agents may interfere with ip, it is still uncertain whether the intrinsic, complex, intracellular alterations of diabetes itself may affect the cellular mechanisms of this cardioprotective phenomenon . Some of these studies have shown that diabetes does not interfere with ip [31, 32], but others have shown negative influences [33, 34]. The great variability in these results is especially due to differences in study protocols, most notably the variability in animal models studied, in the protocols to induce diabetes, the duration of the disease, as well as the variability in myocardial injury protocols . On the other hand, studies in humans are scarce and their results have also been conflicting [12, 13, 35]. Thus, in this study, we aimed at identifying ip in symptomatic multivessel cad patients and compared the results among patients with and without type 2 dm . This was a prospective study that included patients with symptomatic cad followed by the medicine, angioplasty, or surgery study (mass) research group . Briefly, patients were enrolled who had angina symptoms, multivessel cad, preserved left systolic ventricular function, and a recent, positive ischemic treadmill stress test . The diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria . Multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70% obstruction in 2 or more coronary artery territories . The systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was 0.50 . Treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was 1.0 mm, associated or not with thoracic discomfort . Exclusion criteria were single - vessel cad, left main cad, impaired systolic ventricular function (defined as an ejection fraction <0.50), recent and negative treadmill exercise test, high - risk positive treadmill exercise test, limiting angina symptoms, an acute coronary syndrome in the prior 3 months, electrocardiographic signs that could make difficult the interpretation of ischemic changes (left bundle - branch block, st - segment deviation), arrhythmias like atrial fibrillation or flutter, severe valvular disease, cardiomyopathies, or patient refusal to participate in this study . After cardiologic evaluation, all patients were instructed to stop medications with cardiovascular properties 5 days before sets . Diabetic patients were also instructed to stop antidiabetes medications 5 days before the tests . Patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control . A telephone contact was available 24 h a day in case of questions or worsening symptoms . Before treadmill test initiation, all patients underwent 2 sets, symptom limited, with a 30-min interval between them . Tests were conducted during the same period each day, 1 h after lunchtime, on the same treadmill, (mat 2100 treadmill and a fukuda denshi ml8000 stress test system (fukuda denshi; bunkyo - ku, tokyo, japan). A 12-lead electrocardiogram, heart rate, and arterial blood pressure were obtained with the patient in the standing position at baseline . A 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise, at peak exercise, each minute up to 5 min during the recovery phase, at the onset of 1.0 mm st - segment depression, during arrhythmias, and when it was clinically relevant . The level of the st - segment deviation was based on visual assessment of the 0.08 s after the j point by 2 independent cardiologists . In case of disagreement only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation (t-1.0 mm). Criteria for interrupting the exercise test were st - segment depression 3.0 mm, st segment elevation 2.0 mm, maximum age - related heart rate, severe arterial hypotension or hypertension, severe chest pain, physical exhaustion, and sustained arrhythmias . The following parameters were systematically measured: resting heart rate and arterial blood pressure, heart rate and arterial blood pressure at peak exercise, t-1.0 mm in seconds, rate pressure product (rpp) at the onset of t-1.0 mm, and exercise duration in seconds . The improvement in ischemic parameters (t-1.0 mm and rpp) in the second exercise test compared to the first one indicated the presence of ip . Other parameters recorded during sets were the total exercise time, the occurrence and density of supraventricular and ventricular arrhythmias, and st - segment deviation morphology, during exercise and recovery phases . Both arrhythmias and st - segment morphology were graded according to their density and severity . According to the levels of fasting glycemia and glycosylated hemoglobin from baseline, patients were separated into quartiles . For fasting glycemia, the quartiles were defined as quartile 1: <90 mg / dl, quartile 2: 90100 mg / dl, quartile 3: 101125 mg / dl, and quartile 4: 126 mg / dl . For glycosylated hemoglobin, the quartiles were defined as quartile 1: <5.7%, quartile 2: 5.76.3%, quartile 3: 6.46.9%, and quartile 4: 7.0% . Briefly, it was determined by the analysis of studies with similar methodologies that included only diabetic patients and studies that included only nondiabetic patients . By the differences in t-1.0 mm in diabetic and nondiabetic patients, and accounting for an expected loss of 30% of patients who do not demonstrate the ip phenomenon (estimated based on the experience of our research group), 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar (power 0.9 and alpha error 0.05). Continuous variables were compared using an unpaired student s t test or mann whitney test, when appropriate . Data are expressed as mean standard deviation or as absolute frequencies and percentages . All tests were 2-sided, and a value of p <0.05 was considered significant . This was a prospective study that included patients with symptomatic cad followed by the medicine, angioplasty, or surgery study (mass) research group . Briefly, patients were enrolled who had angina symptoms, multivessel cad, preserved left systolic ventricular function, and a recent, positive ischemic treadmill stress test . The diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria . Multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70% obstruction in 2 or more coronary artery territories . The systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was 0.50 . Treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was 1.0 mm, associated or not with thoracic discomfort . Exclusion criteria were single - vessel cad, left main cad, impaired systolic ventricular function (defined as an ejection fraction <0.50), recent and negative treadmill exercise test, high - risk positive treadmill exercise test, limiting angina symptoms, an acute coronary syndrome in the prior 3 months, electrocardiographic signs that could make difficult the interpretation of ischemic changes (left bundle - branch block, st - segment deviation), arrhythmias like atrial fibrillation or flutter, severe valvular disease, cardiomyopathies, or patient refusal to participate in this study . After cardiologic evaluation, all patients were instructed to stop medications with cardiovascular properties 5 days before sets . Diabetic patients were also instructed to stop antidiabetes medications 5 days before the tests . Patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control . A telephone contact was available 24 h a day in case of questions or worsening symptoms . Before treadmill test initiation, all patients underwent 2 sets, symptom limited, with a 30-min interval between them . The modified bruce protocol was applied . Tests were conducted during the same period each day, 1 h after lunchtime, on the same treadmill, (mat 2100 treadmill and a fukuda denshi ml8000 stress test system (fukuda denshi; bunkyo - ku, tokyo, japan). A 12-lead electrocardiogram, heart rate, and arterial blood pressure were obtained with the patient in the standing position at baseline . A 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise, at peak exercise, each minute up to 5 min during the recovery phase, at the onset of 1.0 mm st - segment depression, during arrhythmias, and when it was clinically relevant . The level of the st - segment deviation was based on visual assessment of the 0.08 s after the j point by 2 independent cardiologists . In case of disagreement, only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation (t-1.0 mm). Criteria for interrupting the exercise test were st - segment depression 3.0 mm, st segment elevation 2.0 mm, maximum age - related heart rate, severe arterial hypotension or hypertension, severe chest pain, physical exhaustion, and sustained arrhythmias . The following parameters were systematically measured: resting heart rate and arterial blood pressure, heart rate and arterial blood pressure at peak exercise, t-1.0 mm in seconds, rate pressure product (rpp) at the onset of t-1.0 mm, and exercise duration in seconds . The improvement in ischemic parameters (t-1.0 mm and rpp) in the second exercise test compared to the first one indicated the presence of ip . Other parameters recorded during sets were the total exercise time, the occurrence and density of supraventricular and ventricular arrhythmias, and st - segment deviation morphology, during exercise and recovery phases . Both arrhythmias and st - segment morphology were graded according to their density and severity . According to the levels of fasting glycemia and glycosylated hemoglobin from baseline, patients were separated into quartiles . For fasting glycemia, the quartiles were defined as quartile 1: <90 mg / dl, quartile 2: 90100 mg / dl, quartile 3: 101125 mg / dl, and quartile 4: 126 mg / dl . For glycosylated hemoglobin, the quartiles were defined as quartile 1: <5.7%, quartile 2: 5.76.3%, quartile 3: 6.46.9%, and quartile 4: 7.0% . The sample size calculation has been previously published elsewhere . Briefly, it was determined by the analysis of studies with similar methodologies that included only diabetic patients and studies that included only nondiabetic patients . By the differences in t-1.0 mm in diabetic and nondiabetic patients, and accounting for an expected loss of 30% of patients who do not demonstrate the ip phenomenon (estimated based on the experience of our research group), 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar (power 0.9 and alpha error 0.05). Continuous variables were compared using an unpaired student s t test or mann whitney test, when appropriate . Data are expressed as mean standard deviation or as absolute frequencies and percentages . All tests were 2-sided, and a value of p <0.05 was considered significant . Of 2,140 patients with stable cad followed at our institution, 361 met the inclusion criteria . The main reasons for non - inclusion and exclusion are shown in fig . 1 . Thus, a total of 174 patients completed the 2 sets and had ip assessed . The study population comprised 86 patients with dm and 88 without this diagnosis (fig . The 2,140 initial cad patients screened, 361 met the inclusion criteria and were enrolled, and 174 completed the study protocol . Cad = coronary artery disease; ckd = chronic kidney disease; cva = cerebrovascular accident; ecg = electrocardiogram; ef = ejection fraction; et = exercise test; dm = diabetes mellitus; ip = ischemic preconditioning; set = sequential exercise test (s) study flow chart . Of the 2,140 initial cad patients screened, 361 met the inclusion criteria and were enrolled, and 174 completed the study protocol . Cad = coronary artery disease; ckd = chronic kidney disease; cva = cerebrovascular accident; ecg = electrocardiogram; ef = ejection fraction; et = exercise test; dm = diabetes mellitus; ip = ischemic preconditioning; set = sequential exercise test (s) the main demographic, clinical and biochemical characteristics of the 2 groups are presented in table 1 . Despite the higher prevalence of previous myocardial infarction in the diabetic population and lipid profile (higher ldl - cholesterol levels in nondiabetic patients), both groups had homogeneous characteristics . The duration of diabetes was 11.5 8.8 years (mean sd), with a median of 10 years (interquartiles ranges of 6 and 15 years). Table 2 shows the medications used in the two groups of patients.table 1main demographic, biochemical, and clinical characteristics of the study populationtotal n = 174diabetics (n = 86)non - diabetics (n = 88) p valueage64.1 6.864.3 8.60.84male n (%) 73 (84.9)76 (86.3)0.83hypertension64 (80)74 (84.1)0.54smokers7 (8.8)7 (8.6)previous smokers38 (48.1)33 (40.7)0.6non - smokers34 (43.0)41 (50.6)previous ami36 (46.1)17 (22.7)0.004cabg28 (34.6)23 (26.1)0.25pci24 (30.4)28 (32.2)0.87ef0.61 0.060.62 0.060.2tri - vessel disease38 (52)43 (51.2)1.0bi - vessel disease35 (48)41 (48.8)1.0lad disease63 (87.5)74 (86.0)1.0collateral circulation34 (50.7)38 (46.9)0.64collateral grade 2/330 (88.2)32 (84.2)0.74weight (kg)74.5 11.073.2 12.70.51height (m)1.66 8.11.66 9.10.75bmi26.5 2.826.4 3.20.54fasting glycemia143.5 47.099.0 9.40.0001a1c7.35 1.615.63 0.300.0001bun41.9 13.440.1 9.40.32creatinine1.04 0.231.07 0.200.37total cholesterol156.5 34.5168.9 36.30.02ldl cholesterol90.5 27.4102.0 32.70.01hdl cholesterol38.0 9.640.3 9.00.12triglycerides147.2 88.3132.6 72.30.24data are expressed as means standard deviation or as absolute and relative risks ami stands for acute myocardial infarction, cabg coronary artery bypass surgery, pci percutaneous coronary intervention, ef ejection fraction, lad left anterior descending, bmi body mass index, a1c glycosylated hemoglobin, bun blood urea nitrogen, ldl low - density lipoprotein, hdl high - density lipoproteintable 2classes of medications used by the two groups of diabetic and nondiabetic patientsaspirin / clopidogrelstatinsbeta - blockersacei / arbcalcium blockersdiureticsoadinsulinsdm93.2% 93.2% 89.8% 78.4% 35.2% 37.5% 88.5% 26.5% non - dm96.5% 96.5% 86.2% 78.2% 36.8% 34.5%--dm stands for diabetic patients, non - dm nondiabetic patients, acei angiotensin converting enzyme inhibitors, arb angiotensin receptor blockers, oad oral antidiabetic drugs main demographic, biochemical, and clinical characteristics of the study population data are expressed as means standard deviation or as absolute and relative risks ami stands for acute myocardial infarction, cabg coronary artery bypass surgery, pci percutaneous coronary intervention, ef ejection fraction, lad left anterior descending, bmi body mass index, a1c glycosylated hemoglobin, bun blood urea nitrogen, ldl low - density lipoprotein, hdl high - density lipoprotein classes of medications used by the two groups of diabetic and nondiabetic patients dm stands for diabetic patients, non - dm nondiabetic patients, acei angiotensin converting enzyme inhibitors, arb angiotensin receptor blockers, oad oral antidiabetic drugs among the 86 diabetic patients, 62 (72%) had an improvement in t-1.0 mm consistent with ip . Among the 88 nondiabetic patients, 60 (68%) had an ischemic improvement consistent with ip (fig . 2, p = 0.62).fig . 2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip . Ip = ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip . Ip = ischemic preconditioning the t-1.0 mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0 mm between the 2 sets of 79.4 47.6 s, whereas nondiabetic patients who demonstrated ip had an improvement of 65.5 36.4 s (table 3, p = 0.12).table 3t-1.0 mm in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-)et1et2et2-et1 p valuedm269.2 117.8320.9 132.351.7 63.20.15non - dm275.6 111.9314.6 126.239.0 52.3 dm / ip+274.8 102.8354.3 115.179.4 47.60.12non - dm / ip+296.9 107.7362.4 108.565.5 36.4 dm / ip -254.7 151.6234.7 137.020.0 36.40.80non - dm / ip -230.1 108.7212.4 98.217.7 31.9data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et, exercise test . the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0 mm in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-) data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et, exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp, the group of diabetic patients who demonstrated ip had an improvement of 3,011 2,430 bpm x mmhg, whereas nondiabetic patients had an improvement of 2,081 2,139 bpm x mmhg (table 4, p = 0.01).table 4rpp in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-)et1et2et2-et1 p valuedm / ip+22,841 4,50025,853 4,7423,011 2,4300.01non - dm / ip + 23,094 5,31225,175 5,4852,081 2,139 dm / ip -22,705 4,00022,124 3,686580 2,2500.43non - dm / ip -23,910 5,38022,869 5,3511,050 2,027data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test . the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-) data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table 5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters, heart rate and blood pressure in the 4 groups of patients, diabetic and nondiabetic, according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip + hr (baseline)79.0 15.186.3 17.07.3 11.0hr (peak)139.0 18.2149.7 19.36.8 12.6bp (peak)192.0 25.0191.8 23.90.16 15.4 dm / ip -hr (baseline)79.0 14.784.4 14.95.4 8.0hr (peak)141.7 14.3143.0 13.51.3 7.4bp (baseline)153.3 22.6147.1 19.76.2 14.7bp (peak)183.7 23.2180.6 22.23.12 14.7non - dm / ip + hr (baseline)78.0 13.187.1 13.99.1 7.9hr (peak)139.6 15.4144.3 14.74.7 5.7bp (baseline)146.7 20.6138.7 18.38.0 11.3bp (peak)193.5 23.6190.2 24.53.3 12.4non - dm / ip -hr (baseline)82.5 12.987.1 12.64.6 7.1hr (peak)139.3 12.4139.6 13.00.35 5.2bp (baseline)158.6 26.2147.9 26.110.7 14.6bp (peak)204.6 25.3194.8 28.18.9 dm stands for diabetic patients, non - dm nondiabetic patients, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test hemodynamic parameters, heart rate and blood pressure in the 4 groups of patients, diabetic and nondiabetic, according to the demonstration of ip data are expressed as means standard deviation . Dm stands for diabetic patients, non - dm nondiabetic patients, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients (20 39 s versus 17 36 s, respectively, p = 0.60). The improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip (50% versus 63% of patients demonstrated improvement in arrhythmias, respectively; p = 0.41). The improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients (38.5% versus 48.3%, respectively, p = 0.41). When the total group of patients (n = 174) was stratified according to the demonstration of ip, the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups, as shown in table 6.table 6frequency of diabetes mellitus, and levels of fasting glycemia and a1c in the study population according to the expression of ipip + (n = 122)ip - (n = 52) p valuedm n (%) 62 (50.8)24 (46.1)0.57fasting glycemia121.3 42.6118.9 34.10.72a1c6.63 1.66.32 1.20.25data are expressed as means standard deviation and as absolute and relative frequencies dm stands for diabetes mellitus, a1c glycosylated hemoglobin, ip + ischemic preconditioning present, ip - ischemic preconditioning absent frequency of diabetes mellitus, and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means standard deviation and as absolute and relative frequencies dm stands for diabetes mellitus, a1c glycosylated hemoglobin, ip + ischemic preconditioning present, ip - ischemic preconditioning absent in addition, when we stratified patients by quartiles of glycemia and glycosylated hemoglobin, there was no statistical difference in terms of ip demonstration among the different quartiles (fig . 3graphs showing the percentage of patients who demonstrated ischemic preconditioning (ip+ in blue) and who did not demonstrate ischemic preconditioning (ip - in red) stratified into quartiles of a1c (graph a) and fasting glycemia (graph b). Ip = ischemic preconditioning; q = quartile (s). X axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning (ip+ in blue) and who did not demonstrate ischemic preconditioning (ip - in red) stratified into quartiles of a1c (graph a) and fasting glycemia (graph b). Ip = ischemic preconditioning; q = quartile (s). X axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia among the 86 diabetic patients, 62 (72%) had an improvement in t-1.0 mm consistent with ip . Among the 88 nondiabetic patients, 60 (68%) had an ischemic improvement consistent with ip (fig . 2, p = 0.62).fig . 2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip . Ip = ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip . Ip = ischemic preconditioning the t-1.0 mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0 mm between the 2 sets of 79.4 47.6 s, whereas nondiabetic patients who demonstrated ip had an improvement of 65.5 36.4 s (table 3, p = 0.12).table 3t-1.0 mm in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-)et1et2et2-et1 p valuedm269.2 117.8320.9 132.351.7 63.20.15non - dm275.6 111.9314.6 126.239.0 52.3 dm / ip+274.8 102.8354.3 115.179.4 47.60.12non - dm / ip+296.9 107.7362.4 108.565.5 36.4 dm / ip -254.7 151.6234.7 137.020.0 36.40.80non - dm / ip -230.1 108.7212.4 98.217.7 31.9data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et, exercise test . the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0 mm in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-) data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et, exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp, the group of diabetic patients who demonstrated ip had an improvement of 3,011 2,430 bpm x mmhg, whereas nondiabetic patients had an improvement of 2,081 2,139 bpm x mmhg (table 4, p = 0.01).table 4rpp in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-)et1et2et2-et1 p valuedm / ip+22,841 4,50025,853 4,7423,011 2,4300.01non - dm / ip + 23,094 5,31225,175 5,4852,081 2,139 dm / ip -22,705 4,00022,124 3,686580 2,2500.43non - dm / ip -23,910 5,38022,869 5,3511,050 2,027data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test . the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 (et1) and exercise test 2 (et2) and the difference between the 2 tests (et2-et1) in diabetic and nondiabetic patients who demonstrated ip (ip+) or who did not demonstrate ip (ip-) data are expressed as means standard deviation . Dm stands for diabetes mellitus, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table 5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters, heart rate and blood pressure in the 4 groups of patients, diabetic and nondiabetic, according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip + hr (baseline)79.0 15.186.3 17.07.3 11.0hr (peak)139.0 15.1142.1 14.73.2 5.6bp (baseline)156.5 18.2149.7 19.36.8 12.6bp (peak)192.0 25.0191.8 23.90.16 15.4 dm / ip -hr (baseline)79.0 14.784.4 14.95.4 8.0hr (peak)141.7 14.3143.0 13.51.3 7.4bp (baseline)153.3 22.6147.1 19.76.2 14.7bp (peak)183.7 23.2180.6 22.23.12 14.7non - dm / ip + hr (baseline)78.0 13.187.1 13.99.1 7.9hr (peak)139.6 15.4144.3 14.74.7 5.7bp (baseline)146.7 20.6138.7 18.38.0 11.3bp (peak)193.5 23.6190.2 24.53.3 12.4non - dm / ip -hr (baseline)82.5 12.987.1 12.64.6 7.1hr (peak)139.3 12.4139.6 13.00.35 5.2bp (baseline)158.6 26.2147.9 26.110.7 14.6bp (peak)204.6 25.3194.8 28.18.9 16.5data are expressed as means standard deviation . Dm stands for diabetic patients, non - dm nondiabetic patients, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test hemodynamic parameters, heart rate and blood pressure in the 4 groups of patients, diabetic and nondiabetic, according to the demonstration of ip data are expressed as means standard deviation . Dm stands for diabetic patients, non - dm nondiabetic patients, ip + ischemic preconditioning present, ip - ischemic preconditioning absent, et exercise test the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients (20 39 s versus 17 36 s, respectively, p = 0.60). The improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip (50% versus 63% of patients demonstrated improvement in arrhythmias, respectively; p = 0.41). The improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients (38.5% versus 48.3%, respectively, p = 0.41). When the total group of patients (n = 174) was stratified according to the demonstration of ip, the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups, as shown in table 6.table 6frequency of diabetes mellitus, and levels of fasting glycemia and a1c in the study population according to the expression of ipip + (n = 122)ip - (n = 52) p valuedm n (%) 62 (50.8)24 (46.1)0.57fasting glycemia121.3 42.6118.9 34.10.72a1c6.63 1.66.32 1.20.25data are expressed as means standard deviation and as absolute and relative frequencies dm stands for diabetes mellitus, a1c glycosylated hemoglobin, ip + ischemic preconditioning present, ip - ischemic preconditioning absent frequency of diabetes mellitus, and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means standard deviation and as absolute and relative frequencies dm stands for diabetes mellitus, a1c glycosylated hemoglobin, ip + ischemic preconditioning present, ip - ischemic preconditioning absent in addition, when we stratified patients by quartiles of glycemia and glycosylated hemoglobin, there was no statistical difference in terms of ip demonstration among the different quartiles (fig 3graphs showing the percentage of patients who demonstrated ischemic preconditioning (ip+ in blue) and who did not demonstrate ischemic preconditioning (ip - in red) stratified into quartiles of a1c (graph a) and fasting glycemia (graph b). Ip = ischemic preconditioning x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning (ip+ in blue) and who did not demonstrate ischemic preconditioning (ip - in red) stratified into quartiles of a1c (graph a) and fasting glycemia (graph b). Ip = ischemic preconditioning; q = quartile (s). X axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia considering that diabetes is an independent risk factor for the occurrence of major cardiovascular events and mortality [5, 6], it is reasonable to consider that diabetes might damage the protective mechanism of ip in cad patients, leading to worse outcomes . However, in this study, the presence of type 2 dm did not have any deleterious effects on the myocardial protective mechanism termed ischemic preconditioning . In this context, our study showed that patients with type 2 dm demonstrated ip in similar frequency and intensity compared with nondiabetic patients and, interestingly, our data indicated an improvement in ischemic parameters associated with diabetes . The analyses of the data showed a better ischemic response evaluated by rpp in diabetic patients . Thus, this study adds clinical information on some questions that have emerged from contradictory experimental studies . The improvement in myocardial oxygen consumption, observed by the analysis of rpp, was more pronounced in patients with compared to those without diabetes, indicating better adaptation of the myocardium of diabetic patients after an ischemic insult . Moreover, diabetic patients had an improvement in t-1.0 mm greater than that in nondiabetic patients, although it did not reach statistical significance . Analyzes of the total exercise time also confirmed the main results of the study, as times were similar among patients with and without dm . Similarly, the st - segment deviation morphology did not differ among diabetic and nondiabetic patients . Because myocardial ischemia is a frequent cause of arrhythmias during treadmill stress tests, we also assessed the occurrence and complexity of arrhythmias and their improvement during set . Thus, the improvement in the occurrence of arrhythmias also confirmed the main findings of the study, as the occurrence did not differ among diabetic and nondiabetic patients . Interestingly, analyzes of the percentage of patients who demonstrated ip among different quartiles of fasting glycemia and glycosylated hemoglobin showed similar rates of ip demonstration . This analyzes infer that the differences in the intensity of glycemic control in our population did not prevent the demonstration of this cardioprotective phenomenon . However, despite our consistent findings, the information from this study is contrary to lee s et al . And ishihara s et al ., who have also studied ip in humans . Studied diabetic and nondiabetic cad patients during percutaneous coronary interventions, and evaluated the action of hypoglycemic agents on ip . Patients underwent consecutive balloon coronary inflations . During the second sequential balloon inflation, patients had less thoracic discomfort, less myocardial lactate production, and lower st - segment deviation . Moreover, the authors observed that diabetic patients treated with glimepiride had higher lactate production compared to nondiabetic patients treated with glimepiride . Despite this finding, an important limitation of this study is that there was not a direct comparison of diabetic and nondiabetic patients who had no drug interference in ip evaluation . Studied patients hospitalized due to an acute myocardial infarction and evaluated the effects of preinfarct angina, on the release of cardiac markers of necrosis, ventricular function, and in - hospital death and compared the results among diabetic and nondiabetic patients . The authors found that in the nondiabetic population, patients with preinfarct angina had a lower release of cardiac markers of necrosis, better recovery of ventricular function, and lower in - hospital mortality, compared to patients with no preinfarct angina . On the other hand, when they analyzed diabetic patients, these variables were not different among patients with and without preinfarct angina . Thus, the authors inferred that diabetes prevented the appearance of ip . Despite their findings, first, this was a retrospective study, which included a small number of diabetic (n = 121) compared to nondiabetic patients (n = 490). Many studies have shown that some of these drugs may block ip, and it has been speculated that this interference with ip mechanisms may partially explain the worse prognosis of diabetic patients hospitalized due to an acute myocardial infarction . On the other hand, other experimental studies that evaluated in vitro human myocardial tissue [41, 42] showed results that match those of the present study . They evaluated the release of cardiac biomarkers of necrosis and the percentage of tissue viability . Among other findings, the authors found a similar intensity in myocardial protection among diabetic and nondiabetic patients . Additionally, cleveland et al . Evaluated the contractile function of isolated right atrial trabeculae from cad patients, resected during coronary artery bypass graft surgery . They showed that the diabetic group that underwent the preconditioning stimuli had similar improvement in contractile function compared to the nondiabetic group . Moreover, a small study conducted by bilinska and colleagues with diabetic patients treated with glibenclamide, gliclazide, and diet compared the demonstration of ip in these groups with that in nondiabetic patients . Besides the main findings of the study, which were related to the effects of sulfonylureas in the warm - up phenomenon, they showed that the group of diabetic patients on diet (n = 15) had similar improvement in ischemic parameters compared to nondiabetic patients (n = 17). Of note, this was a secondary result, and the small sample size did not permit to make a definitive conclusion . This prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism . The group of patients with diabetes was under strict control of hyperglycemia, and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . Assuming that hyperglycemia may interfere negatively with ip, it is possible that the result of this study would be different in a population under less strict hyperglycemic control ., the response of the myocardium to an ischemic insult may probably play an important role in prognosis . One mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon, termed ischemic preconditioning . Because diabetes is an independent risk factor for the occurrence of major cardiovascular events, it is reasonable that it may compromise this cardioprotective mechanism, leading to worse outcomes . Contrary to the initial expectation, the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients . This prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism . The group of patients with diabetes was under strict control of hyperglycemia, and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . Assuming that hyperglycemia may interfere negatively with ip, it is possible that the result of this study would be different in a population under less strict hyperglycemic control ., the response of the myocardium to an ischemic insult may probably play an important role in prognosis . One mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon, termed ischemic preconditioning . Because diabetes is an independent risk factor for the occurrence of major cardiovascular events, it is reasonable that it may compromise this cardioprotective mechanism, leading to worse outcomes . Contrary to the initial expectation, the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients . In this study, diabetes mellitus did not substantially affect myocardial ip in symptomatic cad patients.
Arrhythmias can develop from intraatrial invasion, which can also lead to massive pericardial effusion and tamponade . If the tumor mass is located in the left atrium or ventricle, embolic infarction might occur . A 55-year - old man presented signs of progressive cardiac decompensation during the last 4 months due to tumorous obstruction of the mitral valve . In 2014 the patient underwent resection of a suspected myxoma (824124 mm) in the left atrium . However, in the histological workup, the tumor mass, resected from the posterior mitral annulus, was diagnosed as chondrosarcoma . In 2014, our colleagues in the referring center noticed nodal calcification in the myocardium and the mitral valve . The tumor board in 2014 decided on palliative care with poor sensitivity to chemotherapy and radiation and high risk for organ toxicity . Ct scan and magnetic resonance imaging (mri) showed no signs of malignancy outside the cardiac tissue, and the chondrosarcoma found was the primary tumor . During the course of 12 months, the patient was in a clinically stable state with combined mild mitral valve regurgitation and stenosis and good left ventricular pump function, with ejection fraction (ef) 63% . In january 2016, the patient first presented with recurring tumor mass on the posterior mitral leaflet (pml) max . 23 mm, with moderately decreasing pump function (ef 58%). In april 2016, the patient showed increasing peripheral edema and orthopnea, with no signs of angina pectoris, and nyha iii - iv . The patient presented absolute arrhythmia with atrial fibrillation, pulse 109/min, and blood pressure 82/52 mmhg . Echocardiography showed pleural effusions on both sides and distended vena cava inferior and hepatic veins . Upon admission to our center, the patient presented a 403532 mm mass, originating from the pml (figure 1). A cardio mri could not be performed, as the patient was unable to lie down flat due to major orthopnea . Instead, we decided to perform a cardio ct, which showed atrial roof and interatrial septum invasion . It was not perfectly clear on the ct scan whether the tumor was infiltrating the ascending aorta . A cerebral scan showed no signs of cerebral embolisms or metastasis . Due to clinical instability serum creatinine levels at that point were 1.56 mg / dl and bilirubin 1.10 mg / dl . After careful review of the scan images, we decided on the implantation of a total artificial heart, as the sarcoma had already invaded the whole atrium and the complete mitral valve annulus . A mitral valve replacement was not feasible because there would be no myocardium or endocardium to fixate the stitches in . Implantation of a self - expanding stent valve would also require an intact mitral valve annulus . Furthermore, autotransplantation was rejected for the same reasons . The severe mitral valve stenosis precluded use of a left ventricular assist device (lvad) or biventricular assist device (bivad). The new approach introduced by bruckner et al . Has never been performed in our center and needs experience in the surgical technique as the construction of the new atria presents major challenges because they easily collapse when not prepared properly . We decided on the syncardia total artificial heart (tah) (syncardia, tucson, az). As the ct scan showed no metastases and the sarcoma presented as a recurrence in the left atrium, we hoped that the patient could be listed for htx following recovery after tah implantation . In a similar setting, reich et al . Successfully transplanted a patient after tah implantation, with an occult intracardial malignancy, after a cancer - free period of 14 months . Before the operation, the patient and next of kin were informed that it would be an ultimo ratio intervention because we could not 100% foresee the invasion of the myocardium and the intraoperative circumstances . Zhang et al . Demonstrated a 50% survival rate after initial surgical treatment of primary cardiac sarcomas, depending on histological grade of the tumor . Surgical treatment is the most common approach to improve survival . Even as a palliative measure, reducing the tumor mass through an operation relieves orthopnea and ameliorates signs of decompensation if the patient stabilizes after the intervention . After re - sternotomy following aortic cross - clamping, the apex, ascending aorta, and pulmonary trunk were distally resected, preserving both the aortic and pulmonary valve . The left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve (figure 2). Due to extensive left atrial resection, a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture . Functional valve movements were tested and tah chambers were put in the av - annuli, followed by de - aeration of both tah chambers . To allow postoperative tissue edema, we decided to leave the thorax open . After sufficient volume substitution, the patient was transferred to the icu under moderate catecholamine support and no ventilation . Five days after the initial operation, we planned a secondary thorax closure . During the intervention cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn, with ongoing hemorrhagic transformation . Neurological examination showed the patient was comatose, with slight head movement to the left side . A right - sided facial paralysis was diagnosed . Under continuous anticoagulation for tah, there was a major risk for secondary bleeding due to the size of the media infarction . . The patient showed signs of prolonged low - output syndrome with beginning multiorgan failure . Neurological status was deemed unfavorable and after consultation with the family, the patient died on postoperative day 16 after withdrawal of support . Histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components, with dystrophic calcifications . Chromogenic in situ hybridization could not confirm an amplification of mdm2-gene, and translocation of syt - gene locus was ruled out . Hence, the classification was undifferentiated high - grade spindle cell sarcoma, grade 2 using the french fdration nationale des centres de lutte contre le cancer (fnclcc) grading . Cardiopulmonary bypass was established via the ascending aorta and venae cavae cannulation . Following aortic cross - clamping, the apex, ascending aorta, and pulmonary trunk the left atrium showed infiltration of the intraatrial septum by the sarcoma after resection of the mitral valve (figure 2). The atrial cuffs were prepared and broad felt strips for suture support were placed . Due to extensive left atrial resection, a bovine pericardial patch was implanted to expand the pulmonary vein orifice with a 4 - 0 prolene suture . Functional valve movements were tested and tah chambers were put in the av - annuli, followed by de - aeration of both tah chambers . To allow postoperative tissue edema, we decided to leave the thorax open . After sufficient volume substitution, the patient was transferred to the icu under moderate catecholamine support and no ventilation . Five days after the initial operation, we planned a secondary thorax closure . During the intervention cerebral ct showed vast media infarction with hyper - dense blood appositions and perifocal edema with elapsed grey / white matter differentiation and early compression of the left posterior horn, with ongoing hemorrhagic transformation . Neurological examination showed the patient was comatose, with slight head movement to the left side . Under continuous anticoagulation for tah, there was a major risk for secondary bleeding due to the size of the media infarction . Neurological status was deemed unfavorable and after consultation with the family, the patient died on postoperative day 16 after withdrawal of support . Histological workup showed a malignant mesenchymal neoplasm with spindle cell formations and heterologous chondroid and osseous components, with dystrophic calcifications . Chromogenic in situ hybridization could not confirm an amplification of mdm2-gene, and translocation of syt - gene locus was ruled out . Hence, the classification was undifferentiated high - grade spindle cell sarcoma, grade 2 using the french fdration nationale des centres de lutte contre le cancer (fnclcc) grading . Chondrosarcomas are cartilaginous tissue sarcomas . Our patient was first diagnosed with primary cardiac chondrosarcoma . After initial resection, relapse of the cardiac mass in the left atrium was diagnosed as early as 14 months later . After excision of the heart for tah implantation, our pathologists found an undifferentiated high - grade spindle cell sarcoma, fnclcc g2 . With histological grading through the fnclcc classification, survival, by major correlation cell differentiation, mitotic rate, and percentage of necrosis are evaluated in a systematic score . Since the 4 edition of the who classification of tumors of soft tissue and bone, there is a new category of tumors that cannot be classified in earlier - defined categories . There were no signs of assist malfunction during icu stay . With atrial infiltration and major excision of healthy tissue, implantation of tah traditional bivad implantation as bridge - to - transplant as a short - term solution was not an option due to severe mitral valve stenosis from the atrial mass and mitral valve infiltration . The left atrial tumor was successfully removed . Like bruckner et al ., we acknowledge that tah implantation as bridge - to - transplantation is surgically possible even with atrial wall involvement . Surgical treatment is still the criterion standard for localized cardiac sarcomas, with various perioperative survival rates, ranging from 6 to 17 months . With complete resection of the tumor mass there is no valid data on the benefits of adjuvant chemotherapy or radiation therapy on malignant cardiac soft - tissue sarcomas . Limitations of radiotherapy are cardiac sensitivity to radiation injury and adverse effects on global cardiac functions . Our patient showed good recovery after initial gross resection in 2014 for 14 months without any adjuvant therapy . Bleeding complications due to continuous anticoagulation for device functioning remains a major risk factor in circulatory assist device programs . In a study in 13 patients with tah, ramirez et al . Found that bleeding complications occur generally between postoperative days 1 and 3 . Patients with mechanical assist devices are prone to suffer from thrombotic events [1316], device malfunctioning, wound infections, and bleeding complications . This intervention was the second attempt to help the patient with a long - term solution after first recurrence of an atrial tumor . The surgical options and possibilities were discussed at length with the patient and his family before the tah implantation . A multidisciplinary team and truthfully informed next of kin are hugely important when taking decisions in such a high - risk case.
Cannabis is a prominent political, health, and law enforcement issue in north america that is currently receiving a great deal of attention . As we watch the effects of the legalization of cannabis in colorado, washington, alaska, oregon, and the district of columbia, other jurisdictions are debating the future of this illicit drug . How can we prevent commercialization promoting increased rates of use within a legalization model? Where does marijuana for medical purposes fit into the picture, if at all? A critical piece of information often missing from these debates or not addressed adequately is the scientific evidence about the effects of cannabis use on a person's overall health . This concern is even more pressing when it comes to the cognitive, physical, and mental health harms to adolescents who use cannabis . It is critical we ask these questions because adolescence is a time of rapid development that helps lay the foundation for success later in life . Conversely, it can also set the stage for experiencing challenges in adulthood, as youth are disproportionately more likely than adults to experience greater harms from drug use . Their brains are undergoing rapid and extensive development that can be negatively affected by cannabis use . There is certainly cause for concern about the amount and frequency of cannabis use among youth . According to the 2014 monitoring the future (mtf) survey in the us, 35.1% of high school seniors reported pastyear use of cannabis, with 5.8% reporting daily or near daily use . Comparable prevalence rates are reported in canada, with 22% of 1519yearolds and 26% of 2024yearolds reporting pastyear use of cannabis in 2013 according to the canadian tobacco, alcohol and drugs survey . Moreover, a 2013 unicef report noted that canada's youth are the highest users of cannabis when compared to students in other developed countries . The mtf survey also indicates a growing perception among young people that cannabis is a relatively harmless drug, and there is strong evidence that perception of harm is inversely related to rates of use . Knowing that youth are disproportionately heavy users of cannabis, what specific concerns should this raise about short and longterm consequences and how do we incorporate these concerns in the debate? Recent evidence shows that early and frequent use of cannabis has been linked with deficits in shortterm cognitive functioning, reduced iq, impaired school performance, and increased risk of leaving school early all of which can have significant consequences on a young person's life trajectory.1 of note, the impact of cannabis use on iq has been the topic of recent debate among experts in the field and further research aimed at understanding this relationship will provide much needed clarity . Heavy cannabis use in adolescence is also a risk factor for psychosis, a risk that is heightened if users have a preexisting vulnerability to that condition.1 cannabis use can also lead to dependence, with evidence from longitudinal studies in the us estimating the risk of developing cannabis dependence to be one in six among those users who initiated in adolescence,2 a rate higher than that reported among adults . Recent data from the 2012 canadian community health survey reveal that more than one in 20 young canadians aged 1524 met the criteria for cannabis abuse or dependence during the past year . There is also growing evidence from the human literature that early and frequent use of cannabis can alter structural aspects of the developing brain, including those that are responsible for memory, decisionmaking, and executive functioning.1 however, we do not yet know the full extent of the impact of early cannabis use on changes in brain function and behavior of young people, which underscores the need for further investment in research in this area . Beyond longerterm consequences, cannabis can also acutely impair cognitive and motor functions like perception, coordination, and balance for hours after use . This impairment presents a safety hazard for drivers getting behind the wheel, as well as for other motorists and pedestrians . In fact, the risk of car crashes doubles for drivers who get behind the wheel after using cannabis and this risk is further elevated if cannabis use is combined with other substances, most notably alcohol . Recent data from the national fatality database indicate that cannabis was the most common illicit drug present among fatally injured drivers aged 1524 in canada between 2000 and 2010 . The drug abuse warning network, which monitors the health impact of drugs in the us, estimated that in 2011, there were nearly 456,000 drugrelated emergency department visits in which cannabis use was mentioned in the medical record . Data from the canadian institute for health information reveal that from 2006 to 2011, youth aged 1524 spent the largest number of days in a hospital for a primary diagnosis of mental and behavioral disorders due to the use of cannabinoids . Furthermore, the number of days spent in a hospital increased by almost 40% during the same time period.3 these findings are particularly troubling because recent research from canada has revealed that young people are confused about cannabis and do not have the knowledge they need about the risks associated with this drug to make more informed decisions.4 some have expressed questionable beliefs about the effects of cannabis, indicating that it helps to improve their focus at school and can prevent or even cure cancer . Youth also expressed mixed beliefs as to whether cannabis improves or impairs driving performance, and felt that cannabis and driving was not as dangerous as drunk driving . Youth talked about how cannabis is natural and so they did not really think of it as a drug . The findings from this reviewed research highlight the complexity of the issues surrounding youth consumption of cannabis . Youth are confused with the mixed messages they are receiving from discussions ranging from legalization to the use of cannabis for medical purposes, which points to the need for a coordinated, comprehensive, factual, and consistent approach to increasing awareness of this drug and its impact . As primary care is an important intervention point for youth experiencing harms related to cannabis, there is an urgent need to develop practical, evidenceinformed tools to assist healthcare providers in the early identification of cannabis use and cannabis dependence, as well as tools to deliver interventions to treat potential problems . Examples of such tools may include screening instruments to screen for cannabis use that warrants clinical intervention, as well as brief intervention strategies aimed at addressing the problematic substance use revealed during the screening process . There is also an urgent need for further research aimed at improving the understanding of differences between the short and longterm effects of cannabis use, particularly on the developing adolescent brain . Such research should include mechanistic studies regarding alteration of cellular structure and function related to cannabis to determine the impact on adolescent brain development . Of particular concern are the impacts of the dramatic increase in concentrations of tetrahydrocannabinol (thc), the main psychoactive ingredient of the cannabis plant, over the past 25 years . The average potency of federally seized cannabis in the us has steadily increased from 3.5% in 1985 to 13.2% in 2012.5 in light of such an increase in potency, some historical findings about the health and developmental effects of cannabis use might not be relevant when trying to predict effects on contemporary users . More research is also needed to shed light on the influence of cannabis policy on public health, public safety, and other outcomes . The current understanding of the impact of cannabis policy on market forces, such as healthcare costs, lost productivity costs, and tax revenues, for example, is very limited, as is overall understanding of how perception, use, and outcomes interrelate around this drug . In an effort to help clear the smoke, the canadian centre on substance abuse is currently undertaking a comprehensive review and synthesis of the literature on the effects of cannabis use during adolescence that will highlight key areas for action in policy, practice, and research . This muchneeded work will provide clarity to misperceptions about cannabis, and make sense of some conflicting evidence about the effects of cannabis use and the impact of early or regular use during a time when the brain is still developing . By integrating neuroscience with the behavioral and social context of cannabis use by youth, this report will provide a muchneeded resource for those working with youth and those involved in making decisions about policies, programs, or practices related to cannabis . Aj porathwaller has provided expert testimony at the standing committee on health's (hesa) recent report on marijuana's health risks and harms, october 2014.
Hypertension (htn) is the most common comorbidity in the world with significant public health implications [1, 2]. The overall u.s . Prevalence of hypertension among adults ages 18 years in 20052008 was 30.9% and was highest among persons ages 65 years (69.7%) and non - hispanic blacks (44%). The american heart association estimates that the incurred costs of hypertension are more than $93.5 billion per year, and that cardiovascular disease and stroke for which htn is the predominant risk factor, account for 17% of the total annual health expenditures in the united states . Hypertension and its sequelae, heart failure (hf), are a progressive disease . Evidence shows that less than half of patients with heart failure survive five years (after diagnosis), and less than a quarter of them live ten years after their initial diagnosis . Framingham heart study showed that hypertensive patients were more likely to develop heart failure (142 cases of hf detected during the first 16 years of followup) than those who were normotensive . The lifetime risk for development of hf among people with blood pressure (bp)> 160/90 mm hg is double that of those with bp <140/90 mm hg . Heart failure in comparison to the most prevalent gender malignancies (bowel cancer in men and breast cancer in women), was associated with worse long - term survival . Over a million hospitalizations, contributing to more than 250,000 deaths, and escalating billions of dollars in health care expenditure is the norm . The disastrous duo, htn and hf, should be recognized, and a tailored focus on management in special populations, high - risk ethnic minority groups especially blacks who are disproportionately burdened should be developed . Among racial groups, african - american adults have the highest rates (44%) of hypertension in the world and are more resistant to treatment . In particular, black women have the highest prevalence and the lowest blood pressure control . The relative incidence of hf is 50% higher in african americans, 3% of whom have hf, compared with 2% of the general population [13, 14]. The disease occurs at an earlier age, and usually at a more advanced stage along with increased hospitalization and mortality . Other causes of hf include coronary artery disease, valvular heart disease, diabetes, left ventricular hypertrophy and cardiomyopathies . Overt clinical hf resulting from diastolic left ventricular dysfunction may be clinically indistinguishable from that resulting from systolic dysfunction . Hf with preserved left ventricular function is observed in 30% to 50% of adult cases of hf . Coronary artery disease and myocardial infarction is a principal cause of systolic left ventricular dysfunction followed by hypertension . A variety of neurohormonal systems, especially the renin - angiotensin aldosterone and sympathetic nervous systems are activated in response to the left ventricular dysfunction and such activation leads to abnormal ventricular remodeling . The inexorable progression to more severe stages of left ventricular dysfunction can be significantly reduced by effective therapy with neurohormonal blockade including angiotensin converting enzyme inhibitors (aceis), beta blockers (bbs), and aldosterone antagonists . Current understanding of the pathophysiology of hf has revolved around neurohormonal activation [19, 20]. This endothelial dysfunction may result from insufficient nitric oxide (no) secondary to either reduced endothelial production of no or to increase no inactivation [17, 19, 21]. In hf, a pronounced increase in angiotensin ii, with modestly reduced no is seen among whites . However, african americans with hf may exhibit greater no reduction; conversely, angiotensin ii might be less elevated among whites . Important studies are investigating gene variation among the races that may influence the risk of developing hypertension and its complications, such as single nucleotide polymorphisms in the genes encoding for 1-adrenergic receptors and a2c - adrenergic receptors . African americans who are homozygous for polymorphisms in both the 1-adrenergic and a2c - adrenergic receptor genes are at a 10-fold greater risk for heart failure, which is overexpressed in african - americans . Groups with overexpression of transforming growth factor -1 (tgf-1), a cytokine involved in cardiac fibrosis and remodeling, vascular and renal disease, production of the vasoconstrictor, endothelin-1, and stimulation of renin release, have a worse prognosis . Other genetic polymorphisms in african americans that may influence heart failure risk include elevated production of endothelial nitric oxide synthase, aldosterone synthase, and the g protein 825-t allele . There are two major approaches to classifying the patient's hf stage or clinical severity . The most common is the new york heart association (nyha) classification, mainly based on functional capacity and subjective level of symptoms severity . This scheme is fluid and its class fluctuates in the same patient depending on their subjective assessment and clinical status . The other approach, adopted by the american college of cardiology / american heart association (acc / aha) guidelines, emphasizes disease progression . Classified as stages a, b, c, and d, each of these stages has definable characteristics and recommended interventions . To emphasize the progressive nature of hf and the need to prevent its development or progression to end - stage disease (stage d) [18, 23], current hf guidelines define patients in stage a as those at increased risk for hf without evidence of structural heart disease or symptoms of hf . Stage a represents a critically important group because many of the risk factors including hypertension can be effectively controlled and by doing so may prevent development of cardiovascular diseases including hf . Patients are in stage b when they present with evidence of structural heart disease but without current or prior symptoms of hf . Stage c is recognized by the presence of structural heart disease and current or prior symptoms of hf . Stage d patients are those who have refractory heart failure requiring frequent hospitalization and specialized interventions . Several studies have shown the efficacy of treatment of hypertension in preventing hf [2427]. Treatment of hypertension reduces the incidence of hf by about 50% . In recent years, significant advances have been made in increasing awareness, treatment, and control of blood pressure . Several large, randomized clinical trials have shown that specific classes of medication have mortality benefit in patients with heart failure [2834]. In the most recent guidelines for the diagnosis and management of hf in adults, the american college of cardiology foundation (accf)/american heart association (aha) recognized the need for specific recommendations for special population . Lifestyle modifications have been shown to reduce blood pressure, increase drug efficacy, and decrease cardiovascular risk . Long - term primary prevention of hypertension includes weight reduction in overweight / obese individuals . Weight loss intervention was associated with a 21% reduction in the incidence of hypertension over a 36-month period . Dietary practices, reducing intake of dietary sodium to <1.5 g nacl [3, 36], low potassium, magnesium, and saturated fat intake, increased calcium intake, decreased consumption of alcohol, have demonstrated greater bp reduction in blacks . These practices are most successful when reinforced with comprehensive education and counseling [39, 40]. Exercise training, regular aerobic physical activity for at least 30 minutes per day most days, has been shown to reduce recurrent cardiac events in patients with lv dysfunction from ischemic heart disease and is recommended under close medical supervision . Culturally cognizant and sensitive modalities should be implemented to help guide improved compliance of therapeutic lifestyle changes (tlc) in special populations [43, 44]. Despite lifestyle modifications, most patients with hypertension need pharmacologic therapy for better bp control . In the united states, pharmacological treatment of hypertension before development of structural heart disease or symptoms of hf is mainly based on studies looking at prevention of end - organ damage . Specific classes of medication are recommended for patients in stages b - d that have shown benefit in reducing mortality and number of hospitalizations . The reader should refer to the major guidelines for full treatment recommendations of hf in the general population [18, 23, 46]. . However, ethnic minorities who are at greater risk for developing hf remain underrepresented in most clinical trials . According to a recent paper by mitchell et al . Treatment of hf in african americans a call to action, most of the available evidence is based on limited representation of african americans in these trials or posthoc analysis of limited evidence . These papers once again summarize the available evidence in hf management in african americans and call for more therapeutic trial focused in these high - risk population . The african american heart failure trial (a - heft) was the first heart failure large trial to focus specifically on the african - american population and evidence represented approximately 50% of all african americans ever studied in heart failure trials . This is randomized placebo - controlled double blind study of fixed dose isosorbide dinitrate / hydralazine (isbd / hyd) (figure 2). In an effort to summarize efficacy of available treatments, psaty et al the database included 192,478 patients from 42 trials randomized to 7 treatment strategies including placebo . Low - dose diuretics proved the most effective first - line treatment for preventing cardiovascular disease and mortality including hf . The allhat trial showed that diuretics are as effective as calcium channel blockers or acei in preventing cardiovascular disease outcomes including hf . The allhat study also has suggested that thiazide - diuretic therapy is useful in preventing disease progression . Although, allhat trials and other trials showed diuretics is the first - line of antihypertensive therapy and is still the most commonly prescribed antihypertensive medications, this is challenged by recent studies as published in february jacc and presented in the european society of htn meetings . However, the diuretics used in the allhat trials was chlorthalidone a long acting thiazide (shown to effectively lower bp and improve survival), not really the same as hydrochlorothiazide (hctz) which is the most commonly prescribed antihypertensive thiazide at a dose of 12.525 mg . (st luke roosevelt hospital, new york, ny), published the analysis of 18 trials on hctz where the antihypertensive effect of hctz at the dose that is most often used12.5 to 25 mg a day is consistently inferior to that of most other antihypertensive drug . The other limitation of diuretics as first line therapy in african americans is not only not adequate but also, has multiple side effects like dm, electrolyte abnormalities and urecemia, to mention a few . However, there is still a role of diuretics in combination with neurohormonal (renin angiotensin aldosterone axis) blockade and when there is compelling indication . According to the recent consensus statement of the international society on hypertension in blacks (ishb), chlorthalidone furthermore, the ishb consensus statement reinforces, patient above goal bp (sbp / dbp15/10) with (goal bp <130/80) end organ damage (hf, lvh, kidney disease) or without end organ damage (goal bp <135/85) should be on at least on combination pharmacotherapy . Use of beta blockers (bb) as treatment for hypertension has come under scrutiny . Conducted a meta - analysis of 13 trials, which included 105,951 patients showing that atenolol did not improve outcomes in terms of stroke and cardiovascular events . In stage b chf (nyha class i), defined by the presence of reduced left ventricular ejection fraction (lvef) <40 percent in otherwise asymptomatic individuals, aceis and bbs are recommended . Iii) manifest left ventricular dysfunction and overt symptoms; in these individuals, aceis and bbs are also indicated . Bbs are also recommended in hf because of clinical studies demonstrating decreased morbidity and mortality, and improvement in hf symptoms . Aldosterone antagonists may provide additional benefit in patients with severe left ventricular dysfunction, usually late stage c (nyha class iii low - dose spironolactone (12.525 mg daily), when added to standard therapy, decreased mortality by 34 percent . In the eplerenone postacute myocardial infarction heart failure efficacy and survival study (ephesus), eplerenone reduced mortality by 15% in patients following a recent mi with lvef <40%, 90% of whom had hf symptoms . Patients were excluded who had a serum creatinine of <2.5 mg / dl and hyperkalemia . The v - heft i and ii are a large - scale trial that compared different vasodilators with placebo and among themselves . V - heft i showed, the mortality rate was significantly lower for the total cohort (african americans and whites) in isdn / hyd arm compared to patients treated with prazosin or placebo added to digitalis and diuretics (figure 1). In v - heft ii, though isbd / hyd was statistically inferior to enalapril in effect on mortality, patients taking isbd / hyd experienced similar mortality rate in patients taking isbd / hyd in v - heft - ii as those in v - heft - i taking isbd / hyd . Posthoc analysis of v - heft i showed isbd / hyd significantly reduced mortality in african american patients but not in white patients . The reanalysis of v - heft ii, demonstrated the annual mortality rate african american (12.9) patients receiving isbd / hyd was 12.9% compared with 12.8% for african americans receiving enalapril . Due to difference in outcomes in african americans in v - heft, the african american heart failure trial (a - heft) definitive study was designed . The a - heft was terminated earlier than anticipated completion date due to a significant (43%) reduction in mortality in patients receiving combination (isdn / hyd) compared to the placebo arm . Adding the combination of a fixed - dose of isdn / hyd (20 mg/37.5 mg) to a standard medical regimen for hf, including acei and bbs, is recommended in order to improve outcomes for patients self - described as african americans, with nyha functional class iii or iv chf . Furthermore, genomic evidence from the grahf (genetic risk assessment of heart failure in african americans) arm of the african american heart failure trial (a - heft) showed that blacks with the more common tt phenotype (61%) of the aldosterone synthase gene (cyp11b2) showed greater responsiveness to the combination of isosorbide dinitrate and hydralazine . The vast majority of blacks will have no identified cause for their htn . Past studies elucidating the pathophysiological mechanisms have shown conflicting information and further investigations exploring the complex relationships and interactions are warranted . There are, however, several secondary forms of htn in blacks that are prominent and preventable . We will focus on the most common, preventable and treatable secondary form of hypertension, obstructive sleep apnea (osa). The prevalence of osa in hypertensive populations is estimated to range between (3040%) and in heart failure at 53%; apnea - hypopnea index (ahi) 10/h). It is an independent risk factor for htn, nondipping of blood pressure and increased bp variability and follows a dose - dependent relationship . A prospective study showed that the odds of developing hypertension over 48 years was 2.89 times more likely for participants who had ahi 15/h compared to ahi 0, independent of known confounders . Cross - sectional data has shown 2.38-times increased likelihood of having heart failure in association with osa, independent of confounders (n = 6424) [67, 68]. Osa has been shown to act through various mechanisms, independent of blood pressure, in promoting left - ventricular (lv) hypertrophy, diastolic, and systolic dysfunction, and overt heart failure . These mechanisms include the hemodynamic consequences of repetitive increases in left - ventricular transmutable wall pressure and over time impaired vigil heart rate modulation leading to lv hypertrophy and ventricular remodeling . Additionally, osa patients without overt heart failure have more impaired diastolic relaxation than those without osa . Emerging evidence illustrates patients with osa have more active atherosclerotic disease with a greater degree of vessel involvement and more vulnerable plaques than do patients without the disease, resulting in greater cardiovascular burden . The definite treatment for osa, long - term adherence to continuous positive airway pressure (cpap) therapy, has shown to suppress sympathetic activity, lower blood pressure, reduce right ventricular volume, and improve systolic function in patients with hf . Systematic reviews, meta - analyses, and randomized controlled trials have shown statistically significant net reduction in blood pressure with cpap, especially in patients with increased ahi events [7883]. A recent 3-year study (n = 340) by durn - cantolla et al . Reported that participants assigned to cpap therapy for 3 months had mean 24-hour ambulatory blood pressure decreased by 2.1 mmhg (0.4 to 3.7) mmhg (p = .01) for systolic and 1.3 (0.2 to 2.3) mm hg (p = .02) for diastolic blood pressures . Similar evidence from randomized trials showed up to 9% increase in lvef and increased quality of life (qol) and functional capacity among patients treated with 13 months of cpap therapy . Furthermore, among patients with no overt failure, cpap therapy has been shown to reverse impaired diastolic relaxation . There is a need for further randomized clinical trials with cpap therapy among high - risk ethnic minorities especially blacks to assess whether treatment of osa has similar long - term effects and to evaluate the effect on htn and hf morbidity and mortality . Taking into consideration, lifestyle and pharmaceutical interventions, our changing health care community needs to emphasize the essential nature of the patient - provider relationship and establish trust and collaboration . In the past, this has been accomplished through physician, patients, family member, and other caretaker awareness, increased education of both provider and patients, and establishment of community outreach programs . These measures have contributed to an increase in compliance and a reduction in the risk of hospitalizations [23, 87]. We recommend an additional emphasis on the implementation of specialty care in patients with heart failure in collaboration with the primary care provider . The dramatic improvement in the management of hf and hypertension over the last 50 years has allowed us to start to target specific populations and provide more evidence - based treatment that will lead to an improvement in mortality . There are disparities in the prevalence, treatment, and control of hypertension and the incidence and morbidity and mortality of heart failure between blacks, a diverse and heterogeneous population, and whites . The management team needs to take into consideration new evidence and develop tailored strategies for effective treatment, especially for hypertension, one of the main causes of hf and many other cardiovascular complications in african africans.
Radiological investigations and therapeutics have become an integral part of the management of critically ill patients in the intensive care unit (icu). Patients admitted to the icu routinely undergo bedside imaging procedures such as chest radiographs for diagnosing heart, lung and other pathology, and for confirmation of the position of devices like endotracheal tubes, nasogastric tubes, central venous catheters and intercostal drains . A review article has suggested that among icu patients, up to 65% of daily chest radiographs and 70 - 75% of chest computerized tomography (ct) scans reveal significant or unsuspected abnormalities that may lead to a change in the patient's management . Further, critically ill patients are frequently transported to the ct scan as well as digital subtraction angiography suites for diagnostic and therapeutic procedures such as angiography, embolization and stenting . In most situations, the icu team is responsible for the transport and management of the patient in the radiology department for these procedures . There is, therefore, potential radiation exposure to healthcare workers, particularly for those working for long periods of time in the icu . Epidemiological data indicates that the exposure to even low - dose radiation may be a cause for concern because such exposure can result in leukemia, thyroid malignancies and other cancers . Nonneoplastic effects of radiation include genetic mutation and developmental malformation in children whose mothers were exposed to radiation during pregnancy . A number of studies have looked at radiation exposure in critically ill patients undergoing repeated radiological procedures . One study has specifically focused on the pediatric age group probably in view of the high ratio of exposure to patient size and the potential for long - term sequelae as the radiation effects have a longer period to become manifest . However, the available literature on the extent of radiation exposure to icu personnel is scarce and relates mainly to the level of scattered radiation within the icu . The conclusion of these studies is that the level of radiation exposure is extremely low and does not pose a hazard to icu personnel . None of these studies have considered the additional radiation exposure to icu personnel involved in the management of critically ill patients in the radiology department . In our icu in a tertiary care cancer referral center, resident doctors working on 12 h shifts in the icu are also responsible for the transport and management of icu patients when they undergo diagnostic and therapeutic procedures in the radiology department . We, therefore, carried out this study to determine the total radiation exposure to icu resident doctors involved in the course of their duties . We conducted a prospective, observational study in the icu and postanesthesia care unit (pacu) of a 500-bedded tertiary care cancer referral center in mumbai, india from september 2012 to february 2013 . All resident doctors who gave voluntary written consent to participate in the study were included . Since the study did not involve patient contact, the requirement for obtaining consent from patients was waived by the ethics committee . The study was carried out in accordance with the principles of good clinical research practice . The resident doctors provide 24 h cover, working in 12 h shifts, with four doctors in each shift . In each shift the icu has a total of 14 beds as shown in figure 1 . On another floor is the pacu with 23 beds, subdivided into 14 postoperative recovery room beds and 9 intensive care beds [figure 2]. Residents provide care for patients in the icu and pacu and also accompany patients from these locations for radiological procedures in the ct scan or interventional radiology suites . Location of thermoluminescent dosimeters are shown with filled triangles layout of postanesthesia care unit showing location of thermoluminescent dosimeter in pacu (tld surrounded by beds) and other in long lobby as control tld to detect levels of radiation, we used thermoluminescent dosimeters (tlds) which are a type of radiation detector . The tld measures cumulative dose of ionizing radiation exposure by measuring the amount of visible light emitted from a crystal in the detector when the crystal is heated . Tlds can measure a wide dosimetric range (from 10 gray to 10 grays) of radiation exposure and are routinely used as personal dosimeters because they are small in size, convenient to use and not expensive . A tld was given to each of the four resident doctors; they were handed over to the next team during shift changeover . In addition, three tlds were kept in nursing stations in icu, and one tld was placed in the pacu . One tld was kept in the doctors' duty room which was within the premises of the icu, and the last was kept as a control in the office of the department of anesthesia, critical care and pain, remote from icu and pacu, and where no radiological procedures were performed . The layout of icu and pacu is represented by figures 1 and 2 respectively and locations of tlds are marked with filled triangles . The participating resident doctors were instructed to wear the tlds at all times during their duty hours . Staff not required to be present during radiographic procedures were kept at least 3 m away from the patient (a distance at which exposure from scattered radiation is considered negligible). It was ensured that patients who were shifted from icu to the radiology suite were accompanied by one icu resident and that the resident was wearing his tld throughout . Residents, if required to be directly involved in the radiologic procedure in suites, wore lead aprons, and the tld was underneath the lead apron . Bedside chest x - rays in the icu were done by siemens multimobil 2.5 (manufactured by siemens ltd, goa, india), ct and ct guided biopsies were performed on siemens somatom emotion 16 (manufactured by siemens shanghai medical equipments ltd, shanghai china) and ge light speed 16 (manufactured by general electric co., milwaukee, usa) respectively . Interventional radiology procedures were carried out using ge innova 4100 iq (manufactured by ge medical systems, france). The study was carried out in two phases of 3 months each . At the end of the first phase, the tlds were handed over to department of nuclear medicine for analysis and another set of tlds was issued at the same time . During the period of the study, we maintained a database of procedures performed both in the icu and in the radiological suites along with the bed number and the patient's hospital registration number . The primary outcome of this study was to quantify the cumulative radiation exposure to the resident doctors working in the icu over a period of 6 months in the course of their duties . The secondary outcomes were to measure the cumulative scattered radiation exposure in various areas of the icu calculated as the average of readings of icu tld badges over 6 months and the estimated cumulative radiation exposure to the resident doctors per year . The control tld placed in the department of anesthesia served as a measure of baseline atmospheric ionizing radiation, and all other values are reported above this baseline . The results of dosimetric analysis of residents tlds during the two phases are shown in table 2 . Readings from tlds placed in the nursing station and doctors' duty room during both phases were immeasurable . The mean values in the first and last 3 months were 0.052 and 0.069 msv respectively, though the highest individual value approached 0.1 msv . Since this was recorded in a 3 month period, the projected reading for similar exposure throughout the year, even assuming a 24 h duty period, would be 0.4 msv, which is well below the safety limit of 20 msv / year . Revolutionary progress in the field of medical imaging has given a big leap to advances in medical diagnostics and therapeutics . This development has also infiltrated the field of critical care medicine, and radio - diagnosis and interventional radiological procedures now play a key role in the management of critically ill patients . While this advancement offers the advantages of rapid bedside diagnosis, and cost - effective and minimally invasive treatment options to critically ill patients, it carries the danger of exposure of patient and physician to radiation . The detrimental effects of exposure to even low - level ionizing radiation have always been known; however, there is renewed concern because of its wide - spread use in medical radio - diagnosis and therapeutics in critically ill patients . It is, therefore, natural that there may be concerns about the long - term effects of radiation exposure to doctors working for long periods of time in the icu . In addition, where icu doctors are responsible for transport of patients to the radiology suite, the extent of radiation exposure is increased . It is reassuring that the results of our study confirm that the extent of radiation exposure to critical care physician during the course of his duty is well within acceptable limits . There is considerable literature on occupational hazards of radiation exposure among radiologists; however, it is difficult to directly extrapolate the conclusions of these studies to the icu . Working conditions in the icu do not mimic those in radiology suites - working hours tend to be long, there is a need to accompany mechanically ventilated and hemodynamically unstable patients inside the radiology suite for procedures, and icu doctors may sometimes take radiation safety norms lightly . A study performed in a trauma icu (ticu) has concluded that radiation exposure is not a significant occupational hazard for the ticu personnel . Similarly, another study looked into the radiation exposure to icu nurses and found that the exposure was well below the permissible level . The strength of our study is that it was planned in a more pragmatic way - apart from bedside radiological procedures, we also took into account the radiation exposure to icu residents accompanying the patient to radiological suites for diagnostic and therapeutic procedures . Ionizing radiation from fluoroscopy in the ct scan or interventional radiology suites may be significant and were not considered in the previous studies . We also tried to measure the amount of scattered radiation within the icu and the pacu, which contributes to overall radiation exposure . Even after taking these additional sources of radiation exposure into account, we found that the cumulative radiation exposure was negligible . The number and types of bedside and out - of - icu radiological procedures can vary on a day - to - day basis according to the case - mix of the icu population, and this may affect the overall radiation exposure; however, this study was carried out over a period of 6 months, and the data obtained would have been adequately representative . The other limitation relates to the generalizability of the study; differences in the types of cases, working pattern of resident doctors, quality and maintenance of radiological equipment between hospitals may restrict the applicability of these results to other hospitals . However, given the large margin of safety that our study has shown, it is unlikely that exposure levels would be dangerously high in any other setting . Though, with advances in technology, the number and types of radiological procedures performed on patients are likely to increase . Furthermore, there is a growing trend toward using radionuclide - based positron emission tomography scans for diagnostic procedures in critically ill patients especially when they are admitted to icu during their diagnostic work - up . Some of these patients may continue to emit radiation long after their procedures are completed . Though none of these patients featured in our study, it will add to the radiation exposure to doctors . The results of this study do not in any way underrate the need to follow safety precautions, while carrying out radiological procedures in critically ill patients . The levels of exposure found in this study should be interpreted bearing in mind that standard protection norms were used by all personnel involved in the study . With these precautions in place, radiation exposure to doctors managing critically ill patients in the icu is minimal and acceptable . Literature on radiation exposure among icu doctors is scarce . In addition to bedside radiological procedures, the risk of exposure may be increased if the same doctors accompany icu patients for out - of - icu radiological investigations . However, we found that if standard safety precautions were followed, cumulative radiation exposure to icu resident doctor was well within permissible limits and was not the cause of concern and hence routine personal dosimetric monitoring is not needed for residents in icu . However, in view of changing practice, there is a need to repeat such audits periodically to monitor radiation exposure.
Despite the huge burden of hepatitis b worldwide, with an estimated 350 million people chronically infected, very few patients in low- and middle - income countries are currently receiving antiviral treatment . This is largely to blame on lack of access to viral load quantification, because this is considered a mandatory component of the diagnostic work - up in all international liver society guidelines . Without a viral load measurement, it is virtually impossible to establish whether a hepatitis b surface antigen (hbsag) positive individual has chronic hepatitis b, characterized by a level of hepatitis b virus (hbv) dna> 200020 000 iu / ml and continued necro - inflammation in the liver . These patients are at high risk of progression to cirrhosis and hepatocellular carcinoma in the absence of antiviral treatment, whereas inactive carriers, characterized by hbv dna levels <2000 iu / ml, have an excellent prognosis in the absence of treatment . The situation for hepatitis c is somewhat different because the main obstacle to treatment has been expensive and toxic treatment regimens . With the release of new direct acting antivirals for hepatitis c, opportunities are opening up in less developed countries where the disease burden is often high . An estimated 130150 million people are chronically infected with hepatitis c virus (hcv) globally, and the prevalence is higher in certain areas such as north africa and central and east asia . However, prior to starting treatment of hepatitis c, hcv rna measurements are required to establish the diagnosis of chronic hcv infection, which is a major obstacle in many places in the world . Lack of access to viral load testing and antiviral treatment of hbv and hcv in resource - limited settings is a silent epidemic with major consequences . The world health organization (who) estimates that about 1 million people die from chronic hbv and hcv infection each year, which places viral hepatitis on the top 10 leading causes of mortality globally . First, serological tests are used to screen for hepatitis markers and exclude co - infections . Thereafter, a new battery of tests is needed to distinguish past or inactive infection from chronic hepatitis . This diagnostic work - up involves molecular biology laboratories and advanced target amplification methods such as polymerase chain reaction (pcr) assays . These tests are typically performed at large referral laboratories, because they require sophisticated equipment, highly specialized laboratory personnel and strict quality control measures . In resource - limited settings, furthermore, stringent requirements for storage and shipment of plasma to the referral laboratory are barriers in settings with limited infrastructure . The solution to this challenge would be to develop reliable, cheap, and easy - to - use assays that can be performed at the site of patient care (point - of - care assays). Numerous rapid diagnostic tests (rdts) for hepatitis b and c are commercially available, most of which provide a test result within 530 minutes; however, the diagnostic accuracy varies from excellent to very poor . A recent meta - analysis by shivkumar and colleagues found that the sensitivity of hbsag rdts used to screen for hepatitis b infection varied from 42 to 100% and the specificity from 0 to 100% . The same authors also reviewed anti - hcv rdts used for hepatitis c screening and found sensitivities ranging from 0 to 100% and specificities from 81.6 to 100% . Hence, although high - quality rdts can be used with confidence in screening for hbv and hcv, health authorities should warn against the use of unreliable (and often very cheap) rdts with poor sensitivity and specificity . Even though serological point - of - care assays for hepatitis b and c are widely available, the same is not the case for virological analyses . However, if we look over the fence into the hiv landscape, there has been an active development of point - of - care kits for viral load quantification over the past few years . Because of major international investments in the fight against hiv / aids, there is an attractive commercial market for such kits . The first products were recently launched, including the alere q analyzer (alere inc ., waltham, ma, usa) and the samba (diagnostics for the real world, sunnyvale, ca, usa), both of which have undergone field testing in sub - saharan africa and shown excellent performance . As there are no similar funding mechanisms for viral hepatitis in resource - limited settings, commercial actors have not shown the same interest in low - tech diagnostics for hbv and hcv; however, many of the technological advances for hiv can be modified to detect other viruses . Currently, there is one product, truenat (molbio diagnostics, goa, india), for hbv dna quantification which is advertized as a point - of - care assay . The truenat kit provides a pcr result within an hour; however, it seems rather complex, involving several manual steps and multiple reagents, making it less ideal for outside laboratory settings . . An independent evaluation of the truenat kit under field conditions would be of major interest . The us - based company wave 80 biosciences is developing a point - of - care kit for hbv dna quantification as well as a qualitative assay for hcv rna detection, building on their existing hiv viral load assay eoscape - hiv (wave 80 biosciences, san francisco, ca, usa). Eoscape - hiv is a fully automated, cartridge format system which is said to be robust and easy to operate . However, eoscape - hiv is still not commercially available, so the timing for the release of the hbv and hcv kits is still uncertain . The uk biotechnology company epistem received a prestigious grant award in 2013 to develop an hcv point - of - care device for viral load testing and genotyping . This novel assay will build on their existing genedrive (epistem, manchester, uk) device, which is a rapid, easy to use, sensitive, handheld pcr platform already ce - ivd marked for other genotype tests, such as mycobacterium tuberculosis identification and antibiotic resistance testing . Far too often advanced technology has been shipped to low - income countries with good intentions, but ended up in a storeroom because of insufficient training, lack of maintenance, and shortage of reagents . Partly, this has to be blamed on the manufacturers, who rarely offer training or service agreements to low- and middle - income countries . It is important, therefore, that point - of - care assays should be designed specifically to operate under basic conditions with minimal maintenance requirements and not just be high - tech solutions forced to fit low - tech settings . The international nongovernmental organization mdecins sans frontires published their desired specifications for a point - of - care hiv viral load assay a few years ago, and the same specification would also apply to an ideal hbv or hcv assay: no need for specialized laboratory facilities closed system to avoid contamination long shelf life in tropical climate no need for cold chain transportation or refrigerated storage affordability is an important issue if treatment for viral hepatitis is to be scaled - up globally . Both the initial cost for the machine, but also the consumables thereafter must be priced reasonably . With regard to hbv, repeated tests over time are usually required, and the accumulated cost of viral load testing can be high . For hepatitis c, on the other hand, one viral load measurement prior to treatment and another 24 weeks after treatment would be sufficient if patients are being treated with the new direct acting antivirals . Sustainability is another major concern with all technological devices, and it should be a prerequisite that the manufacturers provide adequate training and service agreements locally . Furthermore, sensitivity of the assay should be high enough to ensure treatment for those who need it, and even more importantly, specificity must be close to 100% to avoid unnecessary and expensive treatment in uninfected individuals . As described by greenman and colleagues in the current issue of jvh, dried blood spots (dbs) can be a feasible and reliable alternative to point - of - care assays for viral hepatitis . The main advantage of dbs is that it solves the problem of storage and shipment of samples in places with poor infrastructure . Dbs can be stored for weeks at ambient temperature without clinically significant degradation of nucleic acids . A drawback with dbs is the delayed reporting back of results . With viral hepatitis, however, this might be less of a concern, as viral load quantification is part of the pretreatment work - up of each patient and not the day - to - day monitoring of treatment effect (as in hiv). And in viral hepatitis, the decision to start treatment is rarely a matter of urgency . Use of dbs is limited by the small amount of plasma per blood spot and less efficient nucleic acid extraction, which gives a reduced sensitivity in samples with low - level viraemia . With regard to hepatitis c, this rarely has any practical consequences, as most untreated patients have viral loads (far) above 1000 iu / ml . For hepatitis b, however, the situation is rather different as hepatitis b e - antigen (hbeag) negative hepatitis is now the main type of chronic hepatitis b worldwide . These patients typically have fluctuating viral load levels in the lower to medium range, and it is often difficult to distinguish them from inactive carriers . A lack of precision in dbs could therefore jeopardize the management of these patients . Previous studies of hbv dna quantification in dbs have shown inconsistent results . A recent study by mohamed and colleagues showed that dbs yielded viral loads 0.65 log10 lower than plasma, and an older study by jardi and colleagues found that viral load levels were 1 log10 lower in dbs compared to plasma . On the contrary, lira and colleagues found no significant difference between dbs and plasma (0.21 log10 lower in dbs). Thus, more studies are needed to evaluate the precision of dbs for hbv dna quantification, especially in the lower range around the decision threshold of 200020 000 iu / ml . In conclusion, dbs can be a valuable tool for the pretreatment evaluation of patients with hepatitis c, but might be more troublesome in hepatitis b due to a reduced sensitivity in the lower range of viral loads . Management of viral hepatitis is going through a revolution with the launch of new direct acting antivirals for hepatitis c treatment . The cost of nearly 100 000 usd per patient, however, keeps these drugs out of reach for most patients worldwide . Recently, the pharmaceutical company gilead announced that they work with generic drug manufactures in india to produce high - quality, low - cost sofosbuvir for developing countries, raising expectations that this game - changing drug might become available in low- and middle - income countries . Drug availability, however, is not the only issue . In the absence of viral load measurements, doctors in resource - limited settings are left virtually blindfolded in the management of their patients with hepatitis . Point - of - care viral load assays for hepatitis b and c have the potential to bridge this gap and prove valuable tools for expansion of treatment globally . However, assay performance under standardized conditions in europe or north america does not necessarily reflect real - life application in sub - saharan africa, and it is crucial to carry out independent field testing of these instruments before large scale use can be recommended . Finally, sustainable funding mechanisms for diagnostics and treatment of viral hepatitis must be established . Scaling up of hiv treatment globally would have been impossible without major donor programs such as pepfar, unitaid and the global fund to fight aids, tuberculosis, and malaria . Then our colleagues in resource - limited settings might be able to treat viral hepatitis in the not so distant future.
The patient was a 41-week - gestational - age infant girl with a birth weight of 3300 g. after an uneventful pregnancy, the patient was delivered spontaneously and healthy until this episode . At 35 days of age, the patient was admitted to our hospital with a one - day history of anorexia, vomiting and recurrent diarrhea . On admission, conscious level was glasgow coma scale e2 v2 m2, and blood pressure was 104/flat . Physical examination revealed a distended and tender abdomen . Laboratory values were significant for a c- reactive protein (crp) of 1.7 mg / dl (normal range was less than 0.3 mg / dl). The patient was suspected intestinal obstruction due to enteritis, antibiotics were started (abpc / mcipc and amk). 4 hours after admission, she was pale with a temperature of 39.2 c and 220 per minutes pulse . Blood pressure was low, and the fevers, vomiting, and diarrhea persisted and worsened despite antibiotics . She failed to respond to medical managements and died 27 hours after admission due to multiple organ failures . The cyst was unilocular, contained watery fluid, did not communicate with the adjacent intestinal lumen (fig . Microscopic finding of the ileocecal duplication cyst had an undefined mucosal lining though it resembled pyrolic glands or duodenal glands . Duplications of the alimentary tract can be located anywhere between the mouth and the anus.4 they are most commonly found in the ileum (30%), ileocecal valve (30%), duodenum (10%), stomach (8%), jejunum (8%), colon (7%) and rectum (5%) with the half of the all lesions may contain ectopic gastric mucosa.5,6 most alimentary tract duplications are cystic (80%90%), with the remainder being tubular.7 they have three common features, hollow structure that has a wall of smooth muscle often shared with the contiguous intestinal tract, lined by mucous membrane similar to some part of the alimentary canal, and usually attached to some part of the alimentary tube.8 the clinical symptoms are highly variable from minor digestive problems to intestinal obstruction, gastrointestinal bleeding, or perforation.9 most patients present before the age of 2 years with obstructive symptoms, pain, or an abdominal mass . The presence of gastric or pancreatic mucosa can lead to peptic ulceration, perforation, and hemorrhage . Early diagnosis and treatment reduces the risk of dangerous complications, such as bowel perforation, bleeding, obstruction, and malignant alteration . Resection of duplication alone is the treatment of choice, and in uncomplicated cases laparoscopic resection is advised . Surgically treated intestinal duplications have good prognosis, however, such duplications can have a fatal consequence if not properly treated . Possible complications like bowel perforation, bleeding, obstruction, and malignant alterations are the reason why all duplications should be surgically treated at the time of diagnosis.10 alimentary tract duplications can be easily mis - diagnosed as other disorders.5 pyloric and duodenal duplications can mimic hypertrophic pyloric stenosis or choledochal cyst.11,12 ovarian cyst should be considered in female patients, and adolescent patients can sometimes have a presumptive diagnosis of crohn s disease.5 yousefzadeh et al13 showed that duplication of the colon and rectum clinically presented as abdominal pain, vomiting and chronic constipation, a significant number of patients were thought to have hirschsprung s disease . In our fatal case, there are two reasons for this diagnostic difficulty: first, fevers, vomiting, and recurrent diarrhea persisted and worsened despite antibiotics . The patient was suspected intestinal obstruction due to enteritis, however, actually the ileocecal duplication cyst led to intestinal obstruction and necrotizing ileum; second, the patient died in an instant due to multiple organ failures . If this patient was considered for exploratory laparotomy, she might be able to be alive . However, the patient was getting worse very quickly due to multiple organ failures and showed intractable psvt . Exploratory laparotomy was impossible . It is demonstrated a high association of other anomalies in these patients including skeletal and urologic abnormalities,13,14 however, in this case, there were no other congenital anomalies . Sudden unexpected death of infants associated with duplication cysts is extremely rare with only 4 cases reported in medical literature to date (table 1). Puligandla ps et al5 and byard rw15 reported the patients presented with group b streptococcal sepsis and clostridium perfringens sepsis, respectively . Kibayashi et al16 reported the patient died at home after being diagnosed as having gastroenteritis . In present case, though blood, stool and cyst cultures were negative, the patient had high fever and crp level elevated . Infection is suspected to be a risk factor for sudden and unexpected death in infants with duplication cysts . In all cases, this potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause, and duplication cyst should be recognized as a fatal cause in infant.
The term presbyopia derives from greek for old eyes and refers to the age - related loss of natural accommodation and resulting reduction of baseline near vision around the age of 40 years . As people continue to work and stay active later in life than ever before, their need for quality vision at both near and distance vision is also growing . In fact, the presbyopic population worldwide is predicted to rise to 1.4 billion by 2020 and to 1.8 billion by 2050 . Presbyopia can be compensated by glasses or contact lenses, but there is increasing interest in surgical options . Since presbyopia is caused by progressive elasticity changes in the biological crystalline lens, presbyopic surgeries may either directly replace the lens through an intraocular approach or modify extraocular structures such as the cornea or sclera . This review will focus on corneal - based surgical strategies to treat presbyopia and in particular how these methods have been or may be used in pseudophakic patients . To improve uncorrected near vision in presbyopia, the two major techniques to alter the cornea generally utilize either an intracorneal inlay device or laser refractive surgery . Corneal inlays are devices that are surgically placed within the corneal stroma of the nondominant eye to change the optical properties of the cornea . Several different types of corneal inlays each take a distinct approach to minimizing presbyopia (table 1). The best studied inlay to date is the kamra inlay by acufocus, which is an opaque polymer ring that employs a pinhole concept to expand the depth of focus . The raindrop near vision inlay by revision optics is a clear hydrogel implant that increases the anterior corneal curvature to add optical power, with a refractive index approximating that of the cornea . Inlays can also confer differing amounts of refractive power as in the flexivue microlens implant by presbia, which creates a multifocal effect via a central plano zone surrounded by circular rings of plus power . The icolens by neoptics ag is another corneal inlay with a bifocal design similar in concept to the flexivue . Corneal inlays are intended to improve uncorrected near vision but may come at the cost of lowered distance vision or increased glare or haloes [7, 8] or rarely infectious keratitis . However, inlays have been promoted as an additive, removable technology unlike laser refractive surgery which ablates corneal tissue; patients usually return to within + /1.00 diopter of their preoperative refractive state after corneal inlay removal . It should be noted that intrastromal corneal ring segments (icrs or intacs) are another type of corneal implant but are indicated for treatment of keratoconus rather than presbyopia . Laser refractive surgery on the cornea uses an excimer laser to remodel the corneal curvature in order to improve uncorrected vision and reduce dependency on eyeglasses or contact lenses . One of the most popular techniques, laser - assisted in situ keratomileusis (lasik), removes corneal stromal tissue under an anterior flap . Lasik can produce either monovision or multifocality, the latter of which has been nicknamed presbylasik when it is used to treat presbyopia . Conventional monovision lasik corrects the dominant eye for distance vision and the nondominant eye for near vision . In central presbylasik, the central area is shaped hyperpositively for near vision, whereas the midperipheral cornea is adjusted for far vision . In peripheral presbylasik, the central area is shaped for far vision and the midperipheral corneal area for near vision . In the third approach, laser blended vision creates a combination of micromonovision and depth of field increase by inducing spherical aberration . There are concerns for the decreased contrast sensitivity, visual quality, and irreversibility of these laser ablation procedures . Laser blended vision treatment seems to provide the best compromise in terms of safety and quality of vision . In contrast to the literature on corneal inlays in phakic patients, there are only a handful of published reports in pseudophakic individuals (table 2). These include only case reports and retrospective case series . The largest study of pseudophakic patients undergoing inlay implantation included 13 patients with monofocal intraocular lenses (iols). Four of these patients also underwent simultaneous lasik to optimize their underlying refractive error prior to insertion of a kamra pinhole inlay . The mean uncorrected near visual acuity (unva) improved 5 lines from j10 to j4 at 3 months postoperatively, without significant change in mean uncorrected or corrected distance visual acuity (udva, cdva) or corrected near visual acuity (cnva). On a postoperative survey, 77% of the patients said they would opt to have the surgery again . In an earlier report by the same group, kamra inlays were inserted in the nondominant eye of 3 patients with phakic iol implants . These patients saw 25 lines of improvement in unva without change in udva at 3 months postoperatively . Both of these studies had a short follow - up period of 3 months, at which point the authors assert that operative results would have stabilized . Nonetheless, longer studies are definitely needed as the mean age of these patients was around 55 years and so the patients would be expected to live with the inlays for several decades . Two case reports with slightly longer follow - up each described a pseudophakic patient who retained improvement of uncorrected near vision at 624 months after a corneal inlay procedure . One young patient at 32 years of age had received a monofocal iol on account of a traumatic cataract in the right eye, and a year later he developed headaches and asthenopia while reading with his right eye . Given his requirement of a + 3.00 reading add in the right eye, he could not tolerate glasses due to anisometropia . After kamra inlay implantation, he reported improved symptoms but also slightly worse reading in dim light compared to bright light conditions . His unva improved from j17 to j3 in the operated eye at the 6-month follow - up . Another pseudophakic patient with a monofocal iol saw a j3 to j1 improvement in unva after implantation of a flexivue microlens inlay 6 months after the cataract extraction . This recovery of uncorrected near vision continued to be maintained at a 2-year follow - up after inlay implantation . Interestingly, this patient developed a symptomatic posterior capsule opacification in the operated eye at 2 years after the cataract surgery, underwent standard neodymium: yag (nd: yag) capsulotomy, and was reported to still be satisfied and spectacle free with unva of j1 6 months later . The authors asserted that the transparent design of this particular inlay enabled visualization for the nd: yag capsulotomy and also fundoscopy . The aforementioned studies demonstrate the efficacy of corneal inlays in presbyopes with a history of prior intraocular and/or refractive surgery . Similar studies done in phakic patients suggest that the improved uncorrected near vision is achieved for at least 1 to 5 years after corneal inlay implantation . A mexican prospective study of 30 phakic patients undergoing inlay implantation (raindrop near vision inlay) and lasik demonstrated improvement of mean unva by 3 lines at one year, suggesting that the improved unva could also be stable at one year in pseudophakes . A larger retrospective study of 277 eyes in japan showed similar results at one year after simultaneous kamra inlay implantation and lasik, with mean unva improving from j8j10 to j2-j3 depending on patient age . The longest published follow - up of inlays involved an austrian prospective cohort of 32 emmetropic phakic patients who retained improved uncorrected near vision at 5 years after kamra inlay implantation . Of note, the mean unva enhancement declined somewhat from j1 to j3 between the 3- and 5-year time points; however, cdva did not change . It is worth mentioning that, by the 5-year follow - up, there were still no biocompatibility concerns and 84% of the patients said they would opt for the surgery again . Several complications occurred, including 2 inlays needed to be recentered, 1 eye that required debridement for epithelial ingrowth, 18 eyes that developed iron deposits by year 3, and 1 inlay that was removed at year 3 due to patient dissatisfaction . There are several case reports describing the opposite sequence of events, where patients with prior corneal inlay implantation subsequently undergo cataract extraction with intraocular lens placement, either with or without initially removing the inlay . These patients had previously received any one of the three major corneal inlay types, including the kamra inlay, presbia microlens inlay, and the raindrop near vision inlay . These anecdotes suggest that cataract surgery can still be successfully performed after inlay implantation, which will be important for those young presbyopes opting for surgical treatment of their presbyopia . An extensive review of the literature on laser refractive surgery for presbyopia revealed very few studies done in pseudophakic patients, although there are numerous published accounts in phakic patients . Similar to the body of literature on corneal inlays, a history of prior intraocular surgery such as cataract extraction typically excluded such patients from studies on laser refractive surgery for presbyopia . Nonetheless, laser refractive surgery has been performed successfully on pseudophakes to correct for ametropias [2225], suggesting that laser refractive surgery to address presbyopia is likely to be equally successful in pseudophakic patients who have monofocal iols . Clearly, the application of laser refractive surgery to presbyopia in pseudophakia merits more research . Various studies examine the efficacy of laser refractive surgery on treating presbyopia in phakic patients . A prospective trial of central multifocal presbylasik on 50 hyperopic - presbyopic eyes resulted in spectacle independence at all distances for 72% of the patients after 6 months, although nearly a third lost 1 - 2 lines of cdva . This and similar studies have suggested that multifocal laser approaches to improving uncorrected near vision in presbyopia may compromise distance vision to some degree [2729]. Another study utilizing peripheral presbylasik improved mean binocular udva and unva with high reported patient satisfaction rates but decreased contrast sensitivity . Presbylasik treatment for presbyopia may be further modified in pseudophakic patients who are not satisfied with the outcome, as described in a recent case report using a wavefront - guided aspheric treatment to reverse the presbyopic treatment and eliminate dysphotopsia up to 6 months later . Lasik and photorefractive keratectomy (prk) are known to be effective for fine - tuning residual ametropias after presbyopia - compensating iol implantation, which suggests that laser refractive surgery could similarly address presbyopia after iol implantation . Two subsequent studies showed efficacy of lasik at up to 6 months in treating pseudophakic ametropia after multifocal iol implantation in 53 eyes and 85 eyes with mixed ametropias . These and other similar studies suggest that laser refractive surgery could also be used in pseudophakes to treat presbyopia . A general consensus is that lasik should be performed at least 612 weeks after intraocular surgery to reduce possible complications related to cataract incisions, postoperative corneal edema, and refractive and iol stability . While prk and lasik have both been performed safely in pseudophakic patients, it has been postulated that neither are as effective as primary refractive surgery . Conductive keratoplasty (ck), a corneal refractive surgical technique that uses radiofrequency energy to reshape the central cornea by shrinking peripheral collagen, has also been used to treat presbyopia . While again the majority of studies only include phakic patients, one chinese study reported that ck improved unva up to one year later in presbyopic pseudophakic patients with monofocal iols . Studies have noted that myopes and hyperopes may express different levels of satisfaction with presbylasik as well as with other presbyopic treatments . Although near acuity results tend to be better in myopes, the majority of hyperopes are satisfied whereas the majority of myopes are not . The decreasing visual quality after presbylasik in myopes who have experienced excellent uncorrected near visual acuity preoperatively can lead to dissatisfaction over unmet expectations . Pseudophakic patients present a unique set of challenges and advantages for presbyopic surgeries as compared to the phakic population . Some of the challenges stem from the prior intraocular surgery itself, such as navigating corneal scars and residual corneal irregularities from prior incisions . Additionally, iol aberrations may alter laser refractive measurements and calculations normally used on phakic eyes . In general, the age difference of approximately two decades between pseudophakic patients and typical refractive patients also renders keratorefractive treatments less predictable and less effective . The older age of most pseudophakic patients correlates with increased prevalence of dry eye symptoms and slower healing rates . Studies have suggested that the final uncorrected visual acuity after excimer laser or corneal inlay surgery is lower in pseudophakic patients than in typical refractive patients . On the other hand, older patients have reported similar or higher levels of satisfaction after presbyopic surgeries as compared with younger patients who may have had higher postoperative acuity expectations [17, 36]. Presbyopia may continue to progress for phakic individuals who undergo refractive or inlay procedures at a younger age, whereas this is not an issue for patients who have had cataract surgery . At the same time, the lack of residual natural accommodative power in pseudophakic patients gives little room for error in presbyopic surgeries . It is worth noting, however, that because the iol treats most of the spherical error in pseudophakic patients, there would be less keratorefractive - induced effect on the corneal prolate asphericity and quality of vision than in nonametropic phakic patients . In all patients, regardless of phakic status, there are certain considerations to optimize a satisfactory outcome from corneal - based presbyopic surgeries . The importance of a thorough preoperative evaluation cannot be understated, and trial frames or contact lenses should be used in various lighting conditions to help develop realistic patient expectations . Patients should be selected appropriately from baseline characteristics including tear film adequacy, normal corneal shape and thickness, and reasonable expectations . There need to be more studies and counseling of patients on possible negative outcomes of presbyopic surgeries, ranging from increased dry eye or glare symptoms to decreased distance and/or night vision and subsequent safety concerns . Presbyopic surgery may not completely eliminate the need for reading glasses . Given the necessity of fundus viewing for diagnosis and treatment of diseases such as age - related macular degeneration, diabetic retinopathy, or retinal detachments, it should be considered that corneal inlays may obstruct the view or hinder treatment unless they are first removed . Although corneal inlays are promoted as removable, long - term follow - up on removed inlays is not available and presbylasik procedures are in general not reversible with the commonly available technology at present . As the first wave of refractive patients begins to require cataract surgery, we may be required to change not only the assessment and technique of cataract surgery for these patients, but also how we counsel these patients who have high expectations for visual acuity and spectacle independence . For patients suffering from presbyopia, there are now exciting surgical alternatives to glasses and contact lenses . Pseudophakic patients who still desire better uncorrected near vision may choose corneal - based surgical therapies, such as corneal inlays or laser refractive surgery . Nevertheless, presbyopic surgeries will not be ideal for all patients, and appropriately screening patients and managing patient expectations are both key to maximizing satisfactory outcomes . Many studies on presbyopic surgeries excluded patients with prior ocular surgery, and the few published studies on pseudophakic patients are limited by short - term follow - up, small numbers, and limited lighting conditions . Undoubtedly, there is a pressing need for more research on presbyopic surgeries in the pseudophakic population.
Enteric duplications (eds) are uncommon anomalies that can occur at any point of the gastrointestinal tract . The small intestine is the most common location; retroperitoneum is an extremely rare site . In general, diagnosed in the neonatal period or during infancy, they are increasingly diagnosed prenatally; early prenatal detection is possible ., there have been seven reported cases of retroperitoneal ed cyst in the english literature . A female newborn, vaginally born at 39 weeks of gestation from a 32-year - old mother, gravid 3, para 3 . Prenatal ultrasound at 22 weeks of gestation objectified an abdominal cystic mass located in the left upper abdominal quadrant, associated with fetal pyelectasis . Birth weight was 4000 g, length was 51 cm, and head circumference was 35 cm . Postnatal ultrasound found a retroperitoneal para - aortic liquid - filled mass measuring 60 mm 33 mm 22 mm . Magnetic resonance imaging (mri) confirmed the presence of a retroperitoneal cyst occupying the upper left retroperitoneal space; with mass effect displacing the left kidney down [figure 1]. Peroperative finding was a retroperitoneal cyst above the left adrenal, displacing the left kidney down, measuring 70 mm 30 mm; with no communication with any portion of the alimentary tract . Histopathologic examination revealed an ed cyst lined by small intestinal epithelium, with no ectopic gastric mucosa . The postoperative period was uneventful; the patient was discharged on the 5 postoperative day . Magnetic resonance imaging showing retroperitoneal cyst occupying the upper left retroperitoneal space; with mass effect displacing the left kidney intraabdominal duplications account for two - thirds of localization, among them, jejunoileal duplications account for 65% . Eds occur early in intrauterine life; their pathogenesis remains unclear; several hypotheses have been proposed . Reported 38 cases of ed cysts; only one was retroperitoneal communicating with a nonfunctioning right kidney . Eds are a part of fetal intraabdominal cysts which evoke several differential diagnoses including ovarian cysts, renal cysts, choledochal cysts, hepatic cysts, and mesenteric cysts . Marchitelli et al . Found high concordance between prenatal and postnatal findings in fetal intra - abdominal cystic lesions (90.4%). Double - wall sign and peristaltism are suggestive of ed and help to differentiate it of differential diagnosis . Postnatal ultrasound helps diagnosis showing an anechoic fluid - filled mass, rarely an echogenic mass is found due to complication (hemorrhage, necrosis). More serious complications may occur: ulceration, perforation, severe hemorrhage, and malignant changes associated with the presence of ectopic gastric mucosa.
Albert hoffa first described this fracture in 1904.1 these fractures usually involve the lateral femoral condyle . Bicondylar hoffa fracture involving both the femoral condyles is a rare injury and has anecdotally been reported in the literature [table 1].234567 most reported cases of bicondylar hoffa fracture have two separate fracture lines and the two condyles are separated from each other . We describe a rare case of conjoint bicondylar hoffa fracture where both the femoral condyles were joined by a bridge of intact bone adjacent to the intercondylar notch . To the best of our knowledge, only one case of such an injury has been reported in a child.7 previously published reports of bicondylar hoffa fractures a 17-year - old male labourer presented to orthopaedic emergency with the complaint of acute pain and swelling in his right knee following trauma . He had a fall from a 10-m - high ladder with direct impact on his semiflexed right knee . Local examination revealed a painful swollen right knee with a 1 cm 1 cm lacerated wound over the anterior aspect of the knee [figure 1]. Plain radiographs revealed fracture distal end femur but were inadequate to define the exact fracture pattern [figure 2]. Noncontrast computed tomographic (ct) scan was performed which established the diagnosis of conjoined bicondylar hoffa fracture [figure 3]. Cm lacerated wound over the anterior aspect of the knee at the point of direct impact plain anteroposterior and lateral radiographs were inadequate to define the exact fracture pattern axial ct scan cuts showing a bicondylar hoffa fracture joined by a bridge of intact bone the patient was operated in supine position under tourniquet and regional anesthesia . The knee joint was then exposed by swashbuckler (modified anterior) approach.8 a lateral parapatellar arthrotomy was done . The vastus lateralis was retracted laterally after lifting it up from the lateral femoral side . The rest of the extensor mechanism along with the patella was retracted medially for unobstructed visualization of both femoral condyles . On exposing the knee, a tangential fracture involving both the femoral condyles was noted [figure 4]. Both the condyles were joined by a thin bridge of intact bone forming the roof of the intercondylar notch [figure 4]. However, reduction was achieved and five 6.5 mm cannulated cancellous screws were passed from anterior to posterior direction through the nonarticular part under fluoroscopy control . Post operatively above knee back splint with 30 of knee flexion was applied for 2 weeks . Partial weight bearing was started at 6 weeks postoperatively and full weight bearing at 3 months when the fracture had united radiologically . At 18 months, patient had 0145 of range of movements without pain and deformity [figure 5]. Radiographs showed no signs of avascular necrosis, osteoarthritis, or implant breakage [figure 6]. Intraoperative photographs showing the swashbuckler approach (a), pattern of fracture (b), provision reduction (c) and fixation with cannulated cancellous screws (d) clinical photographs at 18 months showing range of motion followup radiographs anteroposterior (a) and lateral (b) views at 18 months showing radiological union, without any avascular necrosis, osteoarthritis or implant breakage hoffa fracture usually results from high - velocity trauma following road traffic accidents or fall from height . The specific mechanism of injury that produces hoffa fracture is not known . Though a shearing force to the posterior femoral condyle has been postulated, both direct impact and vertical shear with twisting mechanisms have also been proposed and a single mechanism is not agreed upon.9 because of the physiological genu valgum, the lateral femoral condyle is more likely to sustain a direct shearing force, and hence is more likely to get fractured.9 this injury is usually caused when knee is hyperflexed at the time of impact, as during driving motorcycle.9 the exact mechanism of injury of a bicondylar hoffa fracture has also not been described . We feel that a bicondylar hoffa fracture occurs when the flexed knee is subjected to a posterior and upward directed force without any varus or valgus component and that the proximity of the fracture line and its obliquity depends upon the degree of knee flexion at the time of impact . The greater the degree of knee flexion, more is the distance of fracture line from the posterior femoral cortex [figures 7 and 8]. Our patient had sustained an injury following a fall from a 10-m - high ladder with direct impact on his semiflexed right knee, as evident by a small lacerated wound, probably without any varus or valgus component . Therefore, the coronal shear force resulted in a bicondylar hoffa fracture where both the femoral condyles were joined by a bridge of intact bone adjacent to the intercondylar notch as the fracture line was anterior to the intercondylar notch . Had the knee been flexed more, it would have resulted in a bicondylar hoffa fracture with two separate fragments with the fracture line being posterior to the intercondylar notch . The most widely used classification system developed by muller, updated by the ao group, and adopted by the orthopaedic trauma association (ota) classifies distal femoral fractures into three groups [figure 9].10 we feel that b3.3 should be subclassified into two groups: type b3.3a- conjoint bicondylar hoffa fracture where the two condyles are joined by a bride of intact bone and occurs due to a posterior and upward directed force with a semiflexed knee without any varus or valgus [figure 7]. Type b3.3b- nonconjoint bicondylar hoffa fracture where the two condyles are separate from each other and occurs due to posterior and upward directed force with a hyperflexed knee without any varus or valgus [figure 8]. However, this subclassification needs validation and further evaluation . In view of the rare occurrence of hoffa, further rarer occurrence of bicondylar hoffa, and unreported occurrence of conjoint bicondylar hoffa similarly, in view of limited information and followup, it is not possible to provide information about the prognosis of this fracture . A line diagram showing conjoint bicondylar hoffa fracture following a posterior and upward directed force (f) in a semiflexed knee a line diagram showing nonconjoint bicondylar hoffa fracture following a posterior and upward directed force (f) in a hyperflexed knee classification of distal femoral fractures the diagnosis of hoffa fracture on plain radiographs can present difficulties because the fracture is obscured in the anteroposterior view by the intact anterior part of the femoral condyle.5 if it is minimally displaced, the fracture may also be difficult to define in the lateral view . If the lateral view is not taken in a standard position, then it is further difficult to interpret whether it is the medial or the lateral condyle that is fractured even if displaced . This may result in the injury being missed on initial imaging.6 oblique radiographs have an important role both preoperatively and also intraoperatively for evaluation of the reduction, screw length, and condyle identification . However, a ct scan is most helpful not only in defining the fracture pattern but also in deciding the surgical approach . In our case also, the initial radiographs were inadequate to define the exact pattern of the injury . The ct scan demonstrated the bicondylar hoffa fracture with both fragments joined to each other by a bridge of bone forming the roof of intercondylar notch . It helped us not only in defining the exact nature of the injury but also in the surgical approach . Therefore, like previous authors, we feel a ct scan with 3d reconstruction is the investigation of choice in such patients to define the exact pattern of the fracture and plan the surgical approach . Nonoperative treatment in the form of plaster cast or skeletal traction leads to loss of extension, nonunion, instability, joint contracture, and deformity.239 therefore, anatomical reduction of articular surface, stable fixation, and early mobilization should be the aim of treatment.11 for open reduction of bicondylar hoffa fracture, most authors have used a combined medial and lateral approach . However, we used a swashbuckler (modified anterior) approach as advocated by dua et al.6 this allowed us to expose the back of femoral condyles on both the sides and facilitated unhindered anatomic reduction of the fracture fragments . The more anterior position of the skin incision as compared to the classical lateral approach allows unobstructed visualization of both the femoral condyles and obviates the need of two incisions . We believe that single incision would result in decreased breach of the quadriceps mechanism, lesser fibrosis, and earlier return of quadriceps strength and range of motion . Lal et al.7 described arthroscopic assisted reduction and fixation for such injuries . However, we believe that it would be technically challenging in a case like ours where the fragment was large and displaced . Although there is no consensus, there is some suggestion that posterior to anterior directed screws may be better.12 however, in our case, the unusual fracture pattern required that we use an approach by which both condyles could be exposed adequately . Once the exposure was adequate, the best method was to fix the fractures from anterior to posterior direction under direct vision . Thus, the direction of the screws was determined by the approach required for the fracture exposure . In conclusion, we described a rare case of a conjoint bicondylar hoffa fracture managed successfully by open reduction and internal fixation with good clinical outcome at 18 months of followup . We feel that a bicondylar hoffa fracture occurs when the flexed knee is subjected to a posterior and upward directed force without any varus or valgus component and that the proximity of the fracture line and its obliquity depends upon the degree of knee flexion at the time of impact . Ct scan not only helps in defining the exact pattern of injury but also is invaluable in the surgical planning . The swashbukler (modified anterior) approach allows excellent exposure of both the condyles . Anatomic reduction and rigid internal fixation allows early mobilization and excellent long term outcome.
, neither buccolingual width nor angulation can be properly visualized on the most traditional radiographs . In the following case report, an innovative simplified method without radiation is used to fabricate a three - dimensional model to assess the available bone for implant placement in the mandible . Even a minor variation in comparison to ideal placement causes difficulties in fabrication of final prostheses . The surgical guide is essential to establish a logical continuity among diagnosis, prosthetic planning, and surgical phase . A patient of age 27 years reported to the department of prosthodontics for the replacement of missing left first molar . Following steps were performed to achieve a three - dimensional model: intraoral impression was recorded with addition silicone putty and light body [figure 1]fabrication of acrylic assembly for aligning both intraoral and extraoral impressiona rectangular block of acrylic was made to which impression tray was pressed to make indentations . After applying separating medium, the counter block of acrylic was made to which extraoral impression was attached [figure 2]. In prototype, [figures 24] plastic rod was used for supporting the extraoral impressionreplacing the plastic rod with metal plate and key system the plastic rod was replaced by a metal plate in the first generation [figure 5] for better stability and better customization . A key system was incorporated to adjust the length and angulation according to each patient's mandible . On the top of the metal plate, addition silicone putty placed on the acrylic plate can be customized each time to record the extraoral impression . The extraoral impression should record the lower border of the mandible to the medial extent as much as possible [figure 6]fabrication of three - dimensional model the intraoral impression was poured first to get a cast . After the initial setting was over, the entire assembly was poured, and a three dimensional model of the mandible was fabricated [figure 7]. Intraoral impression was recorded with addition silicone putty and light body [figure 1] fabrication of acrylic assembly for aligning both intraoral and extraoral impression a rectangular block of acrylic was made to which impression tray was pressed to make indentations . After applying separating medium, the counter block of acrylic was made to which extraoral impression was attached [figure 2]. In prototype, [figures 24] plastic rod was used for supporting the extraoral impression replacing the plastic rod with metal plate and key system the plastic rod was replaced by a metal plate in the first generation [figure 5] for better stability and better customization . A key system was incorporated to adjust the length and angulation according to each patient's mandible . On the top of the metal plate, acrylic addition silicone putty placed on the acrylic plate can be customized each time to record the extraoral impression . The extraoral impression should record the lower border of the mandible to the medial extent as much as possible [figure 6] fabrication of three - dimensional model the intraoral impression was poured first to get a cast . After the initial setting was over, the entire assembly was poured, and a three dimensional model of the mandible was fabricated [figure 7]. Intraoral impression with acrylic assembly prototype (extraoral impression) prototype - extraoral and intraoral impression together with acrylic assembly first generation metal plate assembled metal frame three - dimensional model the accuracy of this method was verified with computed tomography (ct) scan . The cross section where the mandibular premolar teeth were disappearing from the ct image was taken (14 mm depth) as a reference and measured to get the available bone width buccolingually (8.7 mm). This measurement was same when compared with the available bone in the sectioned cast at the premolar area, at a depth of 14 mm (premolar root length). On the three - dimensional model, the surgical guide was fabricated [figure 8]. This surgical template can dictate the implant body placement that offers the best combination of support for the repetitive forces of occlusion and esthetics . Three - dimensional model with surgical guide this article had won the best table clinic award in 35 ips conference conducted in new delhi 2007 by army dental corps.
The use of ergotamines causing ischemia of peripheral vessels has been reported, particularly in patients with peripheral vascular disease . Ischemic bowel induced by ergotamines is a much rarer event, with few documented cases in the literature . While colitis is always on the differential diagnosis of hospital patients with abdominal pain, the majority of cases are secondary to infectious colitis associated with antibiotic therapy . When suspected, non - occlusive mesenteric ischemia can be treated with anti - coagulation and blood pressure support with intravenous fluids . The predisposition for ischemia in patients with chronic constipation secondary to opioid use is amplified by concomitant use of vasoactive medications to control symptoms of migraine headache . While abdominal ct scanning is frequently used to assess colonic pathology, findings often lag behind those identified at the time of colonoscopy . Patchy areas of ischemia are often managed non - surgically by discontinuing the offending medication . Widespread necrosis, coupled with clinical deterioration, as in this case, mandated immediate surgical intervention to prevent irreversible septic shock . A 48-year - old woman with a long - standing history of intractable migraine headaches was admitted to our institution's specialty headache unit for aggressive management . She had no known history of peripheral vascular or cardiac disease but has been admitted multiple times for treatment of her headaches since her initial diagnosis in 2003 . During the course of each of these hospitalizations, she had received one to two courses of dihydroergotamine mesylate (dhe) 45 . During this admission she received two courses of dhe 0.5 mg 1, then 1 mg every 8 h for a total of eight doses, on days 13 and the second course of therapy on days 79 . Several hours after the final dose of her second round she became obtunded, minimally responsive and hypotensive with a blood pressure of 80/45 mm hg . On physical examination her abdomen was moderately distended with few bowel sounds . Mmol / l, white blood cell count was 18.0 (with 17% bands), and stool was clostridium difficile negative . The patient was transferred to the intensive care unit, given intravenous fluid boluses, started on vasopressors, and given broad spectrum antibiotics . Her chest radiograph was unremarkable, and her abdominal x - rays showed only mild prominence of the colon . Over the next several hours she became progressively acidotic with a worsening bandemia . A bedside colonoscopy showed diffuse ischemic colitis with fecal impaction and no pseudomembranes (fig . Operative findings showed a peritoneal cavity filled with murky, foul - smelling fluid and gangrene of the descending colon was seen with necrosis in the area immediately distal to the splenic flexure to the sigmoid colon . Final pathology on the surgical specimen showed 86 cm of patchy, dark green / black large bowel, consistent with ischemic necrosis . The final diagnosis was gangrenous large bowel, ischemic colitis with thrombi or emboli noted . A hypercoaguable panel was performed following the operation and the patient was negative for clotting disorders . The differential diagnosis of ischemic colitis includes arterial occlusion, caused by emboli or thrombus, venous thrombosis, and non - occlusive ischemia caused by systemic hypotension or, as is strongly suspected in this case, ergotamine - induced vasospasm . A diagnosis of ergotamine - induced vasospasm is a diagnosis of exclusion, made when all other etiologies of ischemia have been ruled out . In this case, an arterial occlusion is unlikely, because there was no palpable thrombus and there was no clear ischemic demarcation to suggest a major vessel distribution . The patient had no history of cardiac arrhythmias, such as atrial fibrillation, to predispose her to emboli . Furthermore she was a non - smoker and had no other cardiac risk factors such as atherosclerosis or hyperlipidemia . Emboli to inferior mesenteric artery (i m a) are exceedingly rare, and there are no reported cases of spontaneous emboli or thrombosis to i m a . All reported cases of i m a thrombosis are subsequent to trauma [1, 2]. Venous thrombosis as a cause of ischemia in this patient is equally unlikely as there were no signs of venous congestion or stasis at the time of surgery . The patient had no hypercoagulable disorder and on examination the colon was not edematous and no clot was identified in the vasculature . Ischemia secondary to systemic hypotension alone would have resulted in a watershed - type distribution of gangrene . In this case the most probable cause of this event was non - occlusive mesenteric ischemia in the i m a distribution and more specifically the left colic artery due to intense vasoconstriction induced by high - dose ergotamine use . Underlying bowel wall distention from the impacted stool in combination with vasoconstriction from ergotamine is the most likely etiology for this patient's bowel ischemia . Dhe is indicated for the acute treatment of migraine headaches with or without aura and the acute treatment of cluster headache episodes . Its therapeutic activity in the treatment of migraine headaches is generally attributed to the agonist effect at 5-ht1d receptors . According to the neurovascular theory of migraine, it is hypothesized that activation of 5-ht1d receptors on intracranial blood vessels leads to vasoconstriction . The 5-ht1d receptors on sensory nerve endings of the trigeminal are also activated, causing inhibition of pro - inflammatory neuropeptide release . Ergotamine and dhe cause vasoconstriction of both arteries and veins due to alpha - adrenergic agonistic action, and they may also have possible interaction with prostaglandins, calcium and serotonin . Dhe was developed in 1943 for migraine prophylaxis and shown in four studies in 1987 and five studies in 2001 to have less effect on peripheral arteries than ergotamine . As indicated in this case a european consensus report in 2000 concluded that ergotamine is not a drug of first choice in the triptan era, but may be useful in longstanding migraine attacks with multiple recurrences . Ergotamine is known to cause peripheral ischemia, as has been reported in several case studies, most commonly in the lower limbs [5, 6, 7, 8, 9]. A causal relationship has not however been clearly established and is still the subject of some debate . Other reports of ergotamine causing ischemic bowel appear in the literature [13, 14, 15]. In previous reported cases of colitis, expectant management with this is the first reported cases of ischemia progressing to gangrene such that colon resection was required . It should be noted in this case that the patient's dose of dhe was greater than the recommended amount . The recommended dosing of dhe 45 is 1 mg iv / im / sc, repeated once every 24 h for a total dose not to exceed 6 mg in 7 days . In this case, the patient received 17 mg of dhe over the course of 9 days . Although ischemic colitis is considered a rare side effect of high - dose ergotamine use, it is a very real risk of which clinicians need to remain aware . This is the fourth reported case of ischemic colitis secondary to high - dose ergotamine use reported in the literature and the first to require colon resection . While dhe can be useful in patients with refractory migraines, patients should be informed of the risks of both peripheral and visceral ischemia before deciding on this treatment . Finally, ischemic colitis should be considered in the differential diagnosis of all patients taking ergotamines who present with abdominal pain.
We analyzed all 140 mrsa isolates we obtained during a previous study (2) and 50 other isolates selected from our collection of nosocomial mrsa isolates obtained in 2003 . Characteristics of these 190 isolates are shown in table 1 . * ni, nosocomial infection acquired at national taiwan university hospital (ntuh); nc, nosocomial colonization at ntuh; ieoh, mrsa infection detected at ntuh within 48 h after transfer from another hospital . Bl, blood; ur, urine; sp, sputum; pu, pus; wo, wound; ns, nostril; ct, catheter tip; st, stool . Pulsed - field gel electrophoresis patterns were interpreted according to procedures previously reported (7,8). Thirty - four isolates belonged to pulsotype a, 49 to pulsotype b, 69 to pulsotype c, 6 to pulsotype d, 2 to pulsotype e, 3 to pulsotype f, 11 to pulsotype k, 5 to pulsotype l, 8 to pulsotype m, and 3 (all isolated in 2003) to 3 minor pulsotypes . All isolates were tested by sccmec element typing and multilocus sequence typing (mlst) (9) and were analyzed for the panton - valentine leukocidin (pvl) gene (10) and drug susceptibility to erythromycin, clindamycin, gentamicin, amikacin, ciprofloxacin, levofloxacin, tetracycline, trimethoprim - sulfamethoxazole, rifampin, and vancomycin by using the disk diffusion method (11). Isolates with pulsotype a are sequence type 254 (st254); those with pulsotype b are st241; those with pulsotypes c, k, and l are st239; and those with pulsotypes d, e, f, and m are st59, st 254, st30, and st5, respectively . All mrsa isolates with pulsotypes a, d, e, and f have the type iv sccmec element . However, only isolates with pulsotypes d and f, as well as 2 isolates from 2003 with 2 minor pulsotypes, have the pvl gene . Isolates with pulsotypes b and c have the type iii sccmec element, and isolates with pulsotype m have the type ii sccmec element . * p, pulsotype; scc, staphylococcal cassette chromosome; mlst, multilocus sequence typing; pvl, panton - valentine leukocidin; n, no; y, yes . Ox, oxacillin; em, erythromycin; cl, clinidamycin; gm, gentamicin; am, amikacin; cp, ciprofloxacin; lv, levofloxacin; tc, tetracycline; ts, trimethoprim - sulfamethoxazole; rf, rifampin; va, vancomycin . Results of mlst and typing of sccmec elements of the 3 isolates with 3 minor pulsotypes obtained in 2003 are shown in table 2 . The correlation between sccmec element typing and mlst in this study corresponds to findings of previous reports (6,1214). Enright et al . Identified st254-iv mrsa isolates in germany and the united kingdom (12), and chongtrakool et al . Identified st239-iii and st241-iii mrsa isolates in several asian countries (14). We demonstrate that the predominant mrsa clone at ntuh in early 1990s had the type iv sccmec element . However, the predominant mrsa clones at ntuh from 1994 to 2003 had the type iii sccmec element . These findings differ from those of wisplinghoff et al ., who reported that that the sccmec element in predominant mrsa clones at their institute changed from type iii in 1984 to 1988 to type i in 1989 to 1998 (6). Mrsa isolates of pulsotypes b and c are more resistant than isolates of pulsotype a to certain antimicrobial drugs, especially fluoroquinolones and trimethoprim - sulfamethoxazole; mrsa isolates with pulsotype c are more resistant to clindamycin but less resistant to rifampin than those with pulsotype b (table 2). From 1993 through 2000, annual use of fluoroquinolones increased 3 at ntuh; however, use of trimethoprim - sulfamethoxazole, clindamycin, and rifampin did not change (15). Therefore, the shift of predominant mrsa clones, which also led to the shift in types of sccmec elements at ntuh, might be caused by selective pressure from antimicrobial drugs, especially fluoroquinolones . The mrsa clone (pulsotype a) that predominated in 1992 and 1993 at ntuh has the type iv sccmec element . Although the first study of mrsa with the type iv sccmec element reported that this element was found in community - acquired mrsa (ca - mrsa) (5), some studies have reported mrsa isolates with this element in a hospital environment (12). However, to our knowledge, these reports did not demonstrate that mrsa isolates with the type iv sccmec element became predominant among all mrsa isolates in an institution, especially before the mid-1990s . Furthermore, 4 st59 mrsa isolates obtained in 1994 and 1996 and 3 st30 mrsa isolates obtained in 1992 and 1993 have the type iv sccmec element and pvl gene . Recently, st59 mrsa isolates were found to cause ca - mrsa infection in taiwan (13). Among these st59 ca - mrsa isolates, some had the type iv sccmec element, and others had the type v sccmec element (13). Although the type iv sccmec element could be transferred to ca - mrsa clones with other genetic backgrounds, our finding supports the possibility that st59 mrsa isolates with the sccmec element type iv in taiwan may originate from hospital strains but transfer into ca - mrsa strains . Recently reported the results of sccmec typing of 615 mrsa isolates obtained in 1998 and 1999 from 11 asian countries (14). The st239-iii, st241-iii, st254-ii, and st5-ii mrsa isolates were prevalent in many asian countries . However, the st254-iv, st30-iv, and st59-iv mrsa isolates from our study were not found in other asian countries . In addition, st254-iv mrsa isolates have been found in germany and the united kingdom (12). Whether st254-iv mrsa isolates in our study were transmitted from those in germany or the united kingdom by international travel requires further study . The first mrsa isolate with the type iv sccmec element in our hospital appeared as early as 1992 . The sccmec element carried by predominant mrsa clones changed from type iv to type iii sccmec element during the period 19922003 at ntuh . Because the major mrsa clones isolated in 19942003 are more resistant to antimicrobial drugs, especially fluoroquinolones and trimethoprim - sulfamethoxazole, than those obtained in 1992 and 1993, this shift may be caused by selective pressure from indiscriminate use of antimicrobial drugs.
Acute embolic limb ischemia is an urgent clinical condition in which sudden decrease or worsening in limb perfusion causes a potential threat to extremity viability and to life . Thorough removal of all occlusive emboli in the arterial tree results in the best outcomes; hence, some authors favored arteriography after embolectomy for lower limbs . Angiography is traditionally performed with iodinated contrast material, but carbon dioxide (co2) has been studied as an alternative intravascular contrast agent for patients with iodine allergy or renal function impairment . Since it is not nephrotoxic, it was considered reasonable to utilize this contrast in a patient with borderline renal function who would be submitted to the noxious stimuli of the reperfused limb after revascularization . To the best of our knowledge, this was the first case assessing the intraoperative use of co2 as a substitute for iodine contrast in a patient with known chronic kidney disease and an acute ischemic limb . A 79-year - old woman presented to the emergency department with acute limb ischemia in her left leg with a 2-hour history . Past medical history was positive for hypertension, atrial fibrillation and non - dialysis - dependent chronic renal failure . Two years before, she had been operated on for acute aortic embolic occlusion, with bilateral femoral catheter embolectomy . Upon discharge she presented pedal pulses, but no posterior tibial pulses on either limb . Medication use was irregular, and her international normalized ratio (inr) of prothrombin time upon admission was 1.1 . Her pulse was arrhythmic; she was hypertensive (160x110mmhg) and presented with normal femoral, popliteal and pedal pulses on her right leg . On palpation in the left lower limb only a weak femoral pulse was observed; there were no distal pulses . She was classified as grade iib limb ischemia (immediately threatened) and was immediately referred for surgery . Preoperative exams revealed a diminished creatinine clearance (cockroft - gault of 12ml / min). Arteriotomy was performed and a 4-fr embolectomy catheter was utilized (edwards lifesciences corp, irvine, california, usa). The catheter progressed more than 60 cm on the superficial artery, but could not be felt neither on pedal artery topography nor on posterior tibial artery topography . Back bleeding was not significant, and a decision was made to perform an angiography to better assess the infra - popliteal arterial tree . A homemade water seal co2 delivery system was used, similar to another previously described, but with one modification to prevent room - air contamination . Bloomington, indianopolis, usa) inserted in the arteriotomy while the arteries were clamped . Figure 1 shows a patent popliteal artery, a patent fibular artery and an occlusion on the mid - third of the anterior tibial artery . We hypothesized that the embolectomy catheter reached the fibular artery on the first attempt, and thus could not be felt on physical exam . Patent popliteal and fibular arteries, occlusion in the mid - third of the anterior tibial artery a new embolectomy was then performed . The catheter progressed to the foot, on the topography of the pedal artery (i.e., the catheter could be felt on the projection of the artery), and more thrombi were retrieved . Control angiography (figure 2) showed complete resolution of the anterior tibial artery occlusion, with contrast up to the foot (figure 3). A total of 28ml of co2 was used, and no iodine contrast was needed . The arteries were unclamped, and the popliteal and pedal pulses were noted to be present on the limb . Ischemia signs (e.g., pallor, hypothermia) were promptly resolved after arterial flow release . The patient remained in the intensive care unit (icu) until the 11 postoperative day . During this period, the patient did not receive any nephrotoxic agent, such as vancomycin or vasoactive drugs . After icu discharge she developed a urinary tract infection, as well as deteriorating renal function and uremia . The patient developed pneumonia, which progressed to septic shock on the 36 day of her admission . She required vasoactive drugs for 2 days at the icu and, unfortunately, her renal function was never fully recovered . She was discharged from hospital to a hospice care facility with a pedal pulse and no deficit on her limb, though requiring chronic dialysis . Treatment of embolic arterial occlusion with severe limb ischemia is well defined and involves the use of an embolectomy catheter as the best alternative . Co2 has been used electively in both diagnostic and therapeutic procedures in the femoropopliteal tree, and is reported to be safe and non - nephrotoxic . Intraoperative angiography was beneficial because it allowed the diagnosis of the arterial tree occlusion after the initial embolectomy . Additional removal of clots was performed and the result was documented as complete patency of the anterior tibial artery to the foot . Our goal, when using co2, was to decrease the need for postoperative dialysis . Probably, in this case, the deleterious stimuli (surgical stress, urinary tract infection and septic shock due to pneumonia) contributed to renal failure and subsequent need for dialysis . The countermeasure (such as the use of non - nephrotoxic contrast) if we had used the iodinated medium, we might wonder if something could have been done differently to avoid dialysis . We consider co2 to be an alternative to iodine contrast when the decision to perform angiography is made in the acutely ischemic limb . To the best of our knowledge, this agent has never been described in this clinical setting; it presents good quality imaging, is not nephrotoxic and does not increase the risk for the limb or the patient in acute arterial occlusions . This approach may prove to be fruitful in patients with borderline renal function, thereby reducing the risk of short or long - term need for dialysis . A isquemia aguda de membro de origem emblica uma situao clnica de carter urgente, em que ocorre uma diminuio ou piora sbita na perfuso de um membro, e configura um risco viabilidade da extremidade e vida . O tratamento consiste em revascularizao imediata com um cateter de embolectomia . A remoo completa de todos os mbolos oclusivos na rvore arterial leva aos melhores desfechos; por isso, alguns autores defendem a arteriografia aps a embolectomia, para os membros inferiores . A angiografia tradicionalmente realizada com contraste iodado, mas o dixido de carbono (co2) foi estudado como um agente de contraste intravascular alternativo para pacientes com alergia a iodo ou alterao na funo renal . Como o co2 no nefrotxico, julgou - se razovel utilizar esse contraste em uma paciente com funo renal limtrofe (borderline) que seria submetida aos estmulos nocivos de reperfuso do membro, aps a revascularizao . Em nosso entender, este foi o primeiro caso que avalia o uso intraoperatrio de co2 como um substituto de contraste iodado em um paciente com doena renal crnica conhecida e isquemia aguda de membro . Paciente do sexo feminino, 79 anos, chegou ao pronto - socorro com isquemia aguda no membro inferior esquerdo, com durao de 2 horas . Como antecedentes, apresentava hipertenso, fibrilao atrial e insuficincia renal crnica, no dependente de dilise . A paciente tinha sido operada 2 anos antes, por ocluso artica aguda de origem emblica, quando foi realizada embolectomia femoral bilateral com cateter . Alta, os pulsos pediosos eram palpveis, mas os tibiais posteriores eram ausentes, bilateralmente . A paciente fazia uso irregular dos medicamentos, e o tempo de protrombina (inr, sigla do ingls international normalized ratio) era de 1,1 . O pulso estava arrtmico; era hipertensa (160x110mmhg) e apresentava pulsos femoral, poplteo e distal normais, na perna direita . Palpao do membro inferior esquerdo, havia apenas um pulso femoral fraco; os pulsos distais estavam ausentes . A paciente apresentava dor e dfice motor . Ela foi considerada como portadora de isquemia de membro grau iib (risco eminente) e foi encaminhada imediatamente para cirurgia . O clearance de creatinina estava diminudo nos exames pr - operatrios (cockroft - gault de 12ml / min). Sob anestesia geral, foi realizado acesso femoral na cicatriz da inciso anterior do membro inferior esquerdo . As artrias femorais comum, superficial e profunda foram dissecadas e clampeadas . Foi feita arteriotomia e uso de cateter de embolectomia de 4fr (edwards lifesciences corp, irvine, califrnia, estados unidos). Os trombos foram removidos das artrias femorais superficial e profunda . O cateter avanou mais de 60 cm na artria femoral superficial, mas no pde ser palpado . O fluxo retrgrado no foi significativo e decidiu - se realizar uma angiografia para avaliar melhor a rvore arterial infrapopltea . Foi escolhido o co2 como substituto ao contraste iodado para a angiografia . Utilizou - se um sistema home - made com fornecimento de co2 em selo dgua, semelhante a outro descrito anteriormente, mas com uma modificao para evitar a contaminao do ar ambiente . Bloomington, indianpolis, estados unidos) inserido na arteriotomia, quando as artrias foram pinadas . A figura 1 mostra uma artria popltea e uma fibular prvias, e uma ocluso no tero mdio da artria tibial anterior . Levantamos a hiptese de o cateter de embolectomia ter alcanado a artria fibular na primeira tentativa, e por isso no ter sido palpvel ao exame fsico . Artrias popltea e fibular prvias, ocluso no tero mdio da artria tibial anterior realizou - se, ento, nova embolectomia . O cateter avanou at o p, na topografia da artria pediosa (isto, o cateter pde ser palpado na projeo da artria) e mais trombos foram removidos . A angiografia de controle (figura 2) mostrou resoluo completa da ocluso da artria tibial anterior, com contraste fluindo at o p (figura 3). Foram usados 28ml de co2, e no foi necessrio aplicar o contraste iodado . Resoluo completa da ocluso da artria tibial anterior figura 3arteriografia com dixido de carbono . Chegada de contraste na artria tibial anterior at o p foi realizada a arteriorrafia . As artrias foram desclampeadas, os sinais de isquemia (palidez e hipotermia) foram imediatamente resolvidos aps a liberao do fluxo arterial . A paciente no desenvolveu sinais de sndrome compartimental . A paciente permaneceu na unidade de terapia intensiva (uti) at o 11 dia ps - operatrio . Durante esse perodo, no recebeu nenhum agente nefrotxico, como vancomicina ou drogas vasoativas . Aps a alta da uti, desenvolveu infeco urinria, piora da funo renal e uremia . Iniciou dilise no 18 dia ps - operatrio . A paciente teve pneumonia, que evoluiu para choque sptico no 36 dia de internao . Necessitou de drogas vasoativas por 2 dias na uti e, infelizmente, nunca mais recuperou a funo renal . Teve alta hospitalar e foi para instituio de cuidados terminais, com pulso distal sem dfice no membro inferior, mas em dilise crnica . O tratamento da ocluso arterial emblica com isquemia grave de membro est bem definido, sendo o uso de cateter de embolectomia a melhor alternativa . O co2 tem sido empregado de forma eletiva tanto nos procedimentos diagnsticos como nos teraputicos na rvore fmoro - popltea, sendo descrito como seguro e no nefrotxico . A angiografia intraoperatria foi benfica, j que permitiu o diagnstico de ocluso da rvore arterial aps a primeira embolectomia . Mais cogulos foram removidos, e o resultado documentado foi a permeabilidade completa da artria tibial anterior at o p . Nosso objetivo, ao utilizar co2, foi o de diminuir a necessidade de dilise no ps - operatrio . Neste caso, provavelmente, os estmulos prejudiciais (estresse cirrgico, infeco do trato urinrio e choque sptico por pneumonia) contriburam para a insuficincia renal e para consequente necessidade de dilise . A contramedida (como o uso de contraste no nefrotxico) poderia ter prevenido a insuficincia renal, se fosse esta uma situao nica . Se tivssemos usado o contraste iodado, pensaramos se algo diferente poderia ter sido feito para evitar dilise . Consideramos o co2 como uma alternativa ao contraste iodado quando necessria a angiografia para isquemia aguda de membro . Em nosso entender, esse agente nunca foi descrito nessa situao clnica, proporcionando boa qualidade de imagem, no sendo nefrotxico, nem aumentado o risco do membro ou do paciente, em casos de ocluses arteriais agudas . Essa abordagem pode se mostrar til em pacientes com funo renal limtrofe (borderline), reduzindo, assim, o risco de precisar de dilise a curto ou longo prazo.
Emphysematous gastritis, which was first described by fraenkel in 1889 (1), is a rare disease characterized by the presence of air within the wall of the stomach and diffuse gastric wall inflammation by gas - forming bacteria, which may be also life - threatening due to systemic toxicity . Predisposing factors include the ingestion of corrosives, alcohol abuse, recent abdominal surgery, diabetes, and immunocompromised conditions (1 - 3). Since 1889, only 51 cases have been reported worldwide (1 - 8). Mucormycosis is a rare fungal infection that usually involves the nasopharynx (9). Among the several forms, gastrointestinal mucormycosis is very rare, and the manifestations range from colonization of peptic ulcers to infiltrative disease with vascular invasion and dissemination (9). Commonly associated conditions include diabetes mellitus, lymphoma, leukemia, renal disease, septicemia, malnutrition, and long - term treatment with steroids and antibiotics (10). Among the reported cases of emphysematous gastritis, fungal organisms such as candida species have been isolated rarely (4, 11 - 15). However, emphysematous gastritis associated with mucormycotic infection is extremely rare, and only one case has been reported in the english literature (15). Here we report a case of emphysematous gastritis, diagnosed by computed tomography (ct) and confirmed by histopathologic findings associated with invasive gastric mucormycosis that showed angioinvasion, necrosis, ultimately disseminated to the colon and liver, and was fatal despite medical and surgical treatment . To our knowledge, this is the first report of a patient with emphysematous gastritis associated with invasive gastric mucormycosis in korea . A 43-yr old man was admitted to the emergency room because of diffuse abdominal pain, indigestion, and poor oral intake for 4 days . He had had a 1-yr history of hypertension, there was also a history of excessive ingestion of alcohol and heavy smoking for 20 yr, but there was no history of peptic ulcer disease . His blood pressure was 160/100 mmhg, with a body temperature of 36.2, pulse rate of 98/min, and respiration rate of 26/min . Abdominal examination revealed decreased bowel sounds and severe tenderness in the epigastrium, with rigidity and rebound tenderness . Hematological and biochemical examinations on admission showed hemoglobin 13.9 g / dl (normal: 13 - 18), white blood cell 11.7710/l (3.6 - 11), platelet 21010/l (150 - 450), blood sugar 816 mg / dl (70 - 110), aspartate aminotransferase 148 iu / l (5 - 38), alanine aminotransferase 76 iu / l (4 - 43), lactate dehydrogenase 1,320 iu / l (180 - 460), amylase 6 u / l (45 - 108), blood urea nitrogen 37.7 mg / dl (8 - 23), and creatinine 1.6 mg / dl (0.5 - 1.2), and blood acetone - positive . Chest radiography revealed collapse and consolidation in the basal part of the left lower lobe, suggesting pneumonia . Abdominal ct scan revealed gastric wall thickening with a collection of dirty air bubbles and a small amount of pneumoperitoneum (fig . Operation findings showed nearly total necrosis of stomach wall with dark green colored pus collection around the stomach . Grossly, the stomach had a large area of necrosis with black discoloration of the mucosa (fig . The remaining viable stomach wall showed large numbers of neutrophilis in the edematous submucosa and muscle layer . There were several gas - filled bubbles in the submucosa and muscle layer associated with numerous scattered or colonies of gram - negative bacilli, consistent with emphysematous gastritis (fig . 1). At the same time, there were numerous broad - based, non - septate, right angular branched fungal hyphae, morphologically consistent with mucormycosis, detected on hematoxyline and eosin stain as well as silver stains throughout both necrotic and viable stomach wall (fig . Culture of pus in the peritoneum revealed klebsiella pneumonia, staphylococcus aureus, and pseudomonas aeruginosa . Amphotericin b infusion was added after operation . On the fifth hospital day, there was pus discharge from the wound site . However, the patient remained to have non - toxic appearance with stable vital signs . On the seventh hospital day, he complained of diarrhea and vague abdominal discomfort . Over the following 2 days, follow - up ct revealed an approximately 14.012.012.0 cm - sized, irregularly shaped air - containing fluid collection at the gastrectomy site, suggesting air - forming postoperative abscess and anastomotic site leakage . Reoperation was performed, and the operative findings showed multiple perforations with necrosis in the transverse colon, necrotic change in the liver, hematoma with fluid collection in the peritoneal cavity, and leakage in the anastomotic site . Rouxen - y esophagojejunostomy with cecocolostomy and segmental resection of the large intestine were performed . The submitted pathological specimen consisted of segments of colon, small intestine, and a portion of liver . The gross examination of the segment of colon showed multiple perforations, dirty serosa, and necrotic mucosa with greenish black exudates . Microscopic examination of the colon and small intestine showed ischemic necrosis of mucosa with acute and chronic inflammation, and foreign body reaction . Microscopic examination of the liver also showed totally necrotic parenchyma with many angioinvasive mucormycotic fungal hyphae . Auscultation of the lungs revealed decreased breathing sounds with rale on both lung fields, and saturation of arterial blood gas analysis was decreased . He underwent intubation and ventilation . On the 21st - hospital day, the patient expired . Clinically this condition is divided into gastric emphysema and emphysematous gastritis (3, 5, 12). These two conditions should be differentiated because they are characterized by different clinical symptoms, possible etiology, radiologic findings, treatment, and prognosis (3, 5, 12). In gastric emphysema, air enters the stomach wall from the lumen, from the peritoneal surface, or from its connections with the esophagus and duodenum, resulting from barotraumas in the absence of bacterial infection (12). Radiologically, in contrast to emphysematous gastritis, a more linear distribution of gas in the gastric wall is characteristic of gastric emphysema (3). The symptom of acute abdomen as seen in patients with emphysematous gastritis is usually absent in patients with gastric emphysema (5). In general, gastric emphysema is asymptomatic; its course is usually benign and resolves spontaneously without treatment (3, 5, 12). On the other hand, emphysematous gastritis, the gas formed in situ by gas - forming bacteria invading the gastric wall, results in a necrotizing inflammation of the gastric wall . In 1889, fraenkel first reported a young man who died after several attacks of severe abdominal pain, blood vomitus, and diarrhea (1). At autopsy, originally, the stomach wall is well protected from bacterial infection by the close connection between cells, acidic ph, and good blood supply (3, 12). The predisposing factors, leading to the breakdown of these defenses, are ingestion of corrosives, alcohol abuse, diabetes, recent abdominal surgery, gastroenteritis, immunocompromised conditions, and treatment with non - steroidal anti - inflammatory drugs (1 - 3). In the present case, the patient had a history of alcohol abuse, which might have altered the unstirred mucous layer, and diabetes due to chronic pancreatitis, which might have led to a systemic predisposition to infection . Patients with emphysematous gastritis typically develop acute abdominal pain, as was the case in our patient, usually developing 1 week after initiation, accompanied by diarrhea, nausea, vomiting, and occasionally hematemesis and melena (1, 4, 12). Physical examination of the abdomen reveals epigastric tenderness, distension, and decreased bowel sounds (4, 12). Abdominal computed tomography, the best imaging modality to establish the diagnosis, reveals gastric wall thickening and intramural gas (1, 3, 12). In contrast to gastric emphysema, the gas in the wall is in the form of irregular mottled bubbles or spots, especially around the fundus and the greater curvature, and remains in place despite the position of the body or absorption through the gastric tube (3, 4, 12). Organisms that have been cultured from stomach aspirates, blood, and peritoneal fluid include streptococci, e. coli, p. aeruginosa, clostridium . Perfringens, and s. aureus (1, 4). Among the previously reported cases, those associated with fungal infection were rare (4, 11 - 15). Of these, most cases were candida species (4, 11, 12, 15). Pathologically, the gas forming organisms in emphysematous gastritis have been shown to infiltrate the stomach wall diffusely and there are edematous stomach wall - containing gas bubbles (1, 6). The therapeutic approach for emphysematous gastritis included initial stabilization of the septic condition through vigorous fluid resuscitation and early empirical parenteral antibiotic therapy with a broad range of antibiotics covering gram - negative and anaerobic bacteria (2, 3, 12, 14). Specific therapy should be modified according to the results of the gastric fluid culture and sensitivity testing of the isolated organism (12). Indication for emergency surgery include deterioration despite optimal medical management, involvement of a large portion of or the entire stomach, presence of gastric infarction, or perforation (3, 4). Despite meticulous treatment, the mortality rate of emphysematous gastritis is about 60%, and the morbidity near 21% (2). Mucormycosis is a rare, opportunistic fungal infection that occurs almost exclusively in immunocompromised hosts such as patients with diabetes, leukemia, lymphoma, renal disease, septicemia, burns, malnutrition, and following long - term treatment with steroids and antibiotics (10). Clinical manifestations of mucormycosis can be categorized as rhinocerebral, pulmonary, disseminated, gastrointestinal, and cutaneous (9). Among these, rhinocerebral and pulmonary disease are the most common forms, and gastrointestinal involvement is very rare, accounting for only 7% of all cases (16). Other associated conditions are immunosuppressed conditions such as renal failure, hematological malignancies, cirrhosis, and solid organ transplantation (16). It is believed that infection of the alimentary tract is acquired through direct ingestion of and invasion by fungal spores . The gastrointestinal organ most frequently involved is the stomach, followed by the colon, small intestine, and esophagus (16). When they invade, the lesion extends and marked surrounding induration develops, with a shaggy, velvety discolored surface or large plaqe - like areas of green and blacked eschar (16). They also invade blood vessels and thus tend to cause extensive thrombosis, necrosis, and, ultimately, dissemination (10). Association with emphysematous gastritis is an extremely rare, and only one such case has been reported (15). Mucormycosis is not gas - forming, therefore this infection is not a direct cause of emphysematous gastritis . Gastric wall necrosis caused by bacterial infection may be predisposed to the invasion by mucormycosis . Alternatively, it is possible that invasive mucormycosis with subsequent gastric wall necrosis led to secondary bacterial invasion and intramural gas production (15). Successful management of mucormycosis includes aggressive metabolic support, amphotericin b, and surgical debridement of all necrotic involved tissue (15, 16). As in emphysematous gastritis, the prognosis of gastrointestinal mucormycosis is poor . Forty - two cases of gastric mucormycosis have been described in the literature thus for, with a mortality above 98% (15). We report an extremely rare case of emphysematous gastritis associated with invasive gastric mucormycosis in a 43-yr - old man . Heavy alcohol abuse and diabetes mellitus were the predisposing factors . Ct is the diagnostic procedure of choice of emphysematous gastritis and helps in differentiating with gastric emphysema . Moreover, the gastric mucormycosis showed angioinvasion, necrosis, and ultimately, disseminate to colon and liver . In the case of mucormycosis, biopsy of involved areas this patient also had a fatal outcome despite antibiotic and antifungal therapy and surgery . Increased awareness of these disease entities may facilitate early diagnosis, prompt medical therapy, and appropriate surgical intervention, and ultimately improve survival rates.
In accordance with the 1997 documents of the world health organization (who), amoebiasis is the infection by the protozoan parasite entamoeba histolytica with or without clinical manifestations . The only known natural host of e. histolytica is the human body with the large intestine as the major target organ . This parasite has a very simple life cycle in which the infective form is the cyst that is considered a resistant form of the parasite . The asymptomatic cyst passers and the intestinal amoebiasis patients are the natural transmitters; they excrete cysts in their feces, which can contaminate food and water sources . The cysts are round structures around 1016 m in diameter . However, estimation of cyst diameter in entamoeba spp . In the old and recent literature the cyst has four visible nuclei when mature and only one when immature, and the nuclei are spherical with a membrane displaying small chromatin granules and a central karyosome . When in excystation, each cyst produces eight vegetative forms or trophozoites, which are the motile form of the parasite; they are 2040 m in diameter . Life cycle and the relevant structures of both forms of parasite are shown in figure 1 . Cysts are resistant to desiccation in soil and can survive in humid environments and in water for several weeks . Susceptible hosts exposed to the aforementioned infection sources ingest the cysts, which then undergo excystation during their pass through the gastrointestinal tract . Amoebiasis is also considered a sexually transmitted disease, particularly in sexual relationships between men and in individuals with sexual anilingus practices . Clinical and etiological diagnosis of intestinal and extra - intestinal amoebiasis is neither easy or simple in part because of the discovery of two species made from the previously known e. histolytica species, both indistinguishable under microscopy . E. histolytica sensu stricto is the potentially pathogenic species and e. dispar the commensal non - pathogenic entamoeba . D) trophozoites of e. histolytica species with phagocyted erythrocytes (dic 40) knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the twentieth century, which gave rise to the rapid development of diagnosis technology based on molecular and immunological strategies. [59] during the last 10 years, knowledge of the new epidemiology of amoebiasis in different geographic endemic and non - endemic areas has been obtained through the application of mostly molecular techniques. [1014] moreover, these molecular epidemiology studies have unveiled the extraordinary genetic variability[1315] of e. histolytica and e. dispar, allowing the discovery of other entamoeba species, such as e. moshkowskii, which can also infect the human intestine with a significant frequency . However, much of the epidemiology and its contribution to morbidity of entamoeba infections remain unknown . There are excellent recent reviews on the molecular epidemiology and intestinal and extra - intestinal characteristics of amoebiasis in the human host that can be consulted . Nevertheless, the major purpose of the present work is to highlight the novelties in regard to human infection and the disease that can help the general physician from both endemic and non - endemic countries in their medical practice . This is especially critical given that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide . The speciation of entamoeba protozoa has been discussed since 1925 when emile brumpt proposed the existence of two distinct species that could infiltrate the human intestine: one associated with symptoms of diarrhea with or without dysentery and the other excreted with feces from asymptomatic individuals . While years of scientific discussions have left brump's theory behind, no molecular technology prior to the 1990s allowed clear differentiation of the currently known e. histolytica and e. dispar species in terms of pathogenic or non - pathogenic phenotypes . Both species are genetically diverse and this variability allowed for the beginning of studies on molecular epidemiology in different endemic areas . The new data on the epidemiology of amoebiasis based on frontier technology suggests that genetic variability could be an important tool in the study of geographic distribution of both species and particular strains of entamoeba, which may determine the morbidity rates of different forms of amoebic infection in different geographic areas . Some e. histolytica genetic variants (strains) have been isolated from asymptomatic cyst passers, patients with invasive intestinal amoebiasis or from samples of amoebic liver abscess material . However, e. dispar has been mainly isolated in feces samples resulting from asymptomatic cyst passers and displays high genetic polymorphisms, even more than the e. histolytica species . We recently have obtained evidence that in at least two endemic countries (brazil and mexico), e. dispar genetic variants have been detected in patients with invasive amoebiasis . In brazil, the icb - ado e. dispar strain was isolated from a non - dysenteric colitis patient maintained in culture with his own intestinal flora displaying a pathogenic behavior in experimental models of amoebic liver abscess . Dna extracted from hepatic abscess material obtained from six patients in mexico also clearly showed the presence of e. dispar dna sequences . The third species of entamoeba, e. moshkowskii, has been considered a free - living organism since 1940s in contrast to e. histolytica and e. dispar, with a geographic distribution mainly in developing countries . E. moshkowskii has been frequently detected in individuals from developed and highly industrialized countries . Particularly in regard to this species we are at the beginning of the study of its pathogenic potential and the context in which it is expressed and the epidemiologic significance of infection and its contribution to morbidity rates of diarrheic diseases . What doses of cysts are necessary for colonization of large bowel mucosa? For the three entamoeba species this is not known with any certainty . Moreover, we do not know which environmental characteristics are permissive for intestinal colonization, and this could be an interesting field for future research . Finally, until today, the only species recognized as an etiologic agent of amoebic invasive disease is e. histolytica, which, once in the colon, undergoes excystation and the generation of trophozoites . Trophozoites multiply by binary fusion and some of them may encyst and be excreted with stools . Cyst viability under appropriate conditions of humidity may last as long as several weeks and thus they be available for a new susceptible host . We have to stress that more than 90% of infections have an asymptomatic course and are frequently auto - limited at different periods of time . After intestinal infection there is no evidence of induction of a long - lasting protective immune response, and in endemic areas, individuals may have several periods during the year of re - infection and clearance of infection . In relation to the susceptibility of hiv and aids patients to the invasive forms of infection, contradictory evidence exists; however, morbidity seems to be more related to the particular prevalence of the e. histolytica strain with invasive phenotypes than to the specific immunological status of the patient. [2427] as for the invasive behavior of e. histolytica, some authors consider this trait not to be typical . On the contrary the natural history of invasive intestinal amoebiasis is an acute event, characterized by the presence of diarrhea that occurs days or weeks after exposure in our personal experience lasting no more than four to five weeks . Although there are reports of occurrence years after exposure, in this case we presume the cause and effect relationship is extremely difficult to corroborate . Intestinal amoebiasis is basically an acute disease in which the most frequent symptoms are abdominal pain (colic) and the presence of diarrhea with mucus and/or blood, or a clear dysenteric syndrome . However, fever and other systemic symptoms are infrequent . Severe forms of invasive amoebiasis can be observed in young children (<5 years), pregnant woman, the elderly, and particular those with chronic diseases, such as diabetes mellitus, and in individuals being treated with immunosuppressants or those with immunodeficiency disorders . Those severe forms of amoebiasis are colon ameboma, fulminant necrotizing colitis, and toxic mega colon . The appearance of symptoms, such as severe dysentery and pain with signs of peritoneal irritation (rebound), intense tenesmus, fever (> 38c), tachycardia, hypertension, nausea, and anorexia are suggestive of the previously mentioned severe forms of intestinal amoebiasis . The mortality rates of dysenteric syndrome due to e. histolytica are less than 1%, but mortality due to complications increases up to 75%. [2931] fortunately in the last few decades such complications are uncommon . The intestinal amoebiasis form known as chronic non - dysenteric colitis is the most frequent form of amoebiasis in people of all ages, characterized by non - specific symptoms . Symptoms more relevant in this instance are periods of abdominal pain (colic) and auto - limited episodes of diarrhea alternating with constipation . However, both non - dysenteric amoebic colitis and irritable bowel syndrome are controversial themes in the clinical practice . Where endoscopy examination is available, colonoscopy can be of great help in clinical diagnosis of invasive intestinal amoebiasis . This procedure allows for microscopic examination of samples taken directly from the characteristic flask - shaped ulcer produced by e. histolytica and from other sites of mucosal lesions . Microscopic observations of this type of material are described in the diagnostics section and in figure 2 . On the other hand, colonoscopy detects the presence of lesions related to the mentioned severe forms of intestinal amoebiasis and allows for differential diagnosis of other pathologies, such as inflammatory bowel disease or colon carcinoma . D) intestinal biopsy obtained from the edge of flask - shaped ulcer where large numbers of trophozoites (he and pas stained, 60) are clearly visible . E) biopsy obtained from the edge of amoebic liver abscess (he and pas stained, 20). Notice the presence of trophozoites, hepatocytes, and the large number of inflammatory cells . What circumstances define the extra - intestinal invasive behavior of some e. histolytica strains? This remains unknown today . For example, we do not know the frequency of extra - intestinal invasion after intestinal colonization with virulent e. histolytica . However, this seems to be an infrequent event as suggested by the low morbidity rates of amoebic liver abscess and other extra - intestinal forms of invasive amoebiasis compared with the prevalence rates of asymptomatic infections and intestinal disease . While amoebic liver abscess is a disease that can affect individuals of all ages, in some endemic areas the incidence rates are higher in both children under 5 years and young adults (2045 years). Males are also more prone to developing amoebic hepatic abscess than females (1 female for 46 males). After exposure, 80% of patients display symptoms over a few days to 46 weeks . The most common symptoms suggestive of amoebic liver abscess are fever (38c), chills and diaphoresis, anorexia and abdominal pain in the right upper quadrant that increases during inspiration . Pain also frequently radiates to shoulder and back . Nausea has also been referenced but diarrhea is only occasionally mentioned (50% of cases). Hepatomegaly can be detected during digital percussion of the hepatic area and is always related to the dimensions of the abscess; patients can also display peritoneal signs (abdominal guarding or rebound). Absence of intestinal noises, jaundice, and pleural or pericardial rub are symptoms that should elicit alarm related to the rupture or imminence of rupture of the hepatic abscess . In general, the right hepatic lobule is the most frequently affected due to the portal circulatory system of the right colon . Laboratory findings suggestive of amoebic liver abscess are the presence of leukocytosis, neutrophilia, increased globular sedimentation velocity, and high levels of alkaline phosphatase . Thoracic x - ray data useful in the diagnosis of amoebic abscesses, as well as other types of hepatic abscesses, include elevation of the right hemidiaphragm, atelectasis, or pleural effusion [figure 3a]. Ultrasound is the gold standard technique for diagnosis of amoebic liver abscess, as its positive predictive value (ppv) is around 95% (85100% depending on analyzed series). Although contrast computed tomography (ct scan) [figures 3d and 3e] have a ppv up to 95% due to a higher definition capacity, ultrasound is considerably less expensive compared to ct scan technology, which is of tremendous importance to countries with limited medical and economic resources . Ultrasound reveals hypoechoic areas that can be single or multiple with round edges [figure 3b and 3c]. Several authors have mentioned the presence of a large single abscess as a frequent characteristic of a amoebic liver abscess . However, this characteristic is not a sine qua non of amoebic abscesses, and in our medical practice we have seen multiple abscesses more frequently than we had first assumed . Early lesions due to amoebic invasion of hepatic parenchyma are multifocal in nature (micro - abscesses) as a consequence of tissue destruction and necrosis by proteases from e. histolytica and neutrophil recruitment to the site of infection . The advantage of ct scans and magnetic resonance is detection of small abscesses and the high definition of the images . Moreover, other techniques not always available in endemic countries (e.g. Gallium scans) can help differentiate between amoebic (cold images) and pyogenic abscesses (hot images). Thus, the difference is based on the absence (amoebic) or presence (pyogenic) of white blood cells in the abscess . Additionally, we have to mention that in endemic countries the frequency of mixed abscesses (pyogenic and amoebic) is considerable; in our practice this frequency is approximately 17% (non - reported data). Another important fact in medical practice is the coincidence of previous symptoms with the presence of high levels of serum anti - amoebic antibodies more than 90% of amoebic liver abscess patients develop this type of antibody response . However, in cases of fast development of amoebic abscess these antibodies may not be present . A) thoracic x - ray of a patient with amoebic liver abscess showing the elevation of the right hemi - diaphragm . Ultrasound images of: b) single large amoebic abscess and c) three amoebic hepatic abscesses . D) contrasted computed tomography (ct) scan of a single abscess and e) three clear amoebic liver abscesses etiological diagnosis of intestinal parasitic diseases has been mainly performed using direct or concentration techniques for microscopic examination of fecal specimens . While technically simple in approach, these techniques require the expertise of highly qualified technicians in the morphological identification of ova, cysts, and helminths to be feasible, and the sensitivity and specificity is no more than 80% . While this technique cannot differentiate between the three entamoeba species already mentioned, in some endemic communities this is the only diagnostic technology available . On the other hand, during the last 10 years diagnostics in amoebiasis have changed dramatically, considerably improving sensitivity and specificity . Some of the current techniques are based on immunological strategies, such as elisa and different modalities of polymerase chain reaction (pcr), with clear advantages in bedside diagnosis and in clinical laboratories of health institutions . Even though some of these diagnosis procedures are also suitable for large epidemiologic trials, it is mandatory to make a careful selection of the diagnosis test when the test has to be applied in the field . In particular, the election has to be directed to those procedures that do not need special conditions for sample preservation or pretreatment of specimens in the working field . In our experience, immunologically based diagnostics tests for detection of anti - amoebic secretory antibodies in feces or detection of intestinal amoebic antigens through polyclonal or monoclonal specific antibodies (elisa) are excellent tests in hospitals where fresh specimens (feces) can be obtained . Results are reproducible and reliable . In contrast, in epidemiological trials, these techniques can be biased when samples are more than 18 hours old . Tables 1 and 2 have a list of diagnostic tests that have proven to be useful in clinical and laboratory diagnosis of intestinal and extra - intestinal amoebiasis in first and second level health institutions . Microscopy and immunoassays for e. histolytica detection pcr assays for e. histolytica and/or e. dispar detection the who / paho recommendations published in 1997 contain in detail the actions that who country members have to observe with regard to e. histolytica species infection . They highlight the characterization (when possible) of e. histolytica and e. dispar (we now add e. moshkowskii) to be treated properly . In accordance with the who recommendation only e. histolytica it is also important to remember that in some endemic countries, mixed infections (e.g. E. histolytica and e. dispar) are frequent, and that only those infected with e. dispar should be excluded for anti - amoebic treatment . Table 3 shows treatment schedules that have proven to be highly effective in both intestinal and extra - intestinal invasive amoebiasis . In cases of large amoebic abscesses in which imminence of rupture or distances of less than 1 cm between the abscess wall and liver surface prevail, ultrasound - guided puncture is indicated . The procedure allows for the establishment of a differential diagnosis with other liver pathologies, especially pyogenic liver abscess, which is common in clinical practice . Treatment of amoebiasis disease in our experience, patients with amoebic liver abscess may not excrete e. histolytica / e . However, some of these patients are asymptomatic cyst passers after treatment with systemic anti - amoebic drugs, such as metronidazole . In such cases, treatment has to include luminal anti - amoebic drugs . At present, evidence of low susceptibility or resistant strains of either e. histolytica or e. dispar species to metronidazole gastrointestinal infections are responsible for morbidity and mortality rates of children and young adults worldwide . In africa, diarrhea is responsible for 2575% of all childhood illnesses . Infection and intestinal diseases with icd10 code a00-a09 are the second cause of disease in children under 10 years old in mexico, and intestinal amoebiasis ranks with some annual variations 5 and 6 in the list of the 20 major causes of disease in mexico (http://www.dgepi.salud.gob.mx/anuario/index.html #). Causes of diarrhea in endemic areas include a large variety of enteropathogens (viruses, bacteria, and parasites). In mexico, parasitic intestinal infections are multiple infections that constitute approximately 40% of analyzed individuals in which it is possible to detect more than one pathogen together with commensal parasites that are an indicator of fecalism . Prevalence of parasitic infection in three different communities in the state of morelos, mexico, are shown in table 4, one of which is a strictly rural population (amacuzac) and two (tlaltizapan and xoxocotla) are suburban communities . The relevance of these results lies in the high frequency of mixed parasitic infections detected in the studied populations . A remarkable low prevalence of soil - transmitted helminthiasis was also observed and could be the consequence of a biannual anti - parasitic treatment of school children with albendazole . This policy was implemented by the health ministry since the 1990s . However, there are emerging parasites with an increasing prevalence in the last 10 years, including blastocystis hominis and in some areas cryptosporidium spp . In south africa intestinal mixed infection is present in 46% of patients with diarrhea and 33% in school children . Furthermore, mixed intestinal infections due to bacteria, parasites, and viral pathogens are the major forms of intestinal infection in developing countries . However, cheun et al recently published a splendid study on diarrheal patients in hospitals in korea . The study documented the presence of enteropathogenic bacteria, parasites, and viruses in mixed infections, and highlighted the importance of diarrheal disease associated with protozoan infections . The major association of e. histolytica positive samples was with rotavirus type 1[10.3(6.114.6) positivity/100 infected individuals, 95% ci], astrovirus [9.3(5.213.4) positivity/100 infected individuals, 95% ci], pathogenic escherichia coli [7.2 (3610.0) positivity/100 infected individuals, 95% ci] and clostridium perfringens [10.3(6.014.6) positivity/100 infected individuals, 95% ci]. As the authors mentioned, the question is whether mixed infections with protozoa are more likely to induce serious diarrhea . Prevalence rates of parasite intestinal infections in morelos, mexico efforts in the near future have to be directed on studies focusing the interactions of microorganism in the intestinal environment . This knowledge will have a positive impact in clinical and laboratory diagnosis of diarrheic syndrome, its treatment, and thereafter the implementation of more reliable control schedules.
Engaging in high levels of physical activity is a key strategy for successful maintenance of weight loss . A study of nearly 4,000 participants in the national weight control registry (nwcr), the largest longitudinal study of successful weight loss maintainers, indicated participants expend 2,621 2,252 kcal per week through physical activity, which is equivalent to approximately 60 minutes of moderate - intensity physical activity per day . Additionally, long - term followup of participants in behavioral weight loss programs has shown that those who are most successful at maintaining their weight loss report activity levels similar to those of nwcr participants [3, 4]. Many individuals, particularly those who are obese, are insufficiently active, and increasing physical activity can be a challenge [5, 6]. Thus, strategies that assist individuals in adopting and sustaining high levels of physical activity to help facilitate a healthy body weight are needed . Prior studies have identified multiple strategies for increasing physical activity adoption and maintenance, such as providing home or clinic - based exercise programs, increasing access to active behaviors (e.g., adding exercise equipment in the home), and reducing access to sedentary behaviors (e.g., limiting time to watch television), using pedometers to track activity and progress toward physical activity goals, and accumulating exercise throughout the day in multiple short bouts (10 min) [11, 12]. Performing a variety of different types of activities may be another strategy to increase physical activity levels . The national health and nutrition examination survey (nhanes) showed that individuals who reported engaging in a variety of different activities (i.e., walking + other leisure - time activities) were more likely to meet national physical activity recommendations compared to those who reported no variety (i.e., only walking). Likewise, in an 18-month behavioral weight loss intervention, overweight participants who reported physical activity variety (i.e., 2 different activities) at 6 months had higher self - reported activity - related energy expenditure and a lower body mass index (bmi) at 18 months than those who did not report physical activity variety (i.e., only 1 activity). Thus, while the above findings suggest that physical activity variety may contribute to higher physical activity levels within the context of behavioral weight loss treatment, it is unclear whether physical activity variety is associated with higher physical activity in individuals who have achieved long - term success in controlling their body weight . In the current study, we examined the relationship between physical activity variety, defined as the number of different types of self - reported moderate - to - vigorous activities performed in one week, and minutes spent in objectively measured moderate - to - vigorous physical activity (mvpa) in two groups of individuals who have successfully maintained their body weight long - term: (1) weight loss maintainers with previous history of overweight / obesity and (2) normal - weight individuals without a history of overweight . Normal - weight participants were included as a comparison group given that weight loss maintainers represent a unique group of individuals who report strict adherence to multiple behavioral strategies in order to maintain their body weight . Due to their unique characteristics and history, it is possible that the association between physical activity variety and mvpa could be different for weight loss maintainers and normal - weight individuals . However, based on previous research showing a relationship between physical activity variety and higher physical activity levels across diverse groups and settings [14, 15], we predicted that engagement in a greater variety of moderate - to - vigorous activities would be associated with higher mvpa daily minutes in both the weight loss maintainer and normal - weight groups . Participants were enrollees in the cross - sectional lite study that compared weight control behaviors of weight loss maintainers and normal - weight controls . A convenience sample of men and women was recruited through advertisements placed in national and local publications intended for a general audience . Persons interested in participating were asked to either call a toll - free number or to visit a website . Participants were recruited from across the united states, although most were from new england, california, and the washington, dc area . Weight loss maintainers had a history of overweight or obesity (bmi 25) but were currently normal weight (bmi = 18.524.9), having maintained a 10% loss of their lifetime maximum body weight for at least 5 years . Normal - weight participants had a current bmi between 18.5 and 24.9 and no history of overweight or obesity . Participants in both groups were weight stable (10 lb) for at least 2 years prior to enrollment . Of 813 individuals who responded to advertisements and a brief online screening tool, the study protocol was approved by the miriam hospital institutional review board, providence, ri, usa . Participants reported information about age, gender, marital status, ethnicity / race, type of employment, and education . Weight and weight history were assessed via self - report methods that have been previously validated . Questions on the paffenbarger physical activity questionnaire regarding average number of city blocks walked per day, and weekly frequency and duration of sports and recreational activities performed were used to determine variety or number of different activities performed during the past 7 days . Only activities that were performed at a moderate or vigorous intensity and for 10 minutes in duration were included given that engagement in these activities is considered necessary for improving health and achieving a healthy body weight [1, 12]. The intensity of sports and recreational activities was determined using the paffenbarger coding scheme . For walking to be counted as an activity, participants had to report walking the equivalent of at least 12 blocks per day (i.e., 1 mile at a moderate intensity). Treadmill walking reported as a sports and recreational activity was not distinguished as a separate activity from walking 12 blocks / day . Climbing stair flights, walking <12 blocks / day, and sports and recreational activities that were performed for <10 minutes were not considered to contribute to variety . Monrovia, ca, usa) was used to objectively measure daily minutes spent in mvpa . This device converts accelerations or movements from vertical, horizontal, and anterior - posterior planes into counts, with greater magnitude or intensity of acceleration over a given time period generating a higher number of counts . Participants were sent the device in a postage - paid envelope with instructions on how to activate the device and wear it on their waistband during all waking hours for 7 consecutive days, except while bathing or swimming . Each device was programmed with the participant's personal data (sex, age, height, and weight) and set to capture movements continuously in 1-minute intervals . The rt3 has shown to be a strong predictor of oxygen consumption during sedentary and treadmill activities [20, 21] and a more precise measure of physical activity at the group level compared to its tri - trac predecessor . Consistent with previously documented methods for analyzing rt3 data, a minimum of 4 days on which the device was worn for 10 hours each day was required for data to be considered valid [23, 24]. Rt3 nonwear times, defined as periods of 30 consecutive minutes of zero counts (permitting intervals of up to 2 consecutive minutes registering 1100 counts / min), were deleted from analysis . The remaining time was partitioned according to intensity level . Based on previous rt3 validation research and a recent study that used the rt3 to compare mvpa patterns in weight loss maintainers, normal - weight, and obese groups, we computed time spent in mvpa using a threshold of 984 counts / min . Descriptive statistics are presented in tables as means sd for continuous measures and percentages for categorical responses . Chi independent t - tests were conducted to assess differences between the groups on demographic characteristics, weight, accelerometer daily wear time, mvpa minutes / day, and reported variety / number of different types of moderate - to - vigorous activities performed . Linear regression was used to examine the associations of physical activity variety and group status (weight loss maintainers versus normal weight) with objectively measured mvpa minutes / day, adjusting for age, gender, years of education, marital status, bmi, and accelerometer daily wear time . Logistic regression was used to assess whether physical activity variety and group status were associated with achieving the 250 mvpa minutes / week recommendation for optimal long - term weight maintenance . For this analysis, mvpa minutes / week was calculated by multiplying average daily mvpa minutes by 7 (days). Of 413 participants who were sent an accelerometer and the ppaq, 394 (95%) met valid accelerometer wear requirements and provided complete data on the ppaq . The characteristics of these 226 weight loss maintainers and 169 normal - weight participants are presented in table 1 . Both groups were similar in age (48.8 12.9 years), gender (84% female), marital status (66% married), race / ethnicity (94% caucasian), employment status (82% employed), and job type (95% in professional or clerical positions). A greater proportion of normal - weight participants was college educated, compared to the weight loss maintainers . Both groups were normal weight, although the weight loss maintainers had a slightly higher bmi . On average, weight loss maintainers had lost nearly 29 kg and maintained 10% weight loss for 13.7 9.6 years . There were no differences in accelerometer wear time between the groups, with the weight loss maintainers and normal - weight participants wearing the accelerometer for an average of 14.8 2.0 hours / day on 7.5 0.9 days . As reported previously, weight loss maintainers spent an average of 6 minutes more per day in mvpa, compared to normal - weight participants (58 versus 52 min / d). The variety / number of different activities performed by the weight loss maintainers and normal - weight participants was similar (1.8 1.2 versus 1.7 1.2, p = 0.52). Additionally, when participants who reported no moderate - to - vigorous activities were excluded, the variety / number of activities performed by wlm (n = 180) and nw (n = 141) remained similar (2.2 1.0 versus 2.0 1.0, p = 0.10). We next examined the independent and joint associations of physical activity variety and group status with mvpa minutes per day . Given that the physical activity variety group status interaction was not significant (p = 0.73), the results of the main effects linear regression model for mvpa minutes per day are shown (table 2). Greater physical activity variety (p <0.001) and weight loss maintainer status (p <0.05) were independently related to greater daily time spent in mvpa, after adjustment for demographic characteristics (age, gender, educational level, marital status, current bmi) and daily accelerometer wear time . Figure 1 presents estimated mvpa minutes per day in the weight loss maintainer and normal - weight groups by reported number of different moderate - to - vigorous activities performed . Across both groups combined, the number of moderate - to - vigorous activities ranged from 0 to 4, with 73 participants (18.5%) reporting 0 activities, 105 reporting 1 activity (26.6%), 112 (28.4%) reporting 2 activities, 74 reporting 3 activities (18.7%), and 30 (15.4%) reporting 4 or more activities . Each 1 unit increase in the number of different moderate - to - vigorous activities performed was associated with an additional 9.4 1.3 daily minutes spent in mvpa, representing an additional 56.2 8.3 kcal expended per day based on rt3-derived estimates . Similarly, logistic regression analyses showed that greater physical activity variety (or = 1.78 [1.372.30], p <0.001), weight loss maintainer status (or = 0.23 [0.080.69], p = 0.002), and lower bmi (or = 0.81 [0.690.95], p = 0.01) were independently associated with meeting the 250 mvpa minutes / week guideline for optimal weight maintenance . The physical activityvariety group status interaction was not significant (p = 0.25). Given the continuing obesity epidemic and growing evidence that indicates greater amounts of physical activity are needed for successful long - term weight control, it is important to identify strategies that can assist individuals in adopting and maintaining high levels of physical activity . This study examined whether performing a greater variety of different types of moderate - to - vigorous physical activities was related to greater time spent in mvpa among weight loss maintainers and normal - weight individuals without a history of overweight / obesity . We found that, independent of group, greater variety was associated with higher daily mvpa minutes and meeting the 250 mvpa minutes per week recommendation for optimal long - term weight maintenance . These findings are consistent with previous studies of the general population and in overweight / obese individuals undergoing behavioral weight loss treatment . However, the present study is the first to show a relationship between greater physical activity variety and higher objectively measured mvpa duration and energy expenditure in two groups of individuals who have had long - term success in maintaining a normal body weight . We found that for each additional different type of moderate - to - vigorous activity performed, participants on average spent an additional 9 minutes in mvpa and expended 56 more kcal per day . Thus, for example, participants who reported engaging in 3 different activities during the previous week spent on average an additional 18 minutes in mvpa and expended 112 more calories per day compared to participants who reported engaging in only 1 activity . While our findings do not imply causation, they do warrant additional longitudinal research to examine whether incorporating variety into a physical activity routine may be an efficacious strategy to achieve higher mvpa levels for enhanced weight control . The relationship between greater physical activity variety and engagement in higher mvpa levels may be potentially explained by a number of physiological and psychological factors . For example, alternating different physical activities that involve different muscle groups and energy systems (aerobic, anaerobic) might promote greater exercise consistency by affording more time for recovery and decreasing risk of overuse injuries [13, 26]. Participating in a variety of activities may also facilitate greater exercise adherence via increased enjoyment and decreased boredom [13, 26]. Greater access to a variety of activities may increase the likelihood that individuals will find an exercise activity or a combination of exercise activities that they like and will perform regularly . Additionally, research based on the behavioral economics model suggests that motivation to exercise is enhanced when individuals can choose from a variety of physical activities versus only one physical activity [28, 29]. Consequently, adding a variety component to a physical activity prescription may aid individuals in achieving and maintaining high levels of physical activity . Future research is needed to investigate potential mechanisms that underlie the relationship between variety and higher mvpa levels . Whereas increased variety of healthy physical activities is associated with greater time spent in mvpa, it is also possible that decreased variety of unhealthy sedentary activities might contribute to lesser time spent being sedentary and higher overall physical activity levels . Given that sedentary behaviors, independent of physical activity, have shown to be detrimentally associated with bmi and other cardiometabolic risk factors [30, 31], future studies that examine the association between variety and sedentary behaviors are needed . The cross - sectional nature of our study does not allow us to determine whether greater variety contributes to higher mvpa levels or alternatively whether individuals with higher mvpa levels naturally incorporate more variety into their physical activity routine . As variety in physical activity is rarely measured, the importance of factors such as the time frame for assessing variety (i.e., a week, month, year) and frequency of occurrence of different activities within the time frame (i.e., once a week, twice a month) are not known . While the use of an objective measure of physical activity is a strength of this investigation, it is important to note that hip - worn accelerometers like the rt3 used in this study may be limited in their ability to accurately estimate the intensity of activities not performed on flat surfaces . Thus, it is possible that mvpa was underestimated in individuals who more frequently engaged in activities that involved an incline or greater upper body movement . Additionally, the accelerometer count threshold we used to identify mvpa was determined in a leaner, younger male sample and thus may have affected validity in our older, largely female sample . Given the homogeneity of our sample with respect to gender (female) and race (white non - hispanic), our results may not be generalizable to men or other ethnic populations . Similarly, participants in this study were all normal weight and had high physical activity levels on average . Thus, future investigations should examine the potential importance of variety for increasing physical activity in overweight and inactive populations . Finally, it is important to note that just as altering the variety of different types of activities performed could potentially increase mvpa duration and energy expenditure, so could altering the frequency, intensity, and duration of a single activity, independent of changes in physical activity variety . In summary, this study examined the relationship between physical activity variety and objectively measured mvpa levels in weight loss maintainers and normal - weight individuals who both had long - term success in maintaining their body weight . In both groups, physical activity variety was related to greater engagement in mvpa and likelihood of accumulating 250 mvpa minutes / week, consistent with physical activity guidelines for optimal long - term weight maintenance . Future studies are needed to test whether incorporating variety or different types of activities (e.g., walking and cycling) can facilitate engagement in higher levels of mvpa within interventions aimed at promoting and maintaining physical activity.
Recently we reported on an analysis of the infrared spectrum of c2f4 . By observation of many combination and difference bands, including many from two c - containing isotopologues, and by comparison with mp2 and density - functional calculations, all 12 fundamentals could be assigned, although only five are ir active . The geometry was deduced from ro - vibrational analysis of strong ir active bands and by comparison with theoretical calculations . However, it is also instructive to correlate anharmonic constants with some peculiarities of the reactions of c2f4 . Accordingly, in this work more anharmonic constants (altogether 54) are derived from the spectra, and a complete set (78) is calculated . The earlier vibrational assignments of c2f4 were reported at a time when the unusual properties of this molecule were barely known . Explained this property by positing that the electron attraction of fluorine leads to a preference of sp- over sp - type carbon: radical addition converts both c atoms from sp to sp hybridization, which stabilizes the primary product, lowers the transition state, and thus accelerates the reaction . Alder reactions, for example, which was again explained by stabilization of radical intermediates . (borden also presented an alternative but related description based on delocalization of unpaired radicalic electrons to accepting * orbitals of cf groups .) The preference for carbon sp hybridization can also be expected to lower the force constants for the pyramidalization (wagging) vibrations (7, 8) and/or increase their anharmonicity . Furthermore, torsion (4) should be facilitated by rehybridization and in fact quantum chemistry confirmed this for a 90 twist . Another spectacular property of c2f4 is how readily it can dissociate thermally to two cf2 molecules . A c = c bond energy of only 2.95 ev is derived, less than that of a typical c c single bond (4 ev). The low dissociation energy is caused by stabilization of the resulting two difluorocarbenes by back - bonding from nonbonding fluorine electrons to the empty carbon p - orbital . This also explains why cf2 has a singlet ground state which is more stable than the triplet (by 2.458 ev, taken from the band origin of the phosphorescence spectrum). Whereas in the previous work, only cc stretching is considered as the dissociation coordinate, it was argued in ref (16) that pyramidalization must also be involved . This idea is supported in the present work by the large value found for the anharmonic constant x18 coupling cc stretch with trans pyramidalization . The magnitude of x18 as compared to the much smaller x17 (coupling cc stretch with cis pyramidalization) also supports a valence - bond analysis of weak double bonds, which was reviewed in ref (19). The samples in natural isotopic abundance were either taken from commercial c2f4 (hoechst), that contained a trace of the cyclic dimer, or prepared from c2f4br2 + zn in a high - boiling alcohol (diethylene glycol); the comparison allowed for recognizing absorptions of impurities . A sample enriched in c to 50% was prepared from chclf2 by co2 laser isotope separation with enriched cf2 as the primary product . Ir spectra of c2f4 in natural isotopic abundance and isotopically enriched were recorded between 370 and 4000 cm with 0.2 cm resolution by a perkinelmer ftir spectrometer in garching . At the synchrotron lab, the isotopically enriched sample was measured at moderate 0.1 cm resolution in the far ir region from 100 to 600 cm (capturing the band at 210 cm), and at high resolution for the ir active cf stretch bands, see ref (1). The present work focuses on anharmonic constants of c2f4 . Only where the constants were expected to be different for ccf4 and c2f4 (e.g., in the case of fermi resonance) theoretical values of the anharmonic constants xij were obtained from anharmonic vibrational frequency calculations performed using gaussian 03 revision c02 at the mp2 level using dunning - type correlation - consistent (cc - pvtz) orbitals . The molecular force field at this level of theory was shown in the previous study to reproduce the fundamental wavenumbers and the isotopic shifts of c2f4 very satisfactorily (for details, see ref (1)), and hence was an appropriate choice for computing xij values . The calculations were performed for each of the c2f4, ccf4, and c2f4 isotopologues . The spectra were measured previously and the fundamentals extracted from them, using a limited number of anharmonic constants (from combination and hot bands) where necessary . For convenience table 1 lists the fundamentals again . In this work we derived a large set of experimental anharmonic constants from combination bands and in particular for the lower - wavenumber vibrations from hot bands, and report a complete set of theoretically computed values . Symmetry types refer to a d2h molecule with z axis along the cc bond and the y axis perpendicular to the plane, as used in ref (1) for easier comparison with the c2v symmetric ccf4; the more conventional designation with x axis along the cc bond and z axis perpendicular to the plane is given in parentheses . Revised value (instead of 210 cm), based on a more definite assignment of a hot - band structure and x10,10, see supporting information . Band origins from high - resolution spectra; low - resolution values are 1338.4 and 1187.0 . Upright: experimental values . In parentheses: values calculated by mp2/pvtz . Where two values or a range are given for the experimental data, it may indicate (a) a dependence of the effective xij on the involved levels due to perturbations (fermi resonances, section 4.4) or (b) an uncertainty due to error limits (e.g., where broad bands are involved) or an uncertain assignment . A preferred value is indicated by bold face . For further details on the derivation of each xij and some isotopic data, the anharmonic constants xij were calculated from observed combination and difference bands by assuming that anharmonic shifts are additive: for binary combination bands (i j) and for overtones we used1and2with obvious extensions for more complicated combinations . (the xij values are mostly negative, as usual .) The left - hand side of these equations should be read as wavenumber of (name). For hot bands (satellite transitions near i that originate from a thermally populated lower level j) we used3again with obvious extensions . One can often observe sequences of equidistant q branches, corresponding to hot bands starting from higher levels mj, or combination of such sequences (from mj + nk). Equation 3 implies that the distance of a (first) hot band from the fundamental directly indicates the corresponding anharmonic constant . Combination bands allow for a direct assignment, whereas hot bands must be identified by their intensity (controlled by the boltzmann factor). In a number of cases the supporting information (si) compiles the sources for each xij in table s2, whereas table s1 lists the observed bands (with hot bands) and their isotopic shifts . Two examples of hot bands, whose shifts are dominated by the large x18 anharmonicity, are shown in figure 1 . Absorbance spectra demonstrating the large x18 derived shifts for hot - bands originating in 8 . The spectra were recorded in the garching laboratory, with pressure and path length of 2 bar and 10 cm (for the lower trace of each panel), and 140 mbar and 4 m (upper trace), respectively . For levels involving more than two quanta of vibration, anharmonic shifts this helped to decide between different assignments of combination bands in a few cases where the harmonic sums were similar to each other . However, such additivity need not apply when the levels involved are subject to fermi resonance, producing shifts that depend on the separation of nearby interacting levels . We use eqs 13 to obtain the experimental values, and hence, table 2 reports phenomenological xij, some of which show a spread in the values suggestive of fermi resonances (in particular x18, x26, and x77). Fermi resonances can also be recognized if the apparent xij or the hot - band structure depends on the isotopologue . For c2f4, fermi resonances were identified (see section 4.4 and si) between:5 and 2+6 (discussed already in ref (1)),1 and 5+6 (indicated by the hot - band structure of 1-combinations, which are different in the mixed isotopologue),2 and 27 (causing irregularities in overtone levels of 7, also contributing to the large magnitude of x27 and x77).2 and 23, identified from the mp2 calculations . 5 and 2+6 (discussed already in ref (1)), 1 and 5+6 (indicated by the hot - band structure of 1-combinations, which are different in the mixed isotopologue), 2 and 27 (causing irregularities in overtone levels of 7, also contributing to the large magnitude of x27 and x77). 2 and 23, identified from the mp2 calculations . The mp2/cc - pvtz calculations reproduced the observed wavenumbers and isotopic shifts very well . Supporting the overall vibrational analysis exceptions are 15 (x22, x28, x29, x2,10, x2,11, x36, x3,10, x45, x59, x5,11, x66, x68, x77, x88, x10,11) out of 54 cases where an observed value is available for comparison . Some of these will be discussed below . As already described in the introduction, the peculiar reactions of c2f4 have to do with cc stretching and carbon pyramidalization . The discussion will focus on these coordinates (sections 4.14.3). For a planar geometry, cc dissociation correlates c2h4 and c2f4 with a pair of carbenes in their lowest triplet states (figure 2a). Because the energy of forming these products is practically the same for the two molecules, one could also expect that the potential energy curves coincide and the cc stretch force constants are equal . In fact, already early force - field calculations showed no substantial difference (889 n / m for c2f4 and 940 furthermore, the short cc distance (132 pm, which is 1 pm shorter than in ethylene) and the high cc stretch wavenumber (1873.8 cm, compared to 1625.4 cm for ethylene) do not point to a weakened bond . As early as 1965, the low dissociation energy of c2f4 was attributed to switching over to a channel producing two singlet carbenes and to the stabilization (by 2.458 ev, see introduction) of singlet cf2 by back bonding, so that this channel is energetically favored by 4.9 ev . This path hence leads over an avoided crossing of potential surfaces (figure 2a). If the avoidance is strong, one could expect an influence at least on the anharmonicity x11 (1 is dominated by cc stretching), even if not on the force constant at the bottom of the potential energy well . Table 2 shows that the magnitude of x11 (calculated 7.8 cm) is not unusually large for such a high - frequency vibration, though noticeably larger than for ethylene (x22 = 2.3 cm). That the increase in magnitude is only moderate can be taken as a sign that the avoidance along this coordinate is not very strong . Hence, the back reaction (recombination) will have a high barrier in planar geometry . Whereas the measured recombination barrier is not, in fact, zero, it is only minor (9 kj / mol = 0.09 ev). So, a lower - energy path must lead around the barrier, out of the drawing plane of figure 2a, that is, with distortion different from cc stretch . Potentials for ground - state cc dissociation of c2f4, leading over an avoided crossing to two singlet carbenes . In (a) it is assumed that the one - sided pyramidalization (q7 + q8) begins only after overcoming the barrier, whereas (b) shows the preferred path, where this distortion is already fully developed on the barrier and is preceded by a trans pyramidalization (q8), while the cc distance is steadily increasing . It was argued in ref (16) that recombination of two cf2 molecules should be easiest if one of them points with its filled n orbital to the empty p orbital of the other . Hence, the dissociation path will change from pure cc stretch to include also pyramidalization (figure 2b), and the potential should be lowered in energy in the direction of q7 (the coordinate of 7) and/or q8 . In turn in fact x18 has an unusually large magnitude, although x17 is in the normal range (table 2). (the magnitude of x18 is not caused by secondary effects such as fermi resonance, as we shall see in section 4.4.3 .) We can conclude that on stretching the cc bond the molecule first bends toward trans pyramidalization (q8) (figure 2b). The one - sided nonplanar bending (figure 2) is a superposition of q7 and q8 . The reason why q8 is active earlier than q7 can be understood by a valence - bond model for weak double bonds . In this model several symmetric arrangements of carbenes (a c of scheme 2) were considered: whereas with two triplet carbenes all arrangements (a d) lead to bonding interaction, with two singlet carbenes bonding occurs only in the trans - bent case (c); this corresponds to a double donor one can add that the cis - bent case with singlet carbenes would lead only to antibonding (scheme 2d). Malrieu and trinquier derived a criterion for trans - bending (deriving even the angle) by comparing the standard double - bond energy with the singlet their findings were confirmed by many examples, in particular with heavier - element homologues . We can apply this rule not only to the equilibrium geometry of c2f4 but also to its dissociation path, if we consider in the comparison the double - bond energy that is reduced on extension of this bond . The rule thus predicts that on cc extension c2f4 is distorted from planar geometry to a trans - bent structure, and this rationalizes the large magnitude of x18 . In the triplet case, the interaction can be bonding in all arrangements . In the singlet case, (a) is antibonding, (b) nonbonding, (c) bonding, (d) antibonding . More recently, borisov et al . Found evidence for trans pyramidalization on extension of the cc bond also from molecular orbital calculation (at high level: mller plesset perturbation theory of second, third and fourth order): the authors found that the onset is relatively sudden on cc extension to 142 pm and that the distortion lowers the energy appreciably . (the large x18 anharmonicity is an indication that this pyramidalization already begins not far from the s0 minimum . In fact, a cc stretch by 10 pm from 132 to 142 pm is just about the vibrational amplitude in the 1 = 1 level .) Although borisov et al . Did not find an energy minimum with such a distortion, they suggested that it plays a crucial role in the low - energy real intermediate found by buravtsev, kolbanovskii et al . In thermal reactions of c2f4 and recombination of cf2 (see ref (31) and literature quoted therein and section 4.3). One could suppose that in order to lower the activation energy for dissociation the only necessary deviation from simple cc stretching is distortion toward the 8 coordinate (q8). However, there is experimental evidence that the asymmetric pyramidalization (corresponding to superposition of q7 and q8 as in figure 2b) is already crucial in the transition state: the pre - exponential factor found in the rate of thermal c2f4 dissociation (2.8 10 s) is clearly larger than would be typical for a simple bond splitting (10 s). This corresponds to a raised entropy of activation, such as that which happens if an additional degree of freedom is activated in the transition state . The most plausible candidate is free internal rotation, as it can be expected in the asymmetric structure with a single dative bond but not in symmetric geometry with double donor acceptor bond . This picture is also consistent with a stepwise cleavage of the double donator acceptor bond on c2f4 dissociation and its stepwise formation on cf2 recombination . It is worth noting that the molecule with one - sided pyramidalization has an electron distribution (figure 2b) that is zwitterionic and correlates in planar geometry with the 2ag state with two electrons excited to *, as already pointed out in ref (16). A fully analogous dissociation path can also be expected for the other systems considered by malrieu and trinquier . In fact, according to ref (32) ground - state (ir induced) dissociation of diazomethane (h2cnn) follows a path with a pyramidalized methylene group to produce n2 + singlet ch2; the out - of - plane deformation was experimentally confirmed . In the backward reaction, n2 points with its n electron pair to the empty p orbital of the singlet methylene (see refs (32 and 33) for a calculated potential in two dimensions, which would look similar for c2f4). Hence, the out - of - plane deformation on dissociation of c2f4 is not an isolated phenomenon . Applying their energetic criterion to the equilibrium geometry of c2f4, trinquier and malrieu found that this molecule is not far from the limit, where it would become nonplanar, and suggested that the 8 force constant may be significantly lower than that of ethylene . This is in fact confirmed in the following way: analytical expressions for a valence force field model for planar x2y4 were given by herzberg . Besides the masses mx and my, bond lengths l1 (for xx) and l2 (xy) and the angle (half of yxy), both 7 and 8 only depend on the force constant for pyramidalization (k/l2). With equilibrium distances and angles from ref (1) and i = (2ci) one derives this force constant from 7 and 8 (with identical results for c2f4 and c2f4):4both values should be identical in this valence - force field model . In fact for ethylene, the value is 22.9 n / m, whether derived from 7 or 8 . The pyramidalization force constant derived from 7 of c2f4 is slightly higher than in ethylene (by 17%), but the same quantity derived from 8 is only 49% of the ethylene value . (of course, these values are not quantitatively meaningful, as the discrepancy highlights that a pure valence - force field model is not sufficient for wagging of c2f4 .) So in fact the restoring force for the 8 vibration is significantly smaller than in ethylene and than that expected on the basis of the same pyramidalization force constant as for 7 . This is consistent with the prediction of the valence - bond model favoring the trans - bent geometry . The slightly raised force constant for 7 could be due to repulsive nonbonding interaction in cis pyramidalization . Such a geometry would be involved in the hypothetical transition state of the diels alder addition of c2f4 to butadiene, a reaction that has not been observed . It was argued that an increased energy associated with this distortion might contribute to inhibiting the reaction . However, an increase in the force constant of only 17% is hardly sufficient to support this explanation, and the other effects discussed in ref (7), in particular the competing fast radical reactions, are probably more important . Applying again the force - field formulas of herzberg, the force constant for torsion, k/l2, is found to be 30.3 n / m for both ethylene and tetrafluoroethylene . That is, near the potential minimum the c = c bond in c2f4 behaves just like a normal double bond . Consequently, the torsional barrier at a 90 twist (which is a measure of the bond strength) would be identical for both molecules (2.8 ev) if the two c cf2 groups remained planar; this was confirmed by a high - level calculation . However, pyramidalization lowers this barrier by 0.57 ev (at mp2 level, consistent with ref (35)). If this stabilization is activated early along q4, it could give rise to substantially negative values for x44 and especially for x47 and x48 . However, x44 and x47 are actually slightly positive (0.8 and 0.3 cm) and x48 has no unusual magnitude (0.7 to 1.05 cm). Evidently, the stabilizing effect of pyramidalization becomes effective only at high torsional angles . The corresponding values for ethylene are 2.4, 8.9, and 7.2 cm, respectively . The nondiagonal anharmonicities xi4 and x4j are generally smaller in magnitude than in ethylene . But this may simply have to do with the relative vibrational amplitudes, which are much larger for torsion of ch2 than of cf2 due to the relative masses . In the twisted - pyramidalized structure of c2f4, the triplet (t1) is only 0.013 ev lower than the singlet (s0). It can therefore act as a real intermediate and undergo radical - type reactions, further assisted by its low energy that is calculated at 2.1 ev . This has been a widespread interpretation of some peculiarities of c2f4 reactions (see, for example ref (7)). However, buravtsev, kolbanovskii et al . Found in shock - tube experiments (with c2f4 diluted by ar and adiabatically compressed) an intermediate at much lower energy (0.8 to 1 ev) with uv and visible absorption (235 and 500 nm) (see refs (31,36, and 37) and the literature quoted there). They supposed it to be a singlet diradicaloid of c2f4 with a geometry corresponding not to an energy minimum but to a point on a slope of the potential (see, for example ref (31)). However, as these points are not stationary there are too many (nearly) isoenergetic locations with shifted electronic transitions, so that the electronic absorption would be smeared out . It was therefore suggested that the observed intermediate is another low - energy isomer, tetrafluoro - ethylidene cf3cf (electronic absorption in an ar matrix:). However, its energy (1.9 ev) seems to be nearly as high as that of the twisted - pyramidalized triplet . (in one study the twisted - pyramidalized c2f4 triplet is calculated at 0.90 ev, but computational details are not given, and previous theoretical results are not discussed .) Some more anharmonicities of table 1 have unusually large magnitudes or show a range of values and/or deviate from prediction . In many of them fermi resonances play a role, as will be explained here . In section4.4.3, however, it is shown that x18 is hardly influenced by such effects and its large magnitude mainly results from the overall shape of the potential energy surface . Fermi resonances mix two nearby levels of the same symmetry, which differ by three units in the quantum numbers . The fermi - induced repulsion of the levels depends in a nonlinear way on both the magnitude of the fermi interaction parameter and on the original (i.e., unperturbed) energetic distance of the levels . This separation will, in general, change (a) on isotopic substitution and (b) on adding a quantum of another vibration (i.e., in analogous combination bands; similarly also in higher overtones, in which case a quantum number dependence must also be taken into account). Another method of characterizing fermi resonance interactions relies on intensity borrowing caused by the mixing of levels . According to the mp2 calculations, the great majority of xij change by 1 cm on isotopic substitution the few exceptions are listed in table 3 and are compared to experimental values . Identified (fr13) and the interpretation in the line influenced by is based on the assumption that an xij is affected by such a resonance if at least one of the levels i and j is involved in the fermi interaction . The table shows that the calculated isotopic shift is sometimes much larger than in the experiment, for instance x26, x27 . Also x56 for c2f4 is probably too large, as the two observed bands 5+6+11 and 5+6+12 are close to their harmonic positions (table s1 in si). It seems that at the present level of theory, the calculation does not reliably reproduce the fermi resonance induced shifts, at least the stronger ones . This is not surprising given the highly sensitive and nonlinear dependence of fermi shift on the unperturbed level separations . The same deficiency will also affect those xij connected with at least one level (or a combination thereof) involved in a resonance listed above (examples in figure 3a). In fact, 10 of the 15 deviations listed at the end of section 3 can be attributed in this way to the resonances fr1 and fr2 with possible contributions of fr3 . The exceptions are x36, x3,10, x66, x68, and x88 . In all 42 other cases it is worthwhile to consider the two strongest fermi resonances, fr1 (2/27) and fr2 (5/2+6), in c2f4 in some detail . The effect of these resonances is summarized in figure 3 . Some levels involved in fermi resonances and observed transitions . Solid arrows: ir, broken arrows: raman; double arrows (with interaction matrix element) indicate repulsion between level pairs . The anharmonic shifts involving x77 are only nominal, because the fermi perturbation is strong: 6x77 = 18.1 is less than 3 times (= 8.2) and 4x77 is only 9.9 . (but 2x77 + 4x77 = 6x77, as is obvious from the level scheme, and the raman anharmonic shifts practically coincide with those from the ir spectra .) (b) three - level fermi resonance 5/2+6/27+6 and their combinations with 9 for c2f4 and c2f4 . The indicated level energies are from the spectra except those in parentheses, which are calculated . The broken horizontal lines are the estimated unperturbed positions (see the si), and the signed numbers are the calculated shifts . The 16.4 cm magnitude of the interaction term (with its expected dependence on quantum numbers) has been derived from band shifts and intensities for a series of levels built on 2/27 for all three isotopologues . Bands for 27+x (x = 9, 11) borrow their entire intensity from 2+x . The detailed analysis is provided in si . It is also evident that x77, x27 and x22 are affected by the fermi resonance (and probably other x2i, if the level separations differ) and that x77 is level dependent because of the varying separation . The fermi term is almost entirely responsible for the + 8.2 cm shift of 27 for c2f4 and those of the heavier isotopologues (table 3 under fr1). In the latter the fermi - induced shift is increased, because the levels 2 and 27 (before perturbation) are closer together than in c2f4 and are practically degenerate in c2f4 . The success of these calculations shows that the additional mixing of 2 with 23 (fr3) is apparently a weak perturbation at most . The fermi resonance fr2 between 5 and 2+6 was qualitatively discussed in ref (1). A more complete understanding of the resonance shifts and pattern of intensity borrowing requires the three - level scheme shown in figure 3b; this figure also shows combinations with 9, as they are all ir active and were directly observed . Analysis based on this model has enabled the magnitude of the fermi term to be determined as 11 cm; the fr1 term of 16.4 cm was used without change . Significantly, the model predicts varying fr shifts that even differ in sign (originating from the different repulsions in figure 3b) and can thus account for the unusual isotopic shifts observed between c2f4 and c2f4 . It explains a substantial deviation of the observed isotopic shift 5 = 51.9 from the value of 5 = 43.1 expected from the teller redlich product rule, which is valid for harmonic oscillators . Additionally the relative intensities of combination bands and their isotopic dependence are well predicted . It is also remarkable that according to the model the largest part of the anharmonicity x26 is due to fermi resonance . As described in ref (1), the lower symmetry in ccf4 leads to strong mixing of 5 with 9 (which are nearly degenerate in c2f4). The two levels repel each other (even in the harmonic approximation) and give rise to two infrared - active transitions . It is sometimes difficult to disentangle them and their combination bands from the spectra . If the large magnitude of some anharmonicities is caused by fermi resonance, a crucial question is whether this might also be the case for x18 . If so, the conspicuous value of x18 could be largely a matter of the character of the resonance and less a consequence of global features of the potential for dissociation, which we considered in section 4.1 . In fact, weak perturbations of the 1 level and its combinations by the 1/5+6 fermi resonance are indicated by the slight x18 dependence on the level and on isotopic substitution and the change of the hot - band structure of 1 combinations in ccf4, as explained in the si . A quantitative estimate of the strength of the 1/5+6 interaction can be deduced from the intensities of 5+6 combinations relative to those of the corresponding 1 combinations, assuming that the former borrow their full strength from the latter . The 5+6+12 band (2444.3 cm) has 12% the strength of the 1+12 band (2428.7 cm), see figure 1a . Similarly, the 5+6+11 shoulder (3072 cm) is <1% as intense as 1+11 (3056.2 cm) and clearly visible only with longer path length than shown in figure 1b . From both observations, one can estimate a fermi term (w) of no more than 2.5 cm, causing a shift (and consequent change in x18) of <0.5 cm . This is actually an upper bound, because such weak bands can also be expected without intensity borrowing . Hence, fermi resonance affects the magnitude of x18 only by a little (by <3%), and we can maintain the conclusions of section 4.1 on the shape of the potential . A large set of anharmonic constants of c2f4 was derived from spectroscopic data, and a complete set was calculated . Most of the calculated and experimental values agree very well, and nearly all deviations can be attributed to fermi resonances . The values were used to interpret peculiarities of the potential and reactions of this molecule . The facile thermal cc dissociation of c2f4 is due to curve crossing of the ground state (1ag) with the two - electron excited zwitterionic 2ag state . The crossing barely alters the properties of the potential curve (cut purely as a function of cc stretch) near the equilibrium geometry; the force constant for cc stretching (1 in c2f4, 2 in c2h4) is nearly identical for the two molecules . Only a slightly larger magnitude of the anharmonicity x11 compared to the corresponding x22 of ethylene provides a hint of a weakly avoided crossing in planar geometry of c2f4 . However, the large magnitude of x18 leads us to conclude that the dissociation path bends early toward q8, i.e. To trans pyramidalization of the two carbon atoms (figure 2b). This is rationalized by a valence - bond model, which describes the stretched cc bond as a double donor acceptor bond (scheme 2c). It is also consistent with the tendency of fluorine to induce sp hybridization at carbon, although this preference alone would also allow cis pyramidalization, q7, which is not involved early in dissociation . On further cc stretching, one of the two donor acceptor bonds breaks, and the pyramidalization becomes fully one - sided already in the transition state (figure 2b). In this way, free internal rotation of the cf2 groups becomes possible, providing an additional degree of freedom that rationalizes the high pre - exponential factor in the rate constant of dissociation . This structure with perpendicular arrangement of the two cf2 groups appears very plausible for the back reaction . Thus, one cf2 group approaches with its filled n orbital the empty p orbital of the other cf2 group (figure 2b). The easy trans pyramidalization on minor cc stretching is probably also the reason that the corresponding 8 force constant is lowered, although all other force constants are similar to those in ethylene . The preference of fluorine for sp hybridization at carbon has been invoked to explain not only the easy radical addition and other reactions, but also the low - lying, twisted - pyramidalized triplet, which is nearly isoenergetic with the singlet that has a saddle point there . This is indicated by x44, x47, and x48, which have no unusual values . Very recently, high - level theoretical studies investigated electronic ground and excited states of c2f4 and their dependence on some coordinates, mainly to discover the fate of this molecule after electronic excitation and make comparisons with a corresponding time - resolved experiment (ref (16)). For the ground state, the stabilizing effect of pyramidalization in the twisted molecule was confirmed . Interestingly, the excited molecule enters the s1/s0 conical intersection from the 2ag state, where the molecule has a large one - sided pyramidalization, a 90 cc twist and a cc bond length (1.51) similar to that of a single bond . In the present work, the transition state for s0 dissociation was said to result from avoided crossing of the 2ag and 1ag (i.e., s0) states, also with large one - sided pyramidalization but at longer cc distances and perhaps without twist . It would be interesting to check whether these geometrical differences are large enough to generate an excited - state barrier, because evidence was found in ref (16) that cc dissociation does not begin in the excited state.
Cytomegalovirus (cmv) is one of the most common congenital viral infections in many regions . In the united states it occurs in 0.51% of live births, or approximately 40,000 infants annually . The manifestations of cmv infection cover a broad spectrum ranging from asymptomatic to severe systemic disease resulting in significant morbidity and mortality . 90% of infants with congenital cmv are asymptomatic at birth . Despite being asymptomatic at birth, up to 7% of these children will develop sensorineural hearing loss (snhl) that can be unilateral or bilateral, fluctuating or progressive, and range from mild to profound [2, 3]. Approximately 10% of infants with congenital cmv are symptomatic at birth, and 40% of these patients will develop snhl [1, 2]. Hearing loss is the most common manifestation of congenital cmv infection making cmv a leading cause of nonhereditary congenital hearing loss . Given the relatively large number of children potentially affected by cmv - related hearing loss and the wide range of manifestations of congenital cmv infection, it is difficult to predict how a child with symptomatic cmv will perform with a cochlear implant (ci). Previous studies suggest that children with symptomatic cmv derive auditory benefit from cis, albeit at a slower rate than other children . The degree to which these children benefit remains somewhat controversial as there is variation in the development of language skills after ci [59]. The objective of our study was to examine the relationships between intracranial radiographic abnormalities and preimplantation developmental assessment (including head circumference) with postimplant audiometric and language outcomes in children with symptomatic congenital cmv who have undergone cochlear implantation . This study was a retrospective review of children with a diagnosis of symptomatic congenital cmv who underwent cochlear implantation between 2004 and 2010 at a tertiary pediatric cochlear implant center . Congenital cmv was diagnosed for most children via urine culture of the virus during the first two weeks of life . Diagnosis was also made based on the identification of hearing loss coupled with brain image findings . Medical charts of these patients were reviewed for pre- and postimplantation evaluations and central nervous system imaging findings . Data regarding preimplantation developmental assessments included the diagnoses of developmental delay and/or other disabilities, nonverbal intelligence quotients or developmental quotients (iq / dq), and head circumference (hc) at the preoperative visit . Results from computed tomography (ct) and/or magnetic resonance imaging (mri) scans of the brain were reviewed, and the findings were classified by type and location of anomaly . Postimplant audiometric behavioral testing, which was performed using validated and developmentally appropriate testing techniques, was collected . This data included frequency - specific thresholds and speech awareness or speech reception thresholds (sat / srt). Pure tone averages (pta) were calculated by an average of 4 frequencies (0.5, 1.0, 2.0, and 4.0 khz). Children's developmental disability diagnoses were classified according to the areas of cognition, motor, vision, and language . Cognitive disability was diagnosed using nonverbal cognitive measures such as the leiter international performance scale and supported by evaluating adaptive functioning with the vineland adaptive behavior scale . These evaluations were completed by psychologists with knowledge of testing deaf / hard of hearing children . If the nonverbal iq was not available, a nonverbal developmental quotient was used . This nonverbal or adaptive quotient was obtained from a developmental pediatrician with expertise in hearing loss as a part of the precochlear implant assessment . For this study, the diagnosis of cerebral palsy (cp) was made based on neurologic examination, patterns of persistent primitive reflexes, abnormalities in tone and reflexes, and presence of abnormalities on mri of the brain . The diagnosis of pervasive developmental disorder not otherwise specified was made by interdisciplinary evaluations with speech pathology and psychology using standardized measures to evaluate autism spectrum disorders . Standardized language assessment scores, using the preschool language scale (4th edition) were available on eight subjects who returned for speech therapy after implantation . This study was approved by the institutional review board at cincinnati children's hospital medical center . Data distributions were reported using medians with ranges (for continuous data) and frequencies with percentages (for categorical data). Characteristic differences between children with hc <5th percentile and> 5th percentile were tested using wilcoxon rank sums test for continuous data and chi - square or fisher's exact test for categorical data . Biserial correlations were conducted to understand the association between presence and location of brain abnormalities with audiometric and language outcomes . The wilcoxon rank sums test was also used to explore statistical differences regarding factors (e.g., iq / dq) that may be associated with presence and location of brain abnormalities . Due to the small sample size, exact p values are reported . Table 1 lists the fifteen cmv - positive children (9 females and 6 males) who received a ci during the study timeframe . Two additional subjects with cmv were excluded from this analysis: one subject had not returned for any postimplant visits and one child had normal cns imaging (thought not to be symptomatic cmv). The median age at time of identification of hearing loss was 5 months (range 162 months), and the median age at implantation was 28 months (range 1296 months) (table 2). The median iq / dq for the study population was 65 (range 2390); 9 (60%) children had an iq / dq <70 . Seventy - three percent (n = 11) of children had a diagnosis of cognitive impairment and 13 children (87%) had a motor delay or disability; 9 had a diagnosis of cp . Over half (60%, n = 9) of these children carried a diagnosis of more than one developmental disability and nearly half (47%, n = 7) had both a cognitive disability, and cp . Seven children (47%) had a head circumference (hc) less than the fifth percentile (table 2). Children with a hc <5th percentile had a significantly lower median iq / dq than children with a hc 5th percentile (42 versus 77, p = 0.003). All children with hc <5th percentile had cognitive disabilities compared to only 50% of children with a hc 5th percentile . Additionally, the children with a hc <5th percentile had slightly poorer postimplant pta (38 db versus 27 db, p = 0.02) and sat / srt (30 db versus 23 db, p = 0.04) compared to children with larger heads . All children included in the study had central nervous system abnormalities present on imaging studies (table 3). Twelve children had mri imaging (all with identified abnormalities) and 13 ct imaging of the brain with identified abnormalities . Eighty percent (n = 12) had calcifications, which occurred most frequently (53%) in the periventricular region (table 4). Additional imaging findings included ventriculomegaly in 53%, migrational abnormalities in 33% (n = 5), and encephalomalacia in 27% . All four children with encephalomalacia and all 3 children with cerebellar abnormalities had a diagnosis of cp . In addition to cp, 3 of the 4 children with encephalomalacia and all 3 children with cerebellar abnormalities also had a cognitive disability diagnosis . In fact, the children with cerebellar abnormalities had a lower nonverbal iq / dq than children without cerebellar abnormalities (35 (range 2350) versus 68 (range 2790), p = 0.048). Calcifications in the temporal lobes were associated with poorer post - ci pta (biserial correlation rb = 0.57, p = 0.04). The median (range) receptive language score was 21 (327), and expressive language score was 28 (1777). The iq / dq was highly correlated with both receptive (spearman rho = 0.9, p = 0.002) and expressive (spearman rho = 0.86, p = 0.007), as having a head circumference <5th percentile (point biserial correlation rb = 0.75 and 0.68 resp . ). Periventricular calcifications were associated with lower receptive language (rb = 0.75, p = 0.03) and expressive language (rb = 0.84, p = 0.008) among children with post - ci assessments . Because the iq / dq was associated with both periventricular calcifications (rb = 0.53, p = 0.04) and hc <5th percentile (rb = 0.73, p = 0.002), the relationship between these factors and language appears to be, at least in part, driven by iq / dq scores . One of the challenges in children with congenital cmv is predicting the sequelae they will develop and the extent of neurological and developmental deficits they will incur . Previous studies have shown that brain imaging may be a good predictor of adverse neurodevelopmental outcomes . In one study of children with symptomatic congenital cmv, 90% of the children with an abnormal ct scan developed at least one sequela compared to 29% of those with a normal scan . Greater than two - thirds of all children with congenital cmv had mri abnormalities and, of these, cortical malformations, ventriculomegaly and hippocampal dysplasia correlated highly with poor neurologic outcome . Found that microcephaly was the most specific predictor, and abnormality on head ct was the most sensitive predictor of poor cognitive outcomes in this patient population . In a study of children with a diagnosis of snhl, 80% of the cmv positive children had abnormal brain mri scans compared with only 33% of cmv negative children . Children with calcifications had a slightly poorer median post - ci pta compared to children without calcifications . Interestingly, the location of the abnormalities also seemed to correlate with worse language outcomes . The parietal lobe processes sensory information and houses our language abilities, and the temporal lobe regulates emotion, hearing, language, and learning, which could explain why language outcomes are poorer in children with abnormalities in these regions . Children with cerebellar anomalies also had lower receptive and expressive language levels than the other children . Although the cerebellum has been largely thought to control balance, there may be other aspects of learning and development which depend on this entry point of sensory information to the higher cortical areas . The authors have implicated the cerebellum to be involved in temporal processing, language production and comprehension, spatial reasoning, and visual attention [1821]. In our 3 patients with cerebellar lesions, it is not clear if it is the location of the lesions in the cerebellum or the severe cognitive delay that resulted in worse outcomes . With only 3 patients, it is not possible to draw additional conclusions regarding cause and effect in this group . In addition to looking at the associations between cns imaging findings and post - ci outcomes, our study also looked for correlations between preimplantation developmental assessment and cochlear implant performance in these patients . Although there is a relative paucity of relevant studies in the literature, previous studies have demonstrated that children with cognitive delay seem to derive benefit from ci, albeit at a slower rate than nondelayed children [22, 23]. Although children with cochlear implants who have more significant developmental delay have poorer outcomes than children with only mild delay, the biggest predictor of language development among children with implants and disabilities is a measure of nonverbal cognitive ability . Unfortunately, when compared to appropriately matched developmentally delayed children without hearing impairment, children with cis had lower receptive and expressive language skills, which were not commensurate with their cognitive potential . Although children with multiple handicaps may progress slower and to a lesser extent than typically developing children, these children still derive benefit from the auditory stimulation provided by a ci but may require additional post - ci intervention to optimize benefit [2729]. As we found in our study, the majority of children with symptomatic congenital cmv have associated developmental disabilities . The variability of neurocognitive deficits in these children warrants the examination of this group separately from other children with developmental disabilities . As with children with developmental disabilities, children with symptomatic congenital cmv appear to derive benefit from ci albeit at a slower rate . Ramirez inscoe and nikolopoulos demonstrated mixed results for speech perception and intelligibility with 50% of children with congenital cmv performing more poorly than controls, 31% performing similarly, and 19% performing better than controls . These children did, however, derive auditory benefit from ci . In their study of 13 children with symptomatic congenital cmv, 73% of implanted children achieved closed - set word recognition, and 63% achieved open - set word recognition . Children with congenital cmv have also demonstrated both improved pure - tone hearing thresholds and improved language perception and production as well as useful speech comprehension albeit lower than matched controls . Another study of 11 children with congenital cmv found that 9 of the 11 carried a diagnosis of psycho - neurological disorders (including attention deficit hyperactivity disorder, mental retardation, autism, and pervasive developmental disorder). Although hearing thresholds were similar among children in this group, post - ci performance varied widely depending upon the concomitant diagnosis . Our findings were consistent with these previous studies in that our patients did demonstrate overall improvement in post - ci thresholds . Our results indicate that head size was significantly related to iq / dq and that children with microcephaly (hc <5th percentile) were more likely to have cognitive disabilities than those without microcephaly . The microcephalic children also had significantly poorer post - ci pta and sat / srt as well as worse receptive language . These findings indicate that microcephalic children do not achieve the same degree of auditory benefit as the children with normal head size . The mean age at last audiometric followup did vary between the microcephalic and nonmicrocephalic groups (46 versus 59 months). This difference would not be expected to affect the results of audiometric testing as all testing was performed using developmentally appropriate behavioral methods that should account for the difference in age . Given that the majority of the children (73%) had developmental delay and nearly half were considered multiply handicapped, it is notable that head circumference was a better predictor of post - ci outcome than specific developmental delay diagnosis . This is not entirely surprising as head circumference relates to brain growth, which can impact developmental outcomes . Although our study has limitations including its retrospective nature and small sample size, it provides data that may further efforts to identify factors which may help predict which children with congenital symptomatic cmv will benefit from ci . The presence of cerebellar anomalies or greater than one cns abnormality on imaging correlates with poorer outcomes after ci . The location of cns abnormalities, including calcifications, may play a role in audiometric and language outcomes after ci . Early measurements such as brain imaging findings, head circumference, and iq / dq may allow for more accurate counseling of families regarding anticipated postimplantation performance in children with symptomatic congenital cmv.
. There have been significant achievements regarding to the millennium development goals (mdgs) as a global development target . The indicators showed reflection of these achievements, such as decreasing global poverty rate, increasing the number of children going to school, decreasing child death rate, increasing access to clean water, and also increasing malaria, hiv / aids, and tuberculosis control investment . Mdgs were evaluated in the year 2015, and there will be remaining new challenges to sustain the achievement at post-2015 era . Numerous important issues have been raised by united nation (un), which are poverty and hunger elimination, improving health and education, sustainable cities, combating climate changes, and also ocean and forest conservation . In mdgs, hiv / aids, malaria, and major diseases are clearly mentioned as global development goals . In this case, there will be plenty of diseases that are not a major concern of the world . Questions were raised concerning neglected tropical disease (ntd) which is definitely out of the highlight . Will these diseases be included in the ambitious post - mdgs sustainable development? The new sustainable development goals (sdgs), known also as the global goals, had been established after world leaders gathered on 25 september 2015, at the united nations in new york to adopt the 2030 agenda for sustainable development and the broader and further agenda than the mdgs . Mdg explicitly stated that hiv / aids, malaria, and tuberculosis will be efficiently controlled at the end of the year 2015 . The big three are the diseases that attract priority from sponsors, researchers, and policy makers [2, 3]. This situation is not the same as ntd, which has not gained any priority from them and even worsened . The term of neglected in ntd referred to the fact that these tropical diseases are not being considered as important diseases . Ntd result in long life's deformities and handicaps, decrease productivity and economical status, and also end up many social consequences and stigmatization . Though ntd is commonly found in tropical countries, it is not identical with tropical diseases . Poverty as one of ntd determinants frequently occurs in rural area, slums, and marginalized populations living nearby the equator . Ntd in this area are closely associated with poverty and limited resources, such as clean drinking water access, poor sanitation, and healthy housing . Ntd may decrease child health, increase the health expenses and risk of ineffective treatment, decrease productivity, and result in education default . Their potential to spread in developed countries is low, because ntd are closely related with local vector and intermediate host distribution which are specifically associated with geographic region . The technology and resource to control, prevent, and eliminate ntd are available, but not for the developing countries . Who has included 17 diseases caused by bacteria, virus, and protozoa into ntd (figure 1). Helminths caused diseases that dominated ntd: taeniasis, dracunculiasis, echinococcosis, trematodiasis, filariasis, onchocerciasis, schistosomiasis, and soil - transmitted helminthiasis . The ministry of health of indonesia reported only five ntd that can be found in indonesia, that is, leprosy, filariasis, schistosomiasis, soil - transmitted helminth, and yaws . However, this review describes more than those five diseases and aims to address the magnitude of the problem of ntd in indonesia . Leprosy is an infectious disease is caused by mycobacterium leprae, a road shaped bacteria classified in the same genus as mycobacterium tuberculosis leprosy may be complicated with deformity and handicap, which may decrease the ability of the patient to work in daily life . Leprosy was a serious problem in indonesia at 1980, with 126,221 cases in 1985 . It was noted that the significant improvement in leprosy control was because of massive promotion of leprosy prevention and multidrug therapy (mdt) intervention in more than 5,600 primary health centers in indonesia . However, the new leprosy case finding in 2000 was not significantly different with year 2013 (table 1). Fourteen provinces and 160 districts, which mostly are located in java island, reported prevalence of> 1 per 10,000 population in 2009 . Leprosy epidemical indicators were significant, such as deformity grade 2 10.5%, leprosy cases in children 12.01%, and 82.43% multibacillary (mb) cases [10, 1315]. The problems of leprosy in indonesia are summarized as follows: early detection.development of more effective treatment.development of more effective vaccine.understanding immunopathogenesis of peripheral nerve damage.management of chronic erythema nodosum leprosum (enl).deformity and stigmatization.deformity and stigmatization are serious problems among leprosy patients in indonesia . Survey conducted in subang district (west java); gresik and malang district (east java); bone and gowa district (south sulawesi) showed that deformity in leprosy patients occurred in 76.7% of patients . Almost 60% respondents were experienced handicaps during their life, including participating to social activity . Mdt has significant impact to decrease leprosy prevalence globally . However, there are various substantial operational and technical problems among countries and territory . Resistance of m. leprae is a serious problem because of limited effective antimicrobes for leprosy . Resistance against dapsone, that was applied as monotherapy, was initially reported in 1964 at malaysia . Since then, resistance to dapsone and other drugs was reported . Theoretically, combination of more than two drugs with different action mechanism will reduce the probability of resistant in mycobacteria . National surveillance of m. leprae resistance against mdt should be established to provide accurate data . Sporadic and partial reports were not sufficient to measure the problems and establish the programs to reduce the prevalence and spread of resistance against mdt . Phenotypic testing is the basic method for susceptibility testing of antimicrobial agents . Even in the new proof of concept which combines the classical growth - based phenotypic test and dna based approach, pure culture of bacteria efforts were documented to culture m. leprae in artificial media and animal models [24, 25]. Mutations in the folp1, rpob, and gyra genes are responsible for resistance to dapsone, rifampicin, and ofloxacin, respectively . To test the susceptibility of m. leprae against particular antibiotics, screening in north maluku and north sulawesi showed that resistance to dapsone occurs as 0.8% in new cases and 10% in relapse cases, while rifampicin occurs with 3.3% in new cases and 20% in relapse cases . There is higher prevalence of dapsone and rifampicin resistance among relapse cases comparing with new cases . It was noted that the incidence of resistance against dapsone and rifampicin in indonesia almost did not change between the time monotherapy was introduced and after mdt was recommended by who [29, 30]. Yaws is a treponema pallidum subspecies pertenue infection, which may develop as chronic and recurrent disease . Yaws is nonsexually transmitted treponemal infection, which is latent and asymptomatic for years with positive serology result . Small portion (10%) of the patients will undergo bone destruction leading to deformities . Diagnosis of yaws is commonly based on the clinical findings and serology [31, 32]. Confirmatory diagnosis of yaws is using serological test, which includes nontreponemal agglutination tests, such as rapid plasma reagin (rpr) and venereal disease research laboratory (vdrl) test, and also treponemal tests such as treponema pallidum hemagglutination assay (tpha), treponema pallidum particle agglutination tppa, and fluorescent treponemal antibody - absorption (fta - abs). The accuracy of yaws serological test is hampered in the area which syphilis, caused by t. pallidum subspecies pallidum, is endemically circulated . Yaws was reported in 68 (14%) of 497 districts in indonesia, mostly in the eastern part of indonesia . East nusa tenggara province reported 2,800 cases in 2012, which is scattered at 566 small islands in this region . However, it gradually decreases to 5,319 cases in 2011 and 3,476 in 2012 [10, 34]. There are several obstacles to eradicate yaws in indonesia: geographically the yaws patients are in a remote area including rural and small islands which are not easy to handle, difficulty to give benzathine penicillin for children, and also local political condition that is somehow counterproductive to the programs . Who planned to eradicate yaws globally in 2020 with adoption of morges strategies . This program intends to introduce a mass treatment in the endemic regions by using azithromycin . However, benzathine penicillin can be used as back up in case of azithromycin is not indicated, treatment failure, or in places where azithromycin is not available . This treatment followed an active survey every six months to detect and treat the remaining cases [32, 34]. The ministry of health launched yaws eradication program, aimed to eradicate yaws in 2013, one year later than who target . There were several approaches conducted, such as active screening new cases and contacts, empowering community, improving the capacity of health worker to diagnose and manage yaws, and intersectoral collaboration approaches . Access to health service is limited in the yaws endemic area, which obstacle the passive screening program . Reported a survey among health workers in primary health center, elementary school teachers, and parents in muna, southeast sulawesi . They were asked about yaws to measure their knowledge about sign and symptoms, causative agent, and therapy for yaws . Another report from southwest sumba showed clean water and healthy behavior is the risk factor for yaws . These reports showed that comprehensive intervention is needed to boost the community participation for yaws eradication . Reporting system for yaws incidence is very important to support the best intervention for yaws eradication . Learning the fact that their knowledge about yaws is remarkably low, it may diminish the validity of yaws reporting system . Insufficiency of this sector may result in delay of yaws eradication and increasing yaws contact population . Yaws eradication problem in indonesia is summarized as follows: early detection by health workers.geographical problem of endemic area.surveillances and reporting system.inadequate confirmatory laboratory test . Dengue virus consists of four serotypes: denv1, denv2, denv3, and denv4 . These viruses are responsible for infectious diseases with width spectrum of clinical manifestation: dengue fever (df), dengue hemorrhagic fever (dhf), and dengue shock syndrome (dss) [38, 39]. Dengue virus infection is an arbovirus disease, which in indonesia is primarily transmitted by aedes aegypti and aedes albopictus . The incidence of dhf significantly increased since reported initially in 1968 (0.05/100.000) to 3540/100,000 in 2013 . However, in 2010, there was an extreme surge of dhf incidence to 86/100,000 . The significant increasing of dhf incidence was parallel with decreasing of case fatality rate (cfr), which is 41% in 1968 to 0.73% in 2013 . However, it was noticed that from 1999 there was a trend of shifting to adults . Dhf incidence described here is believed not to be able to draw a clear picture of all denv infections . . However, the proportion of those three clinical manifestations still becomes an enigma, although the awareness to the diseases, surveillance programs, diagnostic tools, and management of denv infection in indonesia have been significantly improved . It has been realized that controlling adult mosquitoes is prone to fail reducing denv transmission . The vector control approach should aim to interrupt the transmission cycle at an early phase through immature mosquito control, which is including larvae and pupae stages . Immature mosquitoes control is believed to be more effective . The vector control approach is still considered effective while vaccine and prophylaxis remedies continuously develop . It seems that the socioecological approach is the best candidate to be implemented in an endemic country, such as indonesia . Community and intersectoral approach are significantly important fundamentals of integrated public health strategies for dengue vector control [47, 48]. As dengue infection has wide spectrum of clinical manifestation, the clinical diagnosis is confusing . There are guidelines issued by who to respond to the need of applicable tool for clinician: who classification system and dengue and control study (denco) revised clinical management . The study concerning the two guidelines showed that in surabaya, indonesia, the denco revised clinical management guideline superior in detection of severe dengue cases . However, in a study in semarang, indonesia, the denco guideline has failed to show its superiority . It is true that the denco guideline needs to be adjusted in terms of geographic and age - related variations issues . There are many factors that may be related with clinical manifestation of dengue virus infection, including human genetics involvement which is still controversially related with population, race, and geography [54, 55]. Several methods are available for laboratory test confirmation of denv infection: virus isolation, genome detection, antigen detection, and antibody detection . The first three methods are direct detection methods which were considered as more specific and able to confirm earlier onset of denv infection . The antibody detection is widely used in the field because they are easy to perform and less expensive, though it is not as powerful as direct detection especially in secondary infection [50, 56]. Rabies is an acute, progressive, incurable viral encephalitis disease that is transmitted from animal, mainly dogs, to human . It is caused by rabies virus (rabv), a member of the family rhabdoviridae, genus lyssavirus . The highest incidence is concentrated in asia and africa . Despite the fact that rabies is 100% vaccine - preventable infectious disease, it potentially threatens over 3 billion people in the world . However, poor surveillance, underreporting, frequent misdiagnosis, and poor coordination among sectors may lead to underestimation of the burden of the disease . Ten countries of asean have been declared rabies - free asean by 2020 in the occasion of the thirty - fourth meeting of the asean ministers on agriculture and forestry (34th amaf), on september 2012 . Rabies was reported in 24 provinces out of 34 provinces in indonesia . Reported human rabies in indonesia is small (206 cases in 2010 and significantly decreased to 119 cases in 2013) compared to thousands of cases reported in other countries such as india, china, and other asian countries . To control rabies transmission, indonesian government uses one health approach which facilitates the multidisciplinary participation in management of zoonosis diseases . However, kalimantan and sulawesi, other islands of indonesia, are the most plausible hypothetical origin of the bali rabv strains . Totally there were 133 reported human deaths because of rabies in bali from 2008 to september 2011 . This significantly decrease of rabid human death within 3 years is a good model of rabies control for other regions or countries . It was proposed that this success is mainly because of mass vaccination program for dogs, which is the primary (98%) vector of rabies in bali . However, the number of reported humans bitten by dogs remained over 4,000 per month . The preparedness to human rabies should be maintained, as the threat of human rabies escalation is still high . Lymphatic filariasis (lf) is caused by worms inhabiting the lymphatics [6365]. Approximately 65% of total cases are found in the southeast asian region [6668]. Lf cases were found in all provinces of indonesia . In 2009, there were 11.914 lf cases reported . The number of total cases was steadily reported until 2013 . There were 11.912 cases registered in 2013 . Lf prevalence in indonesia varied from 0.5 to 27.6%, in which the highest rates were found in maluku, papua, west papua, east nusa tenggara, and north maluku provinces . Three lymphatic parasites are prevalently circulating in indonesia: wuchereria bancrofti, brugia malayi, and brugia timori [69, 70], which are transmitted by mosquitoes of five genera the site of adult - worm parasitism is within the lymphatic vessels, most commonly involving the extremities and male genitals [64, 65, 70]. The disease predominantly afflicts poor people in both urban and rural areas with limited resources condition, where mosquitoes as a vector might be found in high density [64, 68]. Who launched global program to eliminate lymphatic filariasis (gpelf), which relies on mass drug administration (mda) approach [68, 71]. The main goal of the program is to hamper transmission of disease between mosquitoes and human beings, mainly through mass drug distribution of diethylcarbamazine (6 mg / kg) or ivermectin (150200 g / kg) combined with albendazole (400 mg) [68, 71]. It is recommended that all people at risk are involved in this program, since patients with asymptomatic infection may have abnormal lymphatics, and that early treatment may prevent subsequent lymphatic damage [66, 69]. The efficacy of six annual rounds of mda was studied in alor island, eastern part of indonesia . Microfilaria rates of b. timori decreased significantly after mda intervention, from 26% to 0.17% . The prevalence of filarial - specific igg4 antibodies was significantly decreased from 80% to 6% . The lf national plan has implemented mda campaigns in 2002 . However, due to financial and human resource constraints, districts often provide only partial coverage of the at - risk population within the district . In 2009, the challenge of mda implementation in indonesia is not the efficacy of the drugs which were given to the community . It seems that the infrastructure is the key of the implementation, which includes the availability of transportation and physical access to the target population, reliable data bases, and competence health workers . The other challenge was to gain the trust from the community member to boost the compliance of drug administration . Currently, mda has been scaled up in a geographically scattered way to address high prevalence areas and political needs . Medical professionals, donors, universities, and ngos have played a critical role in finding new cases, assessing disease burden and supporting trainings and mda campaigns . Prevention of lf depends on mosquito vectors control, which has had limited success because development of mosquitoes resistance against insecticides . Urbanization of vast areas of tropical asia, including indonesia, has resulted in a concomitant rise in the prevalence of both w. bancrofti and b. malayi varieties of filariasis, carried by mosquitoes that breed in nonsylvatic habitats [68, 73]. Schistosomiasis is a chronic water - borne infection caused by trematodes schistosoma, mainly found in developing countries in africa, south america, the caribbean, the middle east, and asia [69, 74, 75]. In indonesia it is found in three isolated areas of central sulawesi province, namely, lindu, napu, and bada valleys . Except for schistosoma haematobium that is responsible for urinary tract disease, the human schistosomes primarily affect the intestine and liver . Clinical manifestations of the disease are recognized as fever with dysenteric symptoms and loss of appetite, as well as physical growth and cognitive delay in children [69, 77, 78]. Disease prevalence fluctuated between 0.3% and 4.8% in napu valley and between 0.8% and 3.6% in lindu valley . However, the prevalence of schistosomiasis in both areas tended to increase during the 20082011 period . The parasite transmission cycle involved domestic and wild animals as reservoir [76, 79]. In 2012, who approved the goal of eliminating schistosomiasis in endemic countries . It focused on improving sanitary conditions and large - scale distribution of the antiparasitic drug, praziquantel to high - risk target groups, such as school - age children, child bearing age women, and individuals involved in frequent contact with contaminated fresh water . Praziquantel is well - tolerated, associated with few side effects, and has a very high therapeutic index . Moreover, a single dose praziquantel administration is usually sufficient to kill all adult worms [69, 78]. Schistosomiasis control in indonesia has faced many difficulties even though the endemic areas are very limited [79, 81]. The core strategy of mda should be coupled with education to the local community, rat and snail surveillance, and support to the environmental management programs including introduction of latrines and suitable water sources . In the future, there should be emphasis to understand the social dynamics and social change related to schistosomiasis, which may provide more information about concrete issues to control transmission of schistosomiasis . Lack of intersectoral coordination and collaboration may have occurred, possibly leading to increase transmission and reinfection rates, and be prone to control failure . Soil - transmitted helminthiasis (sth) is an infection with one or more intestinal parasitic worms: roundworms (ascaris lumbricoides), whipworms (trichuris trichiura), or hookworms (necator americanus and ancylostoma duodenale). The tropical climate of indonesia is highly favourable to support the circulation of soil - transmitted helminths in the country . There are sporadic reports regarding the prevalence of the sth which come from scattered part of the country . The prevalence of sth was reported ranging from 40 to 70% in 80s to 90s . However, the estimated prevalence of sth was decreased in 2005: ascaris (15.2%), trichuris (12.9%), and hookworms (8.4%). The survey conducted by ministry of health in elementary schools located in 33 provinces of indonesia showed prevalence of sth was 31.8% . India (64%) and indonesia (16%) together are contributing 80% of the regional's burden . Drugs used for deworming, albendazole, and mebendazole are effective and inexpensive to control sth transmission and reinfection . The mda programme for elimination of lf which is implemented by using combination of albendazole with ivermectin or diethylcarbamazine seems to have synergistic effect to decrease the prevalence of sth . The program targeted preschool and elementary school age . However, until this moment the country still struggles to combat sth . In conclusion, there were efforts and programs concerning ntd that were planed and implemented in indonesia . Many factors may contribute to the rendering of ntd elimination in indonesia, such as high population, wide range geographic of the archipelago, and limited resources . By the end of 2015 ntd
Walking is one of the recommended modes of exercise for obese individuals because of its merits in terms of safety, accessibility, and popularity, as well as proven efficacy in weight management1, 2 . However, walking over a terrain of repetitive uphill and downhill inclines in activities such as mountain climbing can cause excessive fatigue and muscle damage3 . In particular, repetitive eccentric contractions of the lower limb muscles during downhill walking places sarcomeres under excessive tensile stress . This stress can destroy sarcomeres by extending them beyond their normal length, sometimes involving a local inflammatory reaction, and can thereby lead to delayed onset muscle soreness or exercise - induced muscle damage . The indexes reflecting such conditions are serum creatine kinase (ck) and lactate dehydrogenase (ldh) levels4, 5 . It has been reported that the use of a pole during walking over an uneven terrain could improve gait stability and balance, and that its use during downhill terrain walking can reduce the load and stress on the lower limbs (such as the ankle, knee, and hip joints)6,7,8 . However, previous studies on the effects of a pole during downhill walking have been limited to the kinetic aspects such as the reduction of knee joint forces or the load on the lower extremity joints . Meanwhile, it has been suggested that increases in peripheral cartilage oligomeric matrix protein (comp) levels after exercise could serve as a biomarker of cartilage degradation and damage9, and pruksakorn et al . In addition, according to a comparative study by andriacchi et al . That measured the maximum flexion of the lower extremity joints during various routine activities, the flexion of the knee joint was approximately 4 times greater during walking down stairs than during walking over level ground11 . Taken together, such previous reports suggest that the use of a pole during downhill walking may alleviate the burden on lower extremity joint cartilages; however, thus far, there has been no report examining this potential benefit . Accordingly, this study aimed to investigate the effect of the use of a trekking pole on muscle and cartilage damage and fatigue during downhill walking . The study specifically examined obese women who had less muscle mass than men and whose heavy weights were likely to increase the burden on their lower limb muscles and joints during walking . Eight obese women (average age of 21.1 1.8 years, average height of 164.8 4.9 cm, average weight of 61.8 7.7 kg, average body fat percentage of 33.9 1.4%, and average resting heart rate of 69.6 3.7 beats / min) with body fat percentages ranging from 30.0 to 36.1 volunteered as subjects for this study . The study conformed to the standards set by the latest revision of the declaration of helsinki, and all subjects read and signed a written informed consent statement consistent with the guidelines of the department of physical education at yonsei university . Height and body composition were measured using a stadiometer (seca213; seca, hamburg, germany) and a bioimpedance analysis (bia) device (inbody720; biospace, seoul, korea), respectively . Resting heart rate was measured in a seated position using a heart rate monitor (polar a5; polar, kempele, finland). Each of the eight subjects participated in a total of two trials: one np trial (walking without using a trekking pole), and one tp trial (walking using a trekking pole). With the treadmill angle set to a decline of 15% based on a study by perrey and fabre12, the participants were subjected to 30 minutes of walking at an exercise intensity of 50% of their heart rate reserve . The experiment used a crossover design to minimize the adaption from the repetitive exercise trials, and each trial was separated by 7 days to avoid any transient effects on the physiological and psychological conditions of the subjects . An adjustable - length trekking pole (6342011; leki, hamburg, germany) was used, and the length was initially set at 70% of the user s height so that the elbow angle was maintained at 90 when the user was standing on level ground . When the pole was used for downhill walking, its length was increased by approximately 5 cm . Using a 22-gauge needle, a serum separator tube (becton dickinson, franklin lakes, nj, usa), and a ethylenediamine tetra - acetic acid tube (becton dickinson), 7 ml of blood was collected from the antecubital vein of each subject at the pre - walking baseline (pwb), immediately after walking (iaw), and 2 hours post - walking (2hpw). Collected blood samples were centrifuged for 15 minutes at 3,000 rpm and then stored at 80 c until analysis . The serum ck and ldh levels were determined using a clinical chemistry analyzer (ektachem dtscii; eastman kodak, rochester, ny, usa). The serum comp levels were determined with an enzyme - linked immunosorbent assay (elisa) using a commercially available human comp elisa kit (anamar ab, lund, sweden). The absorbance was measured at 450 nm with a microplate reader (molecular device, palo alto, ca, usa). The plasma lactate levels were determined using a clinical chemistry analyzer (ektachem dt 60; eastman kodak, rochester, ny, usa). Data are presented as the mean standard deviation (sd) unless otherwise stated . For identifying differences in normally distributed results, when significant group by time interactions occurred, simple main effects were assessed using one - way anova and independent t - tests . The biomarker levels measured are presented in table 1table 1.peripheral biomarkers after downhill walking with the use of trekking poles (tp) and without their use (np)variabletrialpwbiaw2hpwck(u / l)np260.3 39.1349.1 37.9302.9 42.0tp256.1 42.0312.5 44.9270.9 34.5ldh(u / l)np351.5 77.8453.4 70.6377.0 84.0tp354.9 79.3385.1 71.2363.5 64.6comp(u / l)np9.6 2.013.6 3.812.0 2.8tp9.9 2.213.2 3.610.0 np: no trekking pole trial; tp: trekking pole trial; pwb: pre - walking baseline; iaw: immediately after walking; 2hpw: 2 hours post - walking; * p<0.05 vs. pwb .. the serum ck, serum ldh, serum comp, and plasma lactate levels were significantly increased iaw when compared with those at the pwb in trials np and tp (p<0.05). In addition, in trial np, the serum ck, serum ldh, and serum comp levels were significantly increased at 2hpw when compared with those at the pwb (p<0.05). Np: no trekking pole trial; tp: trekking pole trial; pwb: pre - walking baseline; iaw: immediately after walking; 2hpw: 2 hours post - walking; * p<0.05 vs. pwb . Exercise - induced muscle damage increases in situations involving eccentric muscle contractions, such as during downhill walking and/or running4 . This study measured the levels of serum ck, ldh, and comp to investigate the effect of the use of a trekking pole on muscle and cartilage damage during downhill walking . The results demonstrated that the levels of ck, ldh, and comp were significantly higher iaw and 2hpw than at the pwb in the case of the np trials . In the case of the tp trials, however, the levels 2hpw were significantly lower than those for the np trials and actually returned to their level at the pwb . The rationale could be that the use of a trekking pole alleviated the temporary muscle and cartilage damage induced after downhill walking . This result is in accordance with those of previous studies; howatson et al . Showed significantly lower serum ck levels at 24 hours after mountain climbing in an experimental group that used trekking poles than in the control group, suggesting that the use of trekking poles could alleviate muscle damage during recovery3 . In addition, exercise - induced increases in joint load have been shown to cause a temporary increase in serum comp levels after walking or running by promoting catabolism, in particular, in cartilages such as the articular cartilage in the knee9, 13 . Bohne and abendroth - smith suggested that the use of a trekking pole during downhill walking could be effective for reducing such loads on the lower extremity joints7 . According to the results of some previous studies, the use of a trekking pole could reduce lower limb joint forces by as much as 25%, enabling a 13 kg reduction of load per stride during downhill walking3, 14 . However, as for the plasma lactate levels in this study, no significant difference was observed with the use of a trekking pole . The rationale is that although the use of a trekking pole reduced forces on the lower limbs, it increased the activity of the upper limbs and consequently maintained a similar fatigue level in the whole body . This interpretation is supported by the report that plasma lactate concentration was a biomarker reflecting peripheral muscle fatigue during exercise15, as well as by the suggestion of fritschi et al . That, although the use of a pole during walking could reduce vertical knee joint forces, it could increase upper limb muscle activation16 . In conclusion, downhill walking can cause muscle and cartilage damage, and it is suggested that the use of a trekking pole can reduce temporary muscle and cartilage damage after downhill walking.
Periampullary diverticula (pad) refer to extraluminal outpouchings of duodenal mucosa that develop within the radius of 2 to 3 cm from the ampulla of vater (1). Pad are largely asymptomatic but they sometimes can cause both pancreaticobiliary and non - pancreaticobiliary complications . Rarely, obstructive jaundice can develop secondary to pad in the absence of choledocholithiasis or tumor and is termed lemmel's syndrome (2). Recently, the authors experienced an unusual case of abdominal pain and obstructive jaundice due to extrinsic compression of mid common bile duct (cbd) by distended pad filled with pus - like material as a result of impacted intradiverticular enterolith at the pad orifice . An 81-yr - old woman presented to the emergency department on august 3, 2012 with nausea, vomiting, fever (38.4), and diffuse abdominal pain of 4 days' duration . She had undergone subtotal gastrectomy with billroth ii anastomosis due to peptic ulcer perforation 10 yr ago . On physical examination, there was tenderness on her right upper quadrant but murphy's sign was equivocal . Her white blood count was increased to 11,170/l (neutrophil 83.0%) and crp was elevated to 2.392 mg / dl . The results of her liver function test were as follows: total bilirubin, 2.37 mg / dl; aspartate aminotransferase, 88 iu / l; alkaline phosphatase, 349 iu / l; and -glutamyl transpeptidase, 571 iu / l . To evaluate the cause of diffuse abdominal pain with liver enzyme elevation in a cholestatic pattern, abdominal ct scan was taken . The axial images of the ct scan demonstrated distal cbd stone with upstream bile duct dilatation (fig . However, on coronal reconstructed images, the stone was not located within the bile duct but inside the pad and the distended diverticulum was compressing the mid cbd (fig . 1b, 1c). Magnetic resonance cholangiopancreatography (mrcp) also revealed mid cbd compression and absence of choledocholithiasis (fig . These findings were confirmed on endoscopic retrograde cholangiopancreatography (ercp) which showed normal biliary orifice (fig . 2a) with impacted dark brown pigment stone (henceforth enterolith) at the pad orifice (fig . When the enterolith was pushed into the diverticulum by cannulation catheter and contrast dye was injected (fig . 2c, 3a), old blood clots and pus - like fluid gushed out from the opening (fig . Biliary cannulation combined with endoscopic sphincterotomy (est) was also performed to explore the cbd for other possible causes of obstructive jaundice but no stone, stricture or obstruction by tumor could be found . On endoscopic nasobiliary drainage (enbd) tubogram, stenosis at mid cbd was also shown to be resolved, likely because the pad had been decompressed (fig . 3b). After confirming that no other pathology was present, intradiverticular enterolith was crushed and removed by dormia basket on the following day (fig . The patient no longer complained of abdominal pain, her liver enzyme was normalized, and she was discharged without any complication . She had been well until 6 months after enterolith removal, when the patient visited the outpatient clinic with vague abdominal discomfort . Laboratory examination only revealed slightly increased total bilirubin to 1.96 mg / dl, but a large cbd stone was found on abdominal ct scan (fig . When the cbd stone was removed by ercp, the stone proved to be brown pigment sludge stone that typically forms in the presence of ascending infection (fig . Follow - up ercp was performed again after 6 months but there was no recurrence of cbd stone or enterolith . Diverticula of the gastrointestinal tract are outpouchings of all or part of the intestinal wall which can occur anywhere throughout the alimentary tract . The most common site of gastrointestinal diverticula is colon followed by duodenum, which was first described by chomel in 1710 (3). The detection rate of duodenal diverticula ranges from 1% to 27% depending on the diagnostic modalities used and the average age at the time of diagnosis (1). Among duodenal diverticula, pad is the most common type comprising about 70% to 75% of all duodenal diverticula (1). Most pad are asymptomatic but complications can occur in about 5% of cases and they include bleeding, perforation, diverticulitis, pancreatitis, choledocholithiasis, cholangitis, jaundice, enterolith or bezoar formation, intestinal obstruction, etc . Among these complications, hepatocholangiopancreatic disease can seldomly occur in the absence of choledocholithiasis and is termed lemmel's syndrome (2). First, diverticulitis or direct mechanical irritation of pad may cause chronic inflammation of ampulla and lead to chronic fibrosis of papilla (papillitis chronica fibrosa) (4). Third, distal cbd or ampulla can be directly compressed mechanically by pad that is usually filled with enterolith or bezoar (6, 7). In our case, enterolith that formed within the pad did not directly compress the distal cbd but it obstructed the pad orifice instead . This obstruction combined with inflammation of the diverticulum and collection of pus - like material within the obstructed pad seems to have expanded the pad with resultant extrinsic compression of mid cbd (fig . Therefore, the enterolith, bezoar, or food material within the pad is frequently evacuated and thus, the symptom could be intermittent . However, pad in our case had a narrow opening, likely due to repeated inflammation of the pad, and this seems to have hindered the clearance of entrapped enterolith out into the duodenal lumen . Enterolith formation within the duodenal diverticula is known to be facilitated in the static environment such as a blind loop after gastrectomy or proximal portion of stricture formed by crohn's disease or tuberculosis (8). In our case, blind loop created by billroth ii anastomosis seems to have provided a static environment favoring enterolith formation within the pad . During enterolith removal, cbd was also explored to search for other possible source of obstructive jaundice such as cbd stone since primary biliary stone is known to occur more frequently in the presence of pad (9, 10). One possible mechanism behind increased occurrence of primary cbd stone in these patients involves colonization and overgrowth of -glucuronidase producing bacteria within the pad that spread into the bile duct, which in turn leads to deconjugation of bilirubin glucuronides and eventually results in precipitation of calcium bilirubinate gallstones (11). Although cbd was explored in our case, no other etiology of obstructive jaundice could be identified other than extrinsic compression by distended pad . However, primary bile duct stone newly developed on follow - up ercp performed 6 months later (fig . The most plausible explanation is that est performed during cbd exploration at the time of enterolith removal has permitted the occurrence of ascending infection with resultant brown pigment stone formation . Diagnosing lemmel's syndrome could be challenging, but being aware of this condition is important to avoid mismanagement and it begins with identification of pad . Scan and mrcp, pad appear as thin - walled cavitary lesions situated on the medial wall of the duodenum 2nd portion that typically contain gas . However, pad are sometimes filled with fluid and can frequently be mistaken for pancreatic pseudocyst, pancreatic abscess, cystic neoplasm in the pancreas head or even metastatic lymph node (12, 13). Therefore, high index of suspicion is mandatory to arrive at a correct diagnosis in these patients . In our case, enterolith within the pad on axial images was at first mistaken for distal cbd stone due to its distal location combined with upstream dilatation of the bile duct (fig . However, upon careful scrutinization of the coronal reconstructed images, it became evident that the stone was located within the pad (fig . Treatment is generally not recommended in asymptomatic patients or would be conservative management in pauci - symptomatic patients . Nevertheless, since most patients with lemmel's syndrome present with symptoms related to biliary obstruction (i.e. Jaundice, abdominal pain, and cholangitis) as a result of extrinsic compression of cbd, some form of treatment is advocated . Therapeutic options in this situation run the gamut from endoscopic extraction of entrapped material, extracorporeal shock wave lithotripsy to surgery (diverticulectomy or biliodigestive anastomosis) (7, 14, 15). The patient in our case was also successfully treated endoscopically by fragmenting and removing enterolith using a dormia basket . It should be kept in mind that not all forms of lemmel's syndrome are caused by extrinsic compression of cbd by bulging pad . If the underlying mechanism of lemmel's syndrome is likely to be due to papillitis chronica fibrosa or sphincter of oddi dysfunction as mentioned above, the simplest and the most appropriate management would be to perform est (16). In conclusion, lemmel's syndrome is a rare cause of obstructive jaundice that should be included in the differential diagnosis of biliary obstruction when pad is present . Maintaining a high index of suspicion is imperative to establish an accurate diagnosis since it can mimic other cystic or solid lesions around the pancreas head . Symptomatic patient can be successfully managed endoscopically in many instances but recourse to surgical management would be necessary in selected cases.
Body mass index is a simple index of weight for height that is frequently used in the assessment of nutritional status . A low bmi, or underweight status, is often associated with an increased risk of mortality in seriously ill or hospitalized older adults [1, 2]. Conversely, a high bmi, indicative of overweight or obesity, is associated with an exacerbation in age - related physical and cognitive decline [3, 4] and with an increased prevalence or risk of many chronic health conditions common in older adults such as diabetes, hypertension, and cardiovascular disease [35]. Such associations are typically determined across the entire spectrum of older adults (aged 60 +), with no further demarcation within this age classification . Our finding of a much higher prevalence of several nutritional deficiencies in centenarians as compared with octogenarians [6, 7], suggests that there is considerable heterogeneity in nutrient status in the older adult age group . Likewise, there may also be considerable heterogeneity within the older adult age group with regard to chronic health conditions . Thus, it is not known whether the associations between underweight or overweight / obesity and chronic health conditions as observed in previous studies of older adults extend to the very old . Dietary intake patterns featuring a high intake of nutrient - dense foods such as cereals, fruits, vegetables, and low - fat meat and dairy products have been associated with a number of favorable health outcomes in adults including a decreased prevalence of obesity [8, 9], lower rates of weight gain over time, and better quality of life and improved survival . In contrast, low - nutrient dense dietary patterns with high intakes of sweets, desserts, and high - fat dairy products have been associated with higher rates of obesity and poor nutritional status in older adults . Studies comparing energy intakes and dietary intake patterns of centenarians to younger older adult cohorts have generally observed lower energy and/or fat intake in the centenarians, while dietary preferences of centenarians are considerably more varied and dependent of the region of study likely reflecting cultural patterns and cohort differences rather than longevity - related differences per se (reviewed). Nonetheless, there can be considerable variation of body weight status within a given group of centenarians, though to our knowledge the extent to which this may be associated with potential differences in dietary intake patterns or selected health conditions has not been explored . Thus, the objectives of this study were to explore associations (1) between bmi and dietary habits and (2) between underweight or overweight / obesity and health status in a population - based multiethnic sample of centenarians (98 years and above) from northern georgia in the usa . It was hypothesized that overweight / obesity, but not underweight, would be associated with poorer dietary habits and that both overweight / obesity and underweight, as well as dietary habits, would be associated with specific health conditions in this population . This study was a secondary analysis of data collected by the georgia centenarian study, a population - based multidisciplinary study conducted in 44 counties in northern georgia (usa) from 2002 to 2005 . The original study included 244 centenarians (defined as age 98 and older). The sampling procedures and data collection methods briefly, recruitment of participants from skilled nursing facilities was based on estimates of the institutionalized population of the area according to the 2000 u.s . Census tabulations . The community dwelling participants resided in private residences and personal care homes and were recruited from voter registration lists . Participants were recruited to match census figures for gender and race / ethnicity (white or black; all were non - hispanic) and were interviewed by trained personnel in their place of residence . All questionnaires and procedures were approved by the university of georgia institutional review board on human subjects . Information regarding age, gender, race / ethnicity, living arrangements, health conditions (cardiovascular disease, diabetes, hypertension, etc . ), and behaviors (including tobacco and nutritional supplement use) were obtained from each participant (or his / her caregiver) by self - report . Questions regarding food intake, appetite, and weight change were adapted from the mini - nutritional assessment [15, 16] and the response categories for food intake represented current frequency of consumption of food groups, including dairy products (milk, yogurt, and cheese); meat, fish, or poultry; orange / yellow vegetables; green vegetables; citrus and noncitrus fruit and juice . The total food score, ranging from 0 to 5, was based on comparisons with the dietary guidelines for americans 1,600-calorie meal pattern for sedentary older adults, as previously described . Body weight and height were measured by interviewers, obtained from charts or via self - report . In addition, knee height was measured on the right leg, unless contraindicated, to the nearest 0.1 centimeter and used to predict stature as per the formulas of chumlea et al . . Body mass index (bmi) was calculated as weight (kg)/height (meters). Bmi calculated from observed / recorded height and weight was highly correlated with bmi calculated from predicted stature and observed / recorded weight (r = 0.877; p <.0001). Bmi calculated from observed / recorded height and weight was used to form three bmi classifications based on the national institutes of health criteria for overweight / obesity and defined as underweight 20 kg / m, normal weight> 20 and <25 kg / m, and overweight / obese 25 kg / m . Triceps skinfold (tsf) was measured on the right arm, unless contraindicated, by caliper to the nearest 0.1 millimeter . Mid - arm circumference (mac) was measured on the right arm, unless contraindicated, to the nearest 0.1 centimeter ., burlington, nc) and anemia was defined as hemoglobin <12 g / dl for females or <13 g / dl for males . Participants missing data for primary variables of interest were excluded from the present analyses . From the original sample of centenarians, 11 individuals were excluded due to missing data for bmi (n = 7; includes one double - amputee), food intake patterns (n = 1), average grip strength (n = 1), and/or systolic / diastolic blood pressure (n = 3). Overall characteristics of the 233 centenarians included in the study are given in table 1 . Compared to the included centenarians (n = 233), the excluded centenarians tended to be older (102.6 3.6 versus 100.5 1.9 yrs; p = .052), but did not differ in gender (90.9% [excluded] versus 84.6% female), race / ethnicity (72.7% versus 79.0% white), or place of residence (36.4% versus 43.4% skilled nursing facility). Means, standard deviations, medians, range of values, and/or frequencies were calculated . Differences between participants with different bmi classifications were assessed with the wilcoxon rank sum test for continuous variables and chi square analysis for categorical variables . The level of significance was set at p <.05 . Because the food groups provide calories, a series of logistic regression analyses were performed with bmi 20 or bmi 25 as the dependent variable and gender, race / ethnicity, living arrangements, and reported intake of specific food groups as the independent variables . In addition, because bmi can play a causative role as a risk factor for chronic disease, a second series of logistic regression analyses was performed with diabetes, anemia, or other chronic health conditions as the dependent variable and race, gender, residence, and bmi 20 or bmi> 25 as the independent variables (model 1). A final series of logistic regression analyses was performed with diabetes, anemia, or other chronic health conditions as the dependent variable and race, gender, residence, total fruit, and vegetable intake and bmi 20 or bmi> 25 as the independent variables (model 2). P - values are unadjusted for multiple comparisons, because all comparisons were preplanned . Approximately one - third (31.3%) of the centenarians included in the final analytical sample had a bmi of 20 (underweight), 43.8% were classified as being in the normal weight range, and 24.9% met the nih classification for overweight / obesity (bmi 25). Triceps skin fold (tsf) and mid - arm circumference (mac) of centenarians in the lowest bmi classification averaged below the 5th percentile for females 80 + years in usa based on 20032006 nhanes data . Both parameters increased with bmi classification in a stepwise manner (data not shown) and were highly correlated with bmi (pearson correlation coefficient between bmi and tsf = 0.482; and between bmi and mac = 0.624; p <.0001 for both). Chi - square analysis indicated that those with a bmi 20 were more likely to be women, live in a skilled nursing facility, eat a modified food diet, have experienced a weight change in the past three months, and have anemia as compared to centenarians classified as normal weight or overweight / obese (table 2). Conversely, those with a bmi 25 were more likely to be black, diabetic, have a higher systolic blood pressure, and/or have a diastolic blood pressure 90 mm as compared to centenarians classified as underweight or normal weight . There were no differences according to bmi classification with regard to history of cvd, cancer, stroke, depression, or past or current tobacco use . Bivariate analysis of diet intake patterns suggested that centenarians with bmi 20 had the highest total food scores and were more likely to report eating two or more servings of meat, fish, and poultry per day and three or more total servings of fruit per day as compared with centenarians in the other bmi classifications . In contrast, those with a bmi 25 were more likely to report eating less than one serving of citrus or noncitrus fruit per day, less than four servings of orange / yellow vegetables per week, or three total servings of fruit and vegetables per day (table 2). A series of logistic regression analyses indicated that when controlled for gender, race, and place of residence the odds of having a bmi 25 were about two to three times higher in centenarians with lower intakes of citrus and noncitrus fruit (less than one serving per day), orange and yellow vegetables (less than four servings per week), or total fruits and vegetables (less than three servings per day), but were not related to intake of the meat group or dairy group (table 3). Similar analyses with bmi 20 as the dependent variable, failed to show any significant association with dietary intake categories (table 3). Finally, associations between bmi classifications and chronic health conditions were determined in a series of logistic regression analyses that included either bmi 20 or bmi 25 as an independent variable (table 4). When controlled for gender, race, and place of residence, the odds of having anemia, based on blood hemoglobin values, were over twofold higher in centenarians with bmi 20 versus those with bmi> 20 whereas the odds of self - reported cvd tended to be reduced in those in the underweight classification (bmi 20; p = .053). The latter finding became significant in regression models further controlled for total fruit and vegetable intake (model 2; p = .048). In analyses controlled for gender, race, and place of residence, the odds of having self - reported diabetes or systolic blood pressure 140 mmhg were approximately three- and twofold higher, respectively, in centenarians with bmi 25 versus those with bmi <25 . Being overweight / obese (bmi 25) also tended to increase the odds of having diastolic blood pressure> 90 mmhg (approximately three - fold; p = .055) or cardiovascular disease (approximately twofold; p = .074). Further controlling for total fruit and vegetable intake (model 2) strengthened the associations between bmi 25 and diabetes and systolic blood pressure> 140 mmhg, and resulted in a significant association between bmi 25 and diastolic blood pressure 90 mmhg . There were no associations between bmi 20 or bmi 25 and stroke, depression, or cancer in any of the regression models . To our knowledge, this is the first study to explore associations between dietary patterns and body weight status in the oldest old segment of the population . Prevalence of overweight / obesity in this population - based study of centenarians was approximately 25%, which was considerably below the prevalence for these conditions in the overall population of older adults, aged 60 and above, in the usa at the time of data collection (69%;). In analyses controlled for gender, race, and place of residence, several parameters indicative of a low frequency of fruit and vegetable intake were associated with overweight / obesity (bmi 25), whereas there were no associations between frequency of intake of meat, dairy, and fruits and vegetables and being underweight (bmi 20). Other findings include strong associations of underweight with anemia and of overweight / obesity with diabetes and high blood pressure, extending knowledge of such associations to the very old . The present research was part of a large, multidisciplinary study across a range of cognitive, mental, physical, and health - associated domains exploring the role of various factors pertinent to the survival and functioning of centenarians . To decrease testing burden for participants, dietary intake data focused on frequency of intake of specific food groups selected based on the dietary guidelines for americans and age - related associations with nutritional deficiencies and chronic diseases [16, 17]. Notably, we observed that based on frequency of intake and regardless of bmi classification, a large percentage of centenarians were not meeting the dietary guidelines for many food groups, with the exception of green and orange / yellow vegetables . This later finding is consistent with an apparent preference for sweet potatoes and green vegetables reported for an earlier convenience sampling of georgia centenarians [23, 24]. Such preferences likely are reflective of the traditional diet of the southeastern usa, rather than longevity - related differences in dietary patterns, and may not be replicable in other regions and cultures . Our initial analysis indicated a greater intake of meat and total fruits in the underweight centenarians, suggesting that they were eating better than those in the normal weight and overweight / obese classifications . The underweight centenarians also had the highest total food scores, suggesting that they were meeting more of the recommended servings for specific food groups . However, almost twice as many underweight centenarians lived in skilled nursing facilities as compared to the community, and there was no association between low bmi and dietary groups after controlling for race, residence, and gender . In a specific comparison between centenarians residing in skilled nursing facilities and in the community, johnson et al . Reported that those in skilled nursing facilities were more likely to eat three or more meals a day and to have a higher frequency of intake of most food groups . They suggested that such differences may be due to (1) the requirement that skilled nursing facilities serve meals that meet dietary guidelines and other federal nutrition policies [25, 26], (2) the inability of the methodology used to distinguish between food that was served and food that was eaten, and (3) barriers faced by community dwelling centenarians or their caregivers in purchasing, preparing, or consuming appropriate food to meet their nutritional needs . Thus, associations in centenarians between low bmi and dietary status may be quite complex and influenced considerably by place of residence . In the present study, there was an increase in the relative risk for being overweight / obese in centenarians reporting the lowest frequency of intake of some nutrient dense foods including orange / yellow vegetables and citrus and noncitrus fruits . These observations are consistent with previous studies finding associations between lower reported or inferred consumption of fruits and/or vegetables and increased prevalence of overweight / obesity in children and young to middle - age adults [5, 2830]. Interestingly, other studies in adults have indicated that increased consumption of fruits and vegetables may be an effective strategy for decreasing energy consumption and for increasing and maintaining weight loss [29, 31, 32]. In addition to potential beneficial effects on body weight, there is considerable evidence in other age groups that high consumption of fruits and vegetables may offer protective effects against and/or to reduce the relative risk of cardiovascular disease, hypertension, diabetes, and certain cancers [3340]. We observed that centenarians in the highest weight category (bmi 25) reported eating lower amounts of certain types of fruits and vegetables than their nonoverweight / nonobese counterparts and also appeared to be at greater risk for diabetes, high blood pressure, and cardiovascular disease . This suggests that a high intake of fruits and vegetables may be beneficial for maintaining optimal weight, and perhaps decreasing risk of chronic disease, even at very advanced age . However, additional, ideally longitudinal, research collecting more detailed food intake data is needed to support this contention . In addition, as social isolation, missing teeth, digestion difficulties, poor self - reported health, cost and preparation issues have been identified as barriers to fruit and vegetable intake in older adults [4143], research is needed to determine if and to what degree these or other potential barriers may be influencing the intake of fruit, vegetables, and other low energy, high nutrient dense foods in the very old . Clinically defined anemia was present in greater than 60% of the centenarians in the lowest bmi grouping . After controlling for demographic covariates, a strong association remained between low bmi and anemia but not with other health conditions and indicators . Accordingly, low bmi had been identified as an independent correlate or risk factor for anemia in some previous studies in older adults and clinical populations [4446], but not in others . Although older adults with a low bmi are considered to be at nutritional risk [47, 48], it cannot be assumed that the anemia observed in underweight older adults is primarily of dietary or nutritional etiology . Indeed, our previous studies indicate a similar prevalence of anemia in vitamin b12-deficient and vitamin b12-adequate in georgia centenarians and a high prevalence of inflammatory anemia, either alone or in combination with nutritional deficiencies, in this population . Nonetheless, as anemia is associated with increased mortality in acute and chronic disease states, particularly in those underweight [50, 51], it is important to monitor and treat this condition, as appropriate, in the very old . In summary, this secondary analysis provides evidence of an inverse association between fruit and vegetable intake and body weight status in a population - based study of centenarians . In addition, both underweight and overweight emerged as potential risk factors for various chronic diseases, emphasizing the importance of monitoring weight and of maintaining a healthy weight, even at very advanced ages . A major strength of the study is the inclusion of a population - based sampling of centenarians with greater diversity in race, place of residence, and functional status than would be typically obtained with a convenience sample . Limitations of the study include the relatively small sample size, lack of information regarding physical activity, and reliance on data of frequency of intake for only a few food groupings rather than the use of a more extensive food frequency questionnaire including individual foods and serving sizes as per our earlier convenience study of centenarians [23, 24]. Absence of intake data on key dietary components including grains / cereals and sweets / desserts necessitated the use a series of nonindependent binary logistic regression models instead of a more complex, single multinomial logistic regression to explore potential associations between bmi and dietary intake patterns . Thus, specifically designed studies including more detailed information of dietary intake, physical activity data, and additional chronic disease indicators are needed to verify associations, or lack thereof, between dietary intake patterns, weight status, and chronic health conditions in the very old . Furthermore, as dietary habits and other characteristics of this sample from georgia likely differ from those of centenarians from other countries and cultures, our findings need replication in other population groups.
Environmental factors play a major role in the etiology of cancers, cardiovascular disease, and other chronic diseases . These factors are diverse in nature and include diet, drugs, cosmetics, household chemicals, pollutants, or infectious agents . Exposures to these factors vary widely between populations, and between individuals within the same population . Therefore, their measurement is essential to: (i) study associations in epidemiological studies with disease outcomes and assess their contribution to disease risk, (ii) monitor exposures to disease risk factors in population studies and (iii) assess subject compliance in clinical trials or large intervention studies (13). Exposure measurements have traditionally relied on the use of questionnaires and self - reporting . However, these methods are known to be error - prone and biased . Molecular biomarkers, on the other hand, are more direct and objective indicators of exposure . Indeed, biomarkers of exposure have been increasingly used since the early 1980s, thanks to rapid progress in analytical techniques and the establishment of large cohorts with extensive biospecimen collections . Biomarkers of exposure can be compounds present in the environment and absorbed in the gut after ingestion, inhaled in lungs, or absorbed through the skin . They can also be metabolic end - products derived from environmental compounds that were metabolized by the liver and other tissues, and the microbiota . They may also be macromolecular indicators of environmental effects (e.g. Enzymes, proteins or rna transcripts related to the status of a nutrient or toxic agent). Over the past 30 years several hundred biomarkers of exposure have been measured and reported in blood, urine and other biospecimens in various populations . However, this information is scattered over hundreds of publications under many diverse titles and subject headings . This makes the identification of these biomarkers along with their comparative performance, their field of application and their concentration ranges in different populations difficult . Historically, most biomarkers of exposure have been measured individually using compound - specific assays . However, with the emergence of various omics technologies there is an increasing tendency to characterize exposures more comprehensively . Indeed, modern mass spectrometry techniques now allow the measurement of thousands of compounds in blood, urine or other biospecimens in a single analytical run . These developments are leading, increasingly, to the reporting of data from multiple markers of exposure . Modern omics technologies should also allow the translation into practice of the concept of the exposome (the totality of exposures of a particular individual over lifetime (4,5)) and the development of exposome - wide association studies (ewas) (2,68). These newly emerging trends in exposure science, combined with the growing volume of comprehensive exposure data being published, make the establishment of a centralized, online database on biomarkers of exposure particularly critical . To date, relatively little effort has been directed to collecting and organizing data on biomarkers of exposure . The expocastdb database contains information on exposure to environmental chemicals such as pahs, pcbs, nonylphenols, or pesticides (http://actor.epa.gov/actor/faces/expocastdb/home.jsp). Expocastdb contains information on compound concentrations in various environmental matrices but very limited data in biospecimens . The comparative toxicogenomics database (ctd) is the only online database containing a large number of concentration values in blood, urine and other biospecimens extracted from the scientific literature (9). The ctd contains about 35 000 concentration values in various biospecimens for 250 organic and inorganic compounds . While expocastdb and ctd are very useful and important databases it contains comprehensive information on almost 500 biomarkers of exposure with concentrations, correlations with exposure estimates and temporal reproducibility, as well as other details on study population and analytical methods . All data in exposome - explorer was acquired from a careful review and analysis of nearly 500 peer - reviewed publications, with a particular focus on dietary and pollution exposures . All data in exposome - explorer was compiled through extensive literature analysis along with manual curation and computer - assisted validation . Literature searches were conducted using the web of science (wos). Only peer - reviewed publications describing original work with biomarker measurements in observational studies conducted in human populations publications on intervention studies, analytical method development, associations of biomarker with biological status or disease, biomarkers of biological status (e.g. Inflammation, oxidative stress, disease), or animal studies were not considered for this initial stage of development . Articles not available online were omitted . For pollutants, general rather than occupational exposures were prioritized . Exposure, biological monitoring) and biospecimens (blood, serum, plasma, adduct). For dietary biomarkers, data on the correlations between food or food compound intake and biomarker concentrations citations were searched for according to several intake synonyms (intake, consumption, diet, recall, citations were searched for by common pollutant chemical group, such as polycyclic aromatic hydrocarbons (pah), polychlorinated biphenyls (pcb), polybrominated diphenyl ethers (pbde), polybrominated biphenyls (pbb), polychlorinated dibenzodioxins / furans (pcdd / f), heterocyclic amines (hca), phthalates and disinfection byproducts (dbp). To search for biomarker reproducibility values, reliability, reproducibility, repeatability, intra - subject, inter - subject, within - subject, between - subject. Full - record citations from the wos were downloaded in the bibtex format (http://www.bibtex.org/) and handled with bibdesk, an open - source bibliography manager for bibtex libraries (http://bibdesk.sourceforge.net/). Those rated as relevant were then manually described using a series of attributes (tags) related to exposures, study design, type of numerical data, populations, biospecimens, biomarkers and confounding variables to facilitate the selection of references for annotators . The bibdesk smart groups functionality and a combination of criteria based on the tags were used to dynamically generate a priority list of publications for further annotation . Full - texts from these articles were then retrieved and submitted to annotators for detailed analysis and upload of the data to exposome - explorer using the annotation interface . A password protected annotation interface was used for the manual uploading of all data from scientific publications to exposome - explorer ., the annotator is guided through successive steps to ensure comprehensive capture of the following information: publication with its bibliographic details (title, authors, year, journal, pubmed id).subject groups studied in the publication, and the populations to which they belong . Each population is named with a short informative summary sentence (e.g. Cases and controls in a case - control study on breast cancer). All) or several (e.g. Soy consumers and soy non - consumers) subject groups according to different criteria (e.g. By gender, by ethnicity, by smoking status). A reference to a cohort (e.g. Epic) can also be indicated in this data field . Age, height, weight, bmi, group size, gender, health condition, exclusion of supplement users, smoker proportion, country of origin and ethnicity are also specified when available.samples defined for each subject group . These describe number and time of collections (e.g. Baseline, 1 year) of different biospecimens . Biospecimens include urine, whole blood, serum, plasma, hair, nails, adipose tissue, breast milk, or fractions such as plasma phospholipids or red blood cell membranes.biomarkers . Chemical information (e.g. Structure, chemical formula, average mass, monoisotopic mass) is automatically generated by the structure server . Identifiers and links to external databases such as cas, pubchem (10), chebi (11), hmdb (12) and foodb (www.foodb.ca) are provided.biomarker measurements . For each sample or biospecimen type, also included is the analytical method, the original measurement units as described in the publication, and specimen - specific concentration value adjustments (e.g. Normalizing to lipid for blood, or a list of regression variables). Arithmetic or geometric mean, as well as median concentration values are compiled along with the minimum and maximum values, standard deviation, percentiles, confidence intervals, measurement size (number of subjects from which the concentration value was calculated), as well as the proportion of subjects for which the biomarker was detected . Information on the inclusion or exclusion of zero values in the calculated concentration is also given . This was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population . Some authors used a threshold (e.g. Detected in at least 30% samples). Below this threshold, the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . In certain cases, the compound used to express the concentration is given if it is different from the compound or compound class being measured (e.g. Polyphenols in urine expressed as gallic acid).correlations . To compile data on the correlations between specific biomarker measurements and food or dietary compound intake, information was collected on the intake of different foods, food groups or dietary compounds together with the method used for dietary assessment (record or questionnaire, food composition database, food coverage and time period). Food items in exposome - explorer are linked to the foodb database (www.foodb.ca). For food groups, the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication . For dietary compounds, the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication . Correlation coefficients (pearson's product moment or spearman's rank - order) are collated together with p - value, confidence intervals, statistical significance (yes/no), and correlation size (number of measurements from which the correlation coefficient was calculated). Adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient (e.g. Energy intake by residual method or creatinine) as well as a list of regression variables included in the calculation (e.g. Age, smoking status, bmi, gender), and the use of measurement error de - attenuation are also indicated.temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker . A high reproducibility is required when only one sample per subject is available for biomarker measurement, as in most large cohort studies . Reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval (generally weeks or months). Reproducibility is principally described by the intraclass correlation coefficient (icc), defined as the ratio of between - subject variance to the sum of within- and between - subject variance . The higher the icc value, the higher the reliability of the measurement . In some cases, within- and between - subject coefficient of variation (cv), as well as within- and between - subject reproducibility size (number of measurements from which the reproducibility value was calculated) and the confidence interval are also indicated in the database . Publication with its bibliographic details (title, authors, year, journal, pubmed i d). Each population is named with a short informative summary sentence (e.g. Cases and controls in a case - control study on breast cancer). All) or several (e.g. Soy consumers and soy non - consumers) subject groups according to different criteria (e.g. By gender, by ethnicity, by smoking status). A reference to a cohort (e.g. Epic) age, height, weight, bmi, group size, gender, health condition, exclusion of supplement users, smoker proportion, country of origin and ethnicity are also specified when available . These describe number and time of collections (e.g. Baseline, 1 year) of different biospecimens . Biospecimens include urine, whole blood, serum, plasma, hair, nails, adipose tissue, breast milk, or fractions such as plasma phospholipids or red blood cell membranes . Biomarkers . Chemical information (e.g. Structure, chemical formula, average mass, monoisotopic mass) is automatically generated by the structure server . Identifiers and links to external databases such as cas, pubchem (10), chebi (11), hmdb (12) and foodb (www.foodb.ca) are provided . Also included is the analytical method, the original measurement units as described in the publication, and specimen - specific concentration value adjustments (e.g. Normalizing to lipid for blood, or a list of regression variables). Arithmetic or geometric mean, as well as median concentration values are compiled along with the minimum and maximum values, standard deviation, percentiles, confidence intervals, measurement size (number of subjects from which the concentration value was calculated), as well as the proportion of subjects for which the biomarker was detected . Information on the inclusion or exclusion of zero values in the calculated concentration is also given . This was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population . Some authors used a threshold (e.g. Detected in at least 30% samples). Below this threshold, the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . In certain cases, the compound used to express the concentration is given if it is different from the compound or compound class being measured (e.g. Polyphenols in urine expressed as gallic acid). Correlations . To compile data on the correlations between specific biomarker measurements and food or dietary compound intake, information was collected on the intake of different foods, food groups or dietary compounds together with the method used for dietary assessment (record or questionnaire, food composition database, food coverage and time period). Food items in exposome - explorer are linked to the foodb database (www.foodb.ca). For food groups, the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication . For dietary compounds, the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication . Correlation coefficients (pearson's product moment or spearman's rank - order) are collated together with p - value, confidence intervals, statistical significance (yes/no), and correlation size (number of measurements from which the correlation coefficient was calculated). Adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient (e.g. Energy intake by residual method or creatinine) as well as a list of regression variables included in the calculation (e.g. Age, smoking status, bmi, gender), and the use of measurement error de - attenuation are also indicated . Temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker . A high reproducibility is required when only one sample per subject is available for biomarker measurement, as in most large cohort studies . Reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval (generally weeks or months). Reproducibility is principally described by the intraclass correlation coefficient (icc), defined as the ratio of between - subject variance to the sum of within- and between - subject variance . The higher the icc value, the higher the reliability of the measurement . In some cases, within- and between - subject coefficient of variation (cv), as well as within- and between - subject variance (var) reproducibility size (number of measurements from which the reproducibility value was calculated) and the confidence interval are also indicated in the database . Exposome - explorer's annotation interface uses a number of features to help complex, heterogeneous, literature - derived data to be easily and systematically translated into organized electronic data . Controlled vocabularies for compounds, foods, experimental methods, specimens, cohorts and units can be created and are fully documented in both the annotation interface and the database . This helps to avoid the inclusion of duplicate vocabulary items, such as different spellings or synonyms for a same item . Hierarchical associations of populations, samples and measurements can be represented through the creation of parent - children relations . For instance, populations stratified into different sub - groups can be easily described, or the association of several samples taken at different collection times . All data uploaded to the database via the exposome - explorer annotation interface is automatically validated, thereby preventing the uploading of erroneous records . Error checks include the usual database integrity verifications such as the presence of mandatory fields, checks on input text size, checks on allowable values or the uniqueness of specific values . The error checking utilities also include application - specific consistency verifications such as correlations cannot be created between measurements of different subject groups or measurement size cannot exceed subject group size. These checks ensure the highest consistency for data collected by different annotators . Newly uploaded data is also manually and systematically checked by the database's chief curator . Repetitive insertion of similar data is facilitated via the support of record duplication and the ability to edit several records at once . Error messages are displayed to guide annotators toward making correct data entries or fixing erroneous entries . Exposome - explorer's public user interface allows intuitive browsing and searching of almost any data type in the database (figure 1). Data can be retrieved through the quick search functionality or through specific searches offered on the website's different menu pages . Distinct areas are shown: structure and chemical information (a), concentrations (b), correlations (c) and reproducibility (d) collated from several publications . Biomarkers menu lists the biomarkers, biospecimens, analytical methods and cohorts documented in the database . Each item has its own summary page with both general details (name and classification) and all data related to this item in the database . Correlations menu lists the correlation values between different food or dietary compound intakes and the corresponding biomarker measurements . These correlations can be searched according to the food type, dietary compound, or biomarker . The data can be filtered according to a variety of parameters including the type of biospecimen or the method used to assess the dietary exposure . Reproducibility menu lists the biomarker reproducibility values, which can also be filtered according to biomarker classification (as well as other parameters) and ranked in decreasing or increasing order . The data search menu lists all biomarker concentration values and allows searches over all data fields . Searches by chemical structure are also possible, as well as browsing biomarker classes and subclasses . The the list can be filtered by title, authors, year or pubmed i d . Full - text for these publications can be accessed via pubmed links or direct publisher urls . For every annotated publication, the totality of collected data including the bibliographic information, detailed subject descriptions, and biomarker data (concentration values, dietary intake values, correlation values and reproducibility values) is displayed in a single page . Tables on exposome - explorer's different webpages can be easily adjusted to suit user - specific needs . Several hidden columns are available and can be shown in order to provide more details for the default display . Every original value is listed with its corresponding bibliographic citation, making it possible to link each value back to its metadata . Default conversions from highly diverse to standardized units are possible while original values are preserved . Tables can be exported in different formats (e.g. Csv, tab) and reused in other programs (e.g. Excel, r) in order to conduct more specific analyses . Ruby on rails is a web framework which employs the model - view - controller (mvc) design pattern . The exposome - explorer data is stored in a mysql relational database (http://www.mysql.com/). Hierarchical associations of records (trees) are implemented as a nested set model inside a single database column of the tables using the rails gem ancestry (https://github.com/stefankroes/ancestry). Highly diverse units and their conversions are transparently handled with the phys - units library (https://github.com/masa16/phys-units/), which is a ruby implementation of the gnu units software (http://www.gnu.org/software/units/). Website global search is implemented using the wishart's unearth gem, which uses elasticsearch indexing (https://www.elastic.co/). The web interface is built with the bootstrap front - end framework (http://getbootstrap.com/). To date, data from a total of 480 publications have been entered into this first release of exposome - explorer . This includes 10 510 concentration values for 692 biomarker entries, among which 8,861 concentrations correspond to 488 single dietary and pollutant biomarkers . Approximately one third of these biomarkers are dietary biomarkers (table 1). For dietary biomarkers, almost half of the concentrations are related to fatty acids, followed by carotenoids and polyphenols . With regard to pollutant biomarkers, about two thirds of the concentration data in exposome - explorer are related to pcbs and pbdes, followed by pahs, pcdd / fs and phthalates . Some concentration data are also available for other biospecimens such as hair, nails, adipose tissue or breast milk . Exposome - explorer currently contains the most complete and comprehensive information on exposure biomarkers ever compiled from peer - reviewed literature . It is also the first publicly available, web - enabled database specifically dedicated to exposure biomarkers . We believe it provides a good starting point for selecting markers of interest or for defining panels of biomarkers that can be used in exposome - wide association studies . Through exposome - explorer, biomarker concentrations can be compared in different cohorts or population groups at different levels of exposure (e.g. Consumers and non - consumers of a particular food), or between different geographical areas . All of the database's manually curated information is fully linked with other online databases and with the original publications . The high granularity of the data in the database should allow users to conduct very diverse and advanced analyses or comparisons across publications . Looking both at the range of studied compounds and the number of corresponding studies, exposome - explorer also allows users to quickly identify poorly studied exposures or biomarkers that may require further work for validation . Plans for future enhancements to exposome - explorer include its extension to other categories of exposures (nutritional status, pesticides, occupational exposures), the addition of other types of biomarkers (dna and protein adducts), and the inclusion of more information characterizing biomarkers such as their half - life and other pharmacokinetic parameters . Plans are also being made to add putative or non - validated biomarkers identified in (pre)clinical studies, but never measured in populations . Overall, we believe exposome - explorer will help in the generation of hypotheses for discovery of new biomarkers to be tested in the laboratory . It should also help in evaluating the performance of existing biomarkers and integrating exposure data based on biomarkers with data collected with other technologies . Exposome - explorer should contribute to the translation of the exposome into practice in epidemiological research . European commission: exposomics fp7-kbbe-2012; nutritech fp7-kbbe-2011 - 5; joint programming initiative foodball 201417.
The aspergilli are filamentous fungi, which are multicellular eukaryotes with a relatively simple life cycle . Many of them have long been used in food production, industrial fermentation, and agriculture . On the other hand, a few, such as a. fumigatus, a. flavus, a. niger, a. nidulans, and a. terreus, are opportunistic fungal pathogens, causing life - threatening invasive aspergillosis (ia) in immunosuppressed patients [1, 2], in which a. fumigatus is the predominant pathogen [35]. The crude mortality for ia is 6090% and remains around 2942% even when treatment is given . The main reasons for patient death are late diagnosis and the low efficiency of the drug therapies available to treat ia . It maintains cell shape and provides osmotic protection [7, 8] and has therefore been recognized for a long time as an ideal drug target . Indeed, several cell - wall - targeted drugs, such as echinocandins, caspofungin, micafungin, and anidulafungin, have been introduced as therapies . For example, echinocandins, which inhibit synthesis of -1,3-glucan, a crucial component of the cell wall are effective in the treatment of invasive fungal infections including ia . Unfortunately, the echinocandins also trigger an increase of chitin [9, 10], which partially compensates for the loss of -1,3-glucan and reduces the efficiency of treatment due to the complicated mechanism of cell wall biogenesis in a. fumigatus . Therefore, a more profound understanding of the mechanisms of cell wall biosynthesis in a. fumigatus would help to improve the efficiency of drug therapies, especially for drugs which target the cell wall . The cell wall of a. fumigatus is composed of a unique -1,3/1,4-glucan skeleton with chitin and galactomannan covalently linked to the nonreducing ends of -1,3-glucan . The cell wall is mainly coated with gpi proteins, which contain n- and o - glycans [11, 12]. While there is no doubt that glycosylation is involved in cell wall organization, the functional importance of protein glycosylation in cell wall organization has, until recently, remained poorly understood . However, during the past few years, it has become increasingly evident that glycosylation is vital for cell wall synthesis and thus vital for growth and morphology of a. fumigatus . Basically, all eukaryotes possess three types of protein glycosylation, n - glycosylation of asparagine residues, o - glycosylation of threonine and serine residues, and glycosylphosphatidylinositol - anchoring (gpi - anchoring) of the c - terminus of some proteins . Humans lacking individual glycosyltransferases suffer from severe congenital diseases, known as carbohydrate - deficient glycoprotein syndromes (cdgs) [1214]. Clearly, the sugar components of proteins play a major role in embryonic and postembryonic development of humans as well as of all higher eukaryotes . However, the molecular details leading to cdgs are only vaguely understood . During the past 20 years it is now known that carbohydrates play increasingly important roles in regulating the development of higher organisms . However, the mechanism by which carbohydrates play a role in development and disease is still unclear . Our knowledge of protein glycosylation comes mainly from investigation of the model yeast s. cerevisiae and of mammalian cells . Although investigation of the model yeast has been very useful in elucidating the biochemical features of protein glycosylation at the cellular level, they cannot reveal the complicated functions of glycosylation in the development of multicellular eukaryotes . Therefore, investigation of glycosylation in the multicellular fungus a. fumigatus not only helps understand the mechanism of cell wall synthesis in this species but also provides insights into the role of glycosylation in the development of multicellular eukaryotes . This paper concentrates on protein glycosylation in a. fumigatus, which will be discussed with respect to the enzymatic pathways involved and their functional importance . Furthermore, the utility of a. fumigatus as a model for glycosylation during development of multicellular eukaryotes will be outlined . The cell wall of a. fumigatus is mainly composed of -1,3-glucans that are highly branched with -1,6 linkages . Together they constitute a three - dimensional network with a large number of nonreducing ends, to which chitin, galactomannan, and -1,3/1,4-glucan are covalently anchored . The cell wall is mainly coated with gpi proteins, which contain n- and o - glycans derived primarily from the process of glycosylation [11, 17]. Deletion of gel2 leads to slower growth, abnormal conidiogenesis, an altered cell wall composition, and reduced virulence [18, 19]. It has been proposed that gel2p is responsible for the elongation of -1,3-glucan side chains of -1,3/1,6 branched glucan to provide new nonreducing ends . Ecm33p is also involved in maintaining proper cell wall architecture though its function is unknown . Disruption of ecm33 results in morphogenetic aberrations such as defects in conidial separation, increase of chitin in conidial cell walls, rapid conidial germination, and increased virulence [20, 21]. It is clear that glycoproteins are directly, as structural components of the cell wall, and indirectly, as enzymes required for cell wall synthesis, involved in maintaining proper cell wall architecture in a. fumigatus . Increased chitin synthesis has been known as an important compensatory response to cell wall stress both in s. cerevisiae and filamentous fungi [2226]. In s. cerevisiae, the cell wall is required to maintain cell shape, which is essential for the formation of a bud and hence cell division . The yeast cell remodels its rigid structure to accommodate cell expansion during vegetative proliferation, mating pheromone - induced morphogenesis, and nutrient - driven filamentation through the cell wall integrity (cwi) signaling pathway . Cell wall defects or damage induces the cells to activate the cwi pathway to survive, and the compensatory mechanism characterized by an increased chitin content is triggered . The cwi signaling pathway in s. cerevisiae is activated in response to low osmolarity, thermal stress, or mating pheromone and polarized growth . It is comprised of a family of cell surface sensors coupled to the small g - protein rho1p, which activates the cwi mapk cascade via protein kinase c (pkc1p). This signaling cascade activates the expression of genes encoding for cell wall proteins that stabilize the cell wall . Meanwhile, activated rho1p also activates a set of additional effectors such as bni1p and bnr1p formin proteins, skn7p transcription factor, and the sec3p exocyst component, which regulate a diverse set of processes including -glucan synthesis at the site of cell wall remodeling, gene expression related to cell wall biogenesis, organization of the actin cytoskeleton, and secretory vesicle targeting to the growth site . A family of cell surface sensors is responsible for detecting and transmitting the status of the cell wall to rho1p . These sensor molecules include wsc1p (hcs77p / slg1p) [2931], wsc2p and wsc3p, and mid2p and mtl1p [32, 33]. Among these cell wall stress sensors, wsc1p and mid2p appear to be the most important and serve a partially overlapping role in cwi signaling . Reduced o - mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the pkc1 pathway and finally causes cell death in the absence of osmotic stabilization . More recently, n - glycan is found to be directly involved in mid2p sensing . It has been shown that both the extent of the n - linked glycan and its distance from the plasma membrane affect mid2p function . These observations demonstrate that n- and o - glycosylation are important for cwi sensing and thus important for cell wall biogenesis and polarized growth in yeast . The presence of a. fumigatus genes encoding for proteins homologous to the yeast rho1p, rho3p, and cdc42p suggests a similar mechanism for the cwi pathway . Indeed, it has been recently shown that afcdc42/cdc42, afrho1/rho1, and afrho3/rho3 are highly expressed in the mutant devoid of cwh41p (glucosidase i), which suggests an activation of these genes induced by cell wall damage . Also, increased expression and activation of the a. fumigatus mpkap, an ortholog of the s. cerevisiae mpk1p, is also induced by cell wall damage [38, 39]. It is becoming clear that, as in yeast, defects in cell wall integrity also trigger the cwi mapk cascade in a. fumigatus . On the other hand, in contrast to yeast, little is known about the cell wall stress sensors in a. fumigatus . In the last release of the a. fumigatus genomic database (http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main), only one protein (afua_5g09020) is annotated as a homologue of the wsc4p, which does not appear to contribute to cwi signaling in yeast . Therefore, the a. fumigatus cell wall stress sensor molecule remains to be identified and investigated . The precursor of all mannose residues found in galactomannan, glycoprotein, and gpi anchor in a. fumigatus is gdp - mannose . The activation of mannose initiates from formation of mannose 6-phosphate (man-6-p), which occurs by one of two routes: direct phosphorylation of mannose by hexokinase or interconversion from fructose 6-phosphate (fru-6-p) via phosphomannose isomerase (pmi), and the latter pathway requires three enzymes: pmi, phosphomannomutase (pmm), and gdp - mannose pyrophosphorylase (gmpp). Fru-6-p is converted to man-6-p by pmi, and then man-6-p is converted to mannose 1-phosphate (man-1-p) by pmm . Subsequently, man-1-p is ligated with the guanosine 5-triphosphate molecule (gtp) to form gdp - mannose by man-1-p guanylyltransferase [4063]. The interconversion of man-6-p and fru-6-p catalysed by pmi is the first committed step in the synthesis of man - containing sugar chains and provides a link between glucose metabolism and mannosylation . Pmi deficiency is the cause of carbohydrate - deficient glycoprotein syndrome type ib (cdg - ib, omim 602579) in humans, but the clinical symptoms and aberrant glycosylation can be corrected with dietary mannose supplements . Genes encoding for pmis have been investigated in several fungal species, such as s. cerevisiae, candida albicans, a. nidulans, and cryptococcus neoformans [4851]. The pmi mutant shows a significantly reduced growth rate at high concentrations of mannose . Biochemical and genome - wide analysis reveals that excess mannose leads to an accumulation of man-6-p, which mainly inhibits the activity of phosphoglucose isomerase (pgi) and thus represses glycolysis, protein biosynthesis, and cell wall biogenesis . The a. nidulans mana1 mutant exhibits abnormal ballooning of hyphal tips and eventually ceases to grow . Disrupted man1 mutant of c. neoformans leads to poor capsule formation, reduced polysaccharide secretion, morphological abnormalities, and attenuated virulence . In a. fumigatus, pmi activity is essential for viability and plays a central regulatory role in both glycosylation and energy production . Deletion of the a. fumigatus pmi1 gene leads to uncoupling of the link between energy production and glycosylation and accumulation of man-6-p, which then results in defects in cell wall integrity, conidiation, and morphology . Although extracellular mannose can rescue the growth of pmi deficient mutants in a. fumigatus, both lower and higher concentrations of mannose lead to a reduction in the levels of -glucan in the cell wall and an accumulation of man-6-p . The phenotypes associated with the mutant under mannose starvation are mainly due to an insufficient supply of gdp - man required for cell wall synthesis . The abnormal morphology associated with the pmi1 mutant under mannose - replete conditions is mainly ascribed to an accumulation of man-6-p, which cannot efficiently enter glycolysis, instead becoming trapped in a cycle of dephosphorylation and rephosphorylation resulting in depletion of intracellular atp . It should be pointed out that the pmi in a. fumigatus mainly catalyzes the conversion of fru-6-p to man-6-p, and its binding affinity for man-6-p is similar to that of yeasts but different from the ones from bacteria or animals (table 1). This suggests that it may be possible to design a specific inhibitor for fungal pmis . Several gmpps have been identified and characterized in different species [5559]. In s. cerevisiae and c. albicans, gmpp is essential [60, 61], while in leishmania mexicana gmpp is not required for viability . Repression of gmpp in yeast leads to pleiotropic phenotypes including cell lysis, failure of cell separation, impaired budding and hyphal switching, clumping and flocculation, and cell wall defects . Repression of expression of a. fumigatus gmpp srb1, a homologue of yeast srb1/psa1/vig9, leads to hyphal lysis, a defective cell wall, impaired polarity maintenance, and branching site selection, as well as rapid germination and reduced conidiation . In contrast to yeast, inducible repression of srb1 expression in a. fumigatus does affect the ability to direct polarity establishment and septation . These reports imply that mannose activation is specifically crucial for the synthesis and organization of the cell wall and thus essential for survival of fungal species . This further suggests that glycosylation is essential for the viability of pathogenic fungal species such as a. fumigates, and inhibitors that specifically block mannose activation in fungi may be potential drugs to treat fungal infections . N - glycosylated proteins contain oligosaccharides that are n - glycosidically linked to the -amido group of asparagine . This type of glycoprotein has been intensively studied in many model systems from yeast to human cells with respect to their structure, biosynthesis, and function . It has been shown that the formation of the highly variable n - linked oligosaccharides is initiated by the assembly of a lipid - linked oligosaccharide glc3man9glcnac2-pp - dol by a series of glycosyltransferases located on the cytoplasmic and luminal faces of the er membrane . The most complete understanding of biosynthesis of the lipid bound precursor has been obtained from s. cerevisiae and from mammals . As far as it is known, the corresponding reactions proceed almost identically in other eukaryotes . Subsequently, the dol - pp - linked glc3man9glcnac2 is transferred as a whole to an asparagine residue within an n - x - t / s consensus sequence of a nascent peptide, which is catalyzed by the oligosaccharyltransferase (ost), and then the n - glycosylated proteins are modified in a species - specific manner and transferred through the secretory pathway to the cell surface where they either get exported or anchored to the plasma membrane, to the extracellular matrix, or to the cell wall (figure 1). The ost complex consists of at least eight different subunits, including ost1p, ost2p, wbp1, stt3p, swp1p, ost4p, ost5p, and ost3p / ost6p [6467]. Although the function of each subunit is still unclear, stt3p is believed to be the catalytic subunit [6870], and its homologues are found in almost all eukaryotes . It appears that the a. fumigatus stt3 is also essential as no viable knockout mutant has been recovered . Repression of the stt3 gene in a. fumigatus leads to a severe retardation of growth and a slight defect in cell wall integrity . Further analysis shows that repression of stt3 upregulates expression of the genes responsible for glucan and chitin synthesis, especially gel1, gel2, fska, chse, and chsg . Indeed, an increase of cell wall mannoprotein and chitin was observed following repression of the stt3 gene . However, this upregulation of chitin is not accompanied by an activation of the mpka kinase . Indeed, only the unfolded protein response (upr) is induced . As the upr has been shown to be involved in cwi signaling in a. fumigatus, it is likely that upr, instead of the mpka - dependent cwi signaling pathway, is the major compensatory mechanism induced by repression of the n - glycosylation in a. fumigatus . Once glc3man9glcnac2 is transferred to proteins, the n - glycan is processed sequentially in the er and golgi . N - glycan processing is initiated by the removal of the glucose residues catalyzed by er glucosidase i and glucosidase ii . In mammalian cells, n - linked glycan plays a decisive role as a quality control (qc) of the folding of secretory proteins, which is composed of calnexin, calreticulin, udp - glucose: glycoprotein glucosyltransferase (gt) and glucosidase ii (gii) and is essential for cellular survival [7577]. N - glycans initially serve to increase the hydrophilicity of the as - yet - unstructured nascent polypeptides . Subsequently, the two outermost glucose residues of the n - glycan are removed by the sequential action of glucosidase i (gi) and gii to the monoglucosylated form, which is recognized and bound by calnexin, a type i er membrane lectin, and calreticulin, its soluble relative . For many glycoproteins, the interaction with calnexin or calreticulin slows down the rate of folding but increases efficiency . Gii - catalyzed removal of the third glucose residue follows the dissociation of folding substrates from calnexin and is required for release of native polypeptides from the er and transport to their final destination . The folding sensor gt adds back a terminal glucose to promote reassociation of nonnative polypeptides released from calnexin, thus prolonging their retention in the er folding environment . Cycles of de-/reglucosylation might be protracted until the polypeptide released from calnexin fulfills quality control requirements . When correct folding is not achieved, an er - specific n - glycan - dependent pathway of degradation removes the misfolded proteins . When n - glycosylation is inhibited, the most commonly observed effect is the generation of misfolded, aggregated proteins that fail to reach a functional state [75, 76]. Before entering the qc system, the outermost glucose residue of the n - glycan is trimmed by er glucosidase i . A human inherited glucosidase i deficiency has been reported to result in neonatal birth with severe generalized hypotonia and dysmorphic features . Unlike mammalian cells, s. cerevisiae lacks a calnexin cycle and gt and only has an effective mannosidase i - dependent erad system [80, 81]. The yeast glucosidase i (cwh1p) is encoded by the cwh41 gene . Mutational defects in the cwh41 gene cause severe and selective instability of glycoprotein kre6p, a putative golgi glucan synthase required for -l,6-glucan synthesis [23, 83, 84]. Some filamentous fungi have been proposed to possess n - glycan - dependent qc of glycoprotein folding based on fungal genome sequence data . Recently, evidence that filamentous fungi possess an n - glycan - dependent qc system has been reported in a. fumigatus [37, 86]. Indeed, calnexin (aas68033), glucosidase ii, and gt have been annotated in the last release of the a. fumigatus genomic database (figure 2). Zhang et al . Reported that deletion of the cwh41 gene in a. fumigatus results in defective n - glycan processing of the proteins secreted by a. fumigatus . Although afcwh41 is not essential for hyphal growth and virulence, a severe reduction in conidial formation, abnormalities of polar growth and septation, and a temperature - sensitive deficiency of cell wall integrity were documented . Also, the genes encoding rho - type gtpases (rho - type gtpase / cdc42) were upregulated, which suggests that the cwi pathway was activated in the mutant . After processing by two er -glucosidases, the n - glycan is further processed by the action of various 1,2-mannosidases, which can remove one or more of the four 1,2-linked mannose residues . In mammalian cells, man9glcnac2 is converted to man5glcnac2 by the action of er and golgi -mannosidases, which is the precursor for complex, hybrid, and high - mannose n - glycans . In s. cerevisiae, a specific er 1,2-mannosidase converts man9glcnac2 into man8glcnac2, which is elongated in the golgi to form an outer chain containing up to 200 residues of mannose [89, 90]. A. saitoi and trichoderma reesei have been found to produce n - glycan structures containing five mannose units (man5glcnac2), suggesting further processing of the man9glcnac2 precursor [91, 92]. The n - glycans on mature secreted glycoprotein produced by a. fumigatus are man6glcnac2, man7glcnac2, and man8glcnac2, in which man6glcnac2 is the major glycoform . These observations demonstrate that n - glycan synthesis in filamentous fungi seems to differ from that in yeast and is similar to that in higher eukaryotes (figure 3). Although small n - glycans are commonly found on glycoproteins of a. fumigatus, hex5 - 13hexnac2 glycans on the galactomannoproteins, and man5 - 9glcnac2 as well as galf1man5 - 7glcnac2 structures on other secreted glycoproteins have been identified in a. fumigatus [93, 94]. The enzyme (udp - galp mutase) required to synthesize the requisite udp - galf donor has been shown to be an important factor in biosynthesis of the cell wall in a. fumigatus, while the gene / enzyme responsible for the transfer of galf has not been identified . Recently, the a. fumigatus och1, a key mannosyltransferase for synthesis of elaborated protein n - glycans in yeast, has been identified . Deletion of the och1 gene results in a reduction of sporulation in the presence of high calcium concentrations . This evidence suggests that polymannosylated n - glycans exist in a. fumigatus and certain proteins engaged in sporulation require n - glycan outer chains to be fully functional . The -mannosidases have been classified into two groups: class i and class ii [102, 120, 121]. Class i -mannosidases include er man9-mannosidase, endomannosidase, and golgi mannosidase i. class ii -mannosidases include the lysosomal mannosidases, golgi mannosidase ii, yeast vascular mannosidase [98, 99], and er -mannosidase ii [122, 123]. Several golgi -mannosidases have been cloned and characterized from penicillium citrinum [124, 125], a. saitoi, a. oryzae, t. reesei, and a. nidulans [102, 129]. These enzymes are all monomeric with a molecular weight of 5060 kda and show the maximal activity in the semiacidic condition (ph 46). Class i -mannosidase is known to play an important role in the processing of mannose - containing glycans . In drosophila melanogaster, deletion of the golgi mannosidase i (mas-1) results in viable progeny, and the null organisms synthesize the same range of oligosaccharides as the wild - type ones, albeit at different ratios . In s. cerevisiae, disruption of the er -mannosidase gene does not prevent outer chain synthesis . In the last release of the tigr database (http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main), nine a. fumigatus genes are annotated to encode -mannosidases, including xp_749038.1, xp_754794.1, xp_751252.1, xp_751819.1, xp_752444.1, xp_752825.1, xp_753592.1, xp_751114.1, and xp_750572.1 . Among them, msdsp (xp_752825.1) has been identified to encode a class i 1, 2-mannosidase and acts on man8glcnac2 to produce man6glcnac2 . Deletion of the msds gene leads to a defect in n - glycan processing, as well as a reduction of cell wall components (including -glucan, -glucan, mannoprotein, and chitin) and reduced conidiation . . In mammalian cells, free oligosaccharides (fos) are generated by ost - mediated hydrolysis of glc3man9glcnac2-pp - dolichol in the lumen of the er or peptide n - glycanase (pngase)-mediated de - n - glycosylation of newly synthesized glycoproteins either in the er or the cytosol . Fos that are liberated in the lumen of the er can be transported into the cytosol, where they are trimmed by an endo--d - n - acetylglucosamine h (endo h)-like enzyme and the -mannosidase man2c1p in order to yield an oligosaccharide, man5glcnac, that can be imported directly into lysosomes to be degraded (figure 4) [122, 123, 131135]. In humans and cattle [137139], a deficiency in -mannosidase results in the lethal disease mannosidosis, a rare lysosomal storage disease with a collection of clinical symptoms including progressive mental retardation, impaired hearing, dysostosis multiplex, immune defects, elevation of serum and urinary oligosaccharide levels, and an enlargement of lysosomes in most cell types resulting from the accumulation of undegraded oligosaccharides . The rat man2c1p is involved in oligosaccharide catabolism of misfolded glycoproteins in the lumen of the er which have been retrotranslocated into the cytoplasm for proteolytic disposal [131133]. A proteolytically cleaved version of the rat man2c1p has been found in the lumen of the er where it is believed to be involved in the early stages of glycoprotein maturation (also called er -mannosidase ii) (figure 4) [122, 123]. The yeast cytosolic -mannosidase ams1p, a counterpart of man2c1p, is also involved in the processing of fos . Since the yeast png1p is mainly localized to the cytosol, it is proposed that the png1p - generated fos may both be generated and processed in the cytosol . The role of the yeast ams1p is to aid in recycling macromolecular components of the cell under nutrient deprivation . Interestingly, after its synthesis in the cytosol, the ams1p is translocated into the vacuole by the cytosol - to - vacuole targeting pathway, which suggests a common feature shared by the s. cerevisiae ams1p and its mammalian counterparts . However, the yeast ams1p only participates in recycling or utilizing of oligosaccharide but not in processing of n - glycan (figure 4). It should also be noted that no structural studies have been performed on the products that can be generated from man8glcnac2 by ams1p, and the ultimate fate of such products remains obscure . On the other hand, two png1p - independent fos pools, man3glcnac2 and man8glcnac2, are also seen in s. cerevisiae . The pool comprising small fos (man3glcnac2) appears to be disposed of by unknown enzymes in the vacuole . The pool containing mainly man8glcnac2 may be generated and disposed of along the secretory pathway . Similarly, a. nidulans -mannosidase iic is also proposed to be involved in oligosaccharide catabolism . Both a. nidulans-mannosidase iic and s. cerevisiae ams1p are not essential for normal cellular function since disruption of these genes has no visible effect on growth or morphology [98, 99, 102]. In contrast to its counterpart in yeast or a. nidulans, the a. fumigatus ams1p is required for normal cellular function . Deletion of the a. fumigatus ams1 leads to a severe defect in conidial formation, especially at a higher temperature . These results show that the afams1 gene is required for morphogenesis and cellular function in a. fumigatus . The involvement of the afams1 gene in polarized growth demonstrates that the processes involved in fos regulation are important for a. fumigatus . It is likely that the ams1p is involved in cell wall synthesis and thus polarity through the cwi pathway . Therefore, probably the afams1 mutant could serve as a simple model to investigate the mechanism of -mannosidosis . Functional analyses of the genes required for n - glycosylation reveal that protein n - glycosylation is important for cell wall synthesis, morphogenesis, and polarized growth in a. fumigatus . 2-d gel analysis reveals that deletion of the cwh41 gene encoding glucosidase i in a. fumigatus leads to er stress, which induces overexpression of hsp70 and calnexin chaperone and activates the erad . Meanwhile, the proteins required for actin rearrangement are found to be underexpressed or missing, which is consistent with the observation of random localization of actin fibers in the mutant . These observations, for the first time, clearly suggest that n - glycosylation contributes to proper folding and trafficking in a. fumigatus . It appears that proteins involved in cell wall biosynthesis in a. fumigatus are more dependent on the n - glycan - dependent folding system . As in yeast, cell wall defects also trigger the cwi signaling pathway in a. fumigatus, which activates downstream effectors that regulate cell wall biogenesis and polarized growth . Zhang et al . [37, 86] have proposed that the proteins required for cell wall synthesis or cell wall stress sensing are substrates of a. fumigatus cwh41p and require glucose trimming for their proper localization and function . Misfolding of these proteins would cause cell wall defects, which then leads to activation of the erad and rho - type gtpases - mediated cwi pathway . Moreover, activation of cdc42 in the cwi pathway also activates sepa, an upstream organizer of actin ring formation at septation sites, and thus causes abnormal polarized growth associated with the afcwh41 mutant (figure 5). Although the phenotypes associated with different n - glycosylation mutants vary, the finding that all of these mutants exhibit phenotypes associated with cell wall defects, abnormal polarization, and morphological changes can all be explained by this proposed model . Obviously, more investigations are needed to identify and characterize all of the proteins affected by n - glycosylation in a. fumigatus . This information would be key to understanding the complex compensatory mechanisms participating in cell wall biosynthesis in a. fumigatus, which would serve as a basis to develop new antifungal therapies, as well as help to elucidate the molecular mechanism of human diseases associated with defects in glycosylation . O - mannose glycosylated proteins were first discovered in yeast and filamentous fungi, and recently this type of glycoproteins has also been described in mammals . The o - mannosylation most likely occurs in all animals, with the exception of nematodes (e.g., caenorhabditis elegans); it is also not detected in plants (arabidopsis thaliana, oryza sativa). However, it has also been discovered in one bacterial species (mycobacterium tuberculosis). In mammalian cells, the inner o - linked mannose is elongated with the first addition of a n - acetylglucosamine and then various sugars . In the case of yeast, the o - mannose type carbohydrate chain starts with a serine / threonine - linked mannose, which is extended to an oligomannose chain . In a. fumigatus, the o - linked glycans on cell wall mannoproteins are found to be glc1, 6man, galf1, 6man1, 6man, galf1, 5galf1, 6man1, 6man and galf1, 5[galf1,5]3 galf1, 6man, while only a single mannose residue was detected on secreted proteins . A further type of protein o - glycosylation, in which a single -o - linked glcnac residue is linked to serine and threonine occurs in animals, plants, and filamentous fungi, but not in s. cerevisiae . For this type of protein modification, protein o - mannosylation is initiated by a family of protein o - mannosyltransferases (pmts) that are evolutionarily conserved from yeast to human [145, 146]. In s. cerevisiae a total of seven pmt family members (scpmt17p) are present [104, 147], which fall into three major groups of homology: (i) pmt1/5/7, (ii) pmt2/3/6, and (iii) pmt4 . Genes with significant homology to pmts have been cloned in humans, mice, and drosophila [148150]. Specific protein substrates that are o - mannosylated by scpmt1p, scpmt2p, or scpmt4p have been described in s. cerevisiae [151153]. In comparison with s. cerevisiae, the pmt family is less redundant in higher eukaryotes . In drosophila only two pmt family members are present (rotated abdomen and twisted) [149, 150]. The same is true for mice and humans (pomt1 and pomt2) [148, 150]. Mutations in human pomt1, a homologue of the yeast pmt4, cause walker - warburg syndrome (wws), which is characterized by severe congenital muscular dystrophy, neuronal migration defects, and structural abnormalities of the eye . Targeted deletion of pomt1 in mice results in embryonic lethality due to defects in the formation of the reichert's membrane, the first basement membrane to form in the embryo . Mutations of the drosophila pmt homologues alter muscle structures and the alignment of adult cuticle . The pmt family is crucial for viability, cell wall integrity, and morphogenesis in several fungal species, such as s. cerevisiae, s. pombe, c. albicans and c. neoformans, a. nidulans, and a. fumigatus [108, 109, 111115, 145, 156]. In s. cerevisiae, single pmt1 mutants fail to grow in anaerobic conditions on some media . The pmt1,2,3-triple mutants grow only in osmotically stabilized medium, whereas the pmt1,2,4- and pmt2,3,4-triple mutants are not viable in any conditions, indicating that pmt protein activity is essential in s. cerevisiae, although individual genes are dispensable . C. albicans contains five pmt genes . The pmt1 mutants are viable, but they are defective in undergoing cellular differentiation from yeast to a true hyphal growth form under some conditions . The virulence of the pmt1 null mutant is significantly attenuated, which is likely due to reduced o - glycosylation of the c. albicans adhesin als1p . The pmt phenotypes are closely linked to alterations in cell wall components, including cell wall mannoproteins and polysaccharides . In s. pombe only one member of each pmt subfamily is present, namely, oma1, oma2, and oma4 . Deletion of oma2, as well as simultaneous deletion of oma1 and oma4 is lethal . Characterization of the viable s. pombe oma1d and oma4d single mutants shows that reduced o - mannosylation results in abnormal cell wall and septum formation, therefore severely affecting cell morphology and cell - cell separation . In c. neoformans, recently, willger et al . Showed that pmt2 is an essential gene, and the double pmt1pmt4 deletion is lethal . Filamentous fungi, such as a. fumigatus, a. nidulans, neurospora crassa, and fusarium gramineum, contain only three pmt genes that belong to the pmt1, pmt2, and pmt4 subfamilies, respectively [107, 108, 113]. All single pmt mutants in a. nidulans are viable but showed reduced growth at elevated temperatures and defects in morphogenesis [111, 112]. The double deletion pmta / pmtc (orthologues of the pmt2 and pmt4) and pmtb / pmtc (orthologues of pmt1 and pmt4) are synthetically lethal . Have shown that a single deletion of a. fumigatus pmt1 results in temperature - sensitive phenotypes . When the a. fumigatus pmt1 mutant was grown on solid complete medium at 37c, no difference was found between the mutant and the wild type . A strongly retarded growth, however, was observed when this mutant was grown at 42c and 50c . This temperature - sensitive phenotype could be complemented by the addition of 1 m sucrose in the media . Further analysis shows that the mannoprotein, -glucan, and chitin in the cell wall of the mutant grown at 37c are increased, while -glucan is reduced . When the a. fumigatus pmt1 mutant was cultured at 42c, the -glucan was increased, while the -glucan was decreased, and the mannoprotein and chitin content remained unchanged . Moreover, deficient conidiation and reduced germination have been documented at 42c . As compared with the s. cerevisiae pmt1 mutants, the a. fumigatus pmt1 mutant, as well as the c. albicans and s. pombe pmt1 mutants, shows more severe defects in cell wall integrity . This significant phenotype could be explained by the fewer members of pmt family presented in a. fumigatus, c. albicans, and s. pombe . However, in a recent study by mouyna et al ., the a. fumigatus pmt1 mutant does not show any visible phenotype . In the report by zhuo et al ., the pmt1 deletion mutant was constructed by replacement of the entire coding region of the pmt1 in a. fumigatus strain cea17 (pyrg) with a pyrg cassette . Therefore, the genetic background of the pmt1 null mutant is pyrg pmt1, while in the report by mouyna et al ., the pmt1 mutant was constructed by transformation of a. fumigatus strain ku80 with a deletion cassette containing the e. coli phleomycin phosphotransferase gene (phle). Therefore, the major differences may be due to the different genetic background of the strains used in these two reports . The single pmt2 or double pmt1pmt4 deletion(s) are lethal [114, 115]. Fang et al . Reported that reduced expression of pmt2 leads to retarded growth, cell wall defects, abnormal polarity, and reduced conidiation; however, no temperature - sensitive growth was found . Interestingly, this is the first time that pmt2p is revealed to be involved in polarized growth . These observations suggest that a. fumigatus pmt2p is required for cell wall synthesis and morphogenesis and its function is distinct from that of a. fumigatus pmt1p . Disruption of a. fumigatus pmt4 leads to abnormal mycelial growth, poor conidiation, and abnormal polarity . Although an increased sensitivity to echinocandin, a 1,3-glucan synthase inhibitor, was observed in the a. fumigatus pmt4 null mutant, glucan synthase activity and 1,3-glucan content were not affected . In contrast to its counterpart in c. albicans, a. fumigatus pmt4 is not required for full virulence . The different functions associated with different a. fumigatus pmts are likely due to their different substrate specificities . Further investigation of the pmt mutants will be helpful for understanding their molecular mechanism, which will not only increase our understanding of the function of o - mannosylation in a. fumigatus, but also may deepen our understanding of the molecular basis of the human walker - warburg syndrome (wws) which features mutations in pomt1, a homologue of a. fumigatus pmt4p, and results in a failure of polarized growth during neuronal migration . Gpi anchoring is a conserved glycosylation process in eukaryotes, which enables many cell surface proteins such as cell surface enzymes, receptors, and adhesion molecules to be covalently anchored to the cell membrane . The core structure of the gpi anchor consists of a lipid group, myoinositol, glucosamine, several mannose residues, and a phosphoethanolamine group, which ultimately connects the gpi anchor to the protein via an amide bond . Although the number of mannose groups and the position of side - chains on the gpi anchors vary widely between species, a common core structure of etnman3glcn - pi is conserved in all gpi - anchored proteins found in protozoa, yeast, plants, and mammals (figure 6). Gpi anchoring is not essential in mammals at a cellular level as several gpi - deficient cell lines have been established . However, an acquired gpi - anchoring deficiency in haematopoietic stem cells causes paroxysmal nocturnal haemoglobinuria, a rare but serious human disease . Also an overexpression of pig - p, a protein of unknown function required for gpi anchor synthesis, has been noted in fetal down syndrome brain . In contrast to mammals, gpi anchor synthesis is essential in s. cerevisiae . In s. cerevisiae, many gpi - anchored proteins are known to be involved in morphogenesis and cell wall organization . One type is the gpi - mannoproteins covalently linked to cell wall -1,6-glucan which play important biological functions in filamentation, mating, flocculation, or adhesion to the external matrix [161167]. The second type are the gpi proteins associated with the plasma membrane which possess enzymatic activities able to modify cell wall polymers and are involved in altering cell morphology, such as -glucanase and -glucosyltransferase [168170]. Recent studies in a. fumigatus suggest that at least nine gpi - anchored proteins are common to filamentous fungi and yeast . Five of them are homologues of putative gpi - anchored yeast proteins that have been shown to play a role in cell wall morphogenesis . The gpi anchor is assembled at the er in multiple steps catalyzed by the concerted actions of approximately 20 proteins . The first step of gpi anchor synthesis is initiated by the transfer of n - acetylglucosamine (glcnac) from udp - glcnac to phosphatidylinositol (pi), which is catalyzed by the glycosylphosphatidylinositol - n - acetyl - glucosaminyltransferase (gpi - gnt) complex . The mammalian gpi - gnt complex consists of seven proteins, including pig - a, pig - h, pig - c, pig - p, gpi1, pig - y, and dpm2 . All except dpm2 have structural and functional counterparts in s. cerevisiae, where they are known as gpi3p, gpi15p, gpi2p, gpi19p, gpi1p, and eri1p, respectively . Pig - a / gpi3p is believed to possess the catalytic domain because gpi3p binds a photoactivatable udp - glcnac analog and is a member of glycosyltransferase family 4 of retaining glycosyltransferases . The roles of the other subunits in the gpi - gnt complex are as yet unclear, but they may mediate regulatory interactions . In yeast, gpi anchoring is essential for viability and plays an important role in the biosynthesis and organization of the cell wall . A gpi3 temperature - sensitive mutant is not viable at 37c [116, 117]., it is postulated that the introduction of mutations in gpi3/gpig-1 genes allows for minimal level of product function and survival when growing the mutant cells below the restrictive temperatures . However, the mechanism, by which the defect in gpi anchoring leads to a lethal phenotype in these two species, is poorly understood . It has been shown that a. fumigatus gpi anchors possess five mannose residues with a phosphoethanolamine linked on the first three residues [174, 175]. The a. fumigatus pig - a gene, the homologue of the gpi3/pig - a gene in yeast, has been investigated . Also, an increased content of -glucan and mannoprotein was observed in the mycelial cell wall of the afpig - a mutant . Unlike the temperature - sensitive or conditional lethal phenotype seen in the yeast gpi3 mutant, afpig - a can survive at temperatures from 30c to 50c . Completely blocking gpi anchor synthesis in a. fumigatus afpig - a leads to cell wall defects, abnormal hyphal growth, rapid conidial germination, and aberrant conidiation . In vivo assays therefore, the gpi anchor seems not essential for viability, but required for cell wall integrity, morphogenesis and virulence in a. fumigatus . Indeed, this is the first report that a deficiency in gpi - anchor synthesis does not lead to a temperature - sensitive or conditional lethal phenotype in microbes, which provides an opportunity to identify the basic function of gpi anchoring in fungi . During the past 50 years, proteins and nucleic acids have dominated the field of biology . The enormous advances in the analysis of complex carbohydrates have enabled us to investigate the structure and function of carbohydrates, and the field has developed enormously . It is now known that carbohydrates play very important roles, especially in the regulation of development of higher organisms . However, the mechanisms by which carbohydrates play a role in development and diseases are still poorly understood . Our knowledge of protein glycosylation comes mainly from the investigation of s. cerevisiae and mammalian cells . Although investigations of the model yeast and mammalian cells have been very useful in elucidating the biochemical features of protein glycosylation, these investigations at the cellular level cannot reflect the complicated functions of glycosylation in the development of multicellular eukaryotes . Therefore, more model systems have been introduced, such as caenorhabditis elegans, drosophila melanogaster, and mice . However, these model systems are still too complex since deletion of individual glycosyltransferase genes in these systems sometimes leads to fetal death or nonvisible phenotypes . As compared with s. cerevisiae, c. elegans, d. melanogaster, or mice, a. fumigatus seems to be an ideal model for investigation of the function of glycosylation since a. fumigatus is a multicellular eukaryote with a relative simple life cycle, in which it undergoes a series of developmental events that require polarized growth . Recent progress shows that a. fumigatus has evolved an intact n - glycan - dependent qc system, which is present in mammalian cells but not in yeast . Disruption of either processing or degradation of n - glycan in a. fumigatus leads to phenotypes such as cell wall defects and abnormal polarity . Based on investigations of s. cerevisiae and filamentous fungi, it is likely that glycosylation first evolved to ensure synthesis of the fungal cell wall and only later did the n - glycan - dependent qc system evolve to ensure precisely controlled cell wall synthesis and polarized growth which are important for multicellular development . However, this hypothesis is controversial . . Showed that the n - glycan - dependent qc system is functional in entamoeba, trichomonas, cryptococcus, and s. pombe, but is not functional in some fungi such as giardia and plasmodium, theileria, encephalitozoon, toxoplasma, cryptosporidium, and tetrahymena . They proposed that the n - glycan - dependent qc system was likely present in the common ancestor of extant eukaryotes and was secondarily lost from some eukaryotes . For example, the s. cerevisiae kre5 is believed to be the gt ortholog that no longer glucosylates misfolded glycoproteins but is instead thought to be involved in -1,6-glucan synthesis . Of course, the possibility that the s. cerevisiae kre5 is the ancestor of gt cannot be excluded . It remains unclear where is the evolutionary origin of glycosylation, what is the basic function of glycosylation at the early stages of evolution, and how glycosylation is regulated . Definitely, the answers to these questions will enable us to understand the basic function and regulation of glycosylation in the development of multicellular eukaryotes and help to understand more complex functions in higher eukaryotes . On the other hand, the investigation of a. fumigatus is also a key to understanding complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development . During the past few years, the framework of the biosynthetic pathways of glycosylation in a. fumigatus has been delineated . Functional analyses of some of the genes in this pathway have shown that glycosylation is required for cell wall synthesis, polarity, morphogenesis, and cellular function in a. fumigatus (figure 7 and table 2). However, a detailed understanding of this pathway remains unknown, such as details regarding the synthesis of the n - glycan precursor, the precise molecular mechanism of n - glycan processing, qc of protein folding, and modification of the gpi anchor . Moreover, the molecular mechanisms by which glycosylation plays a role in morphogenesis and development of a. fumigatus are vaguely understood . Therefore, the future direction would be looking for those key proteins that are affected by glycosylation and identifying the signal transduction pathways that link glycosylation and development, through genetic, biochemical, cell biological, and proteomic studies.
In patients with hf, increased raas contributes to the pathogenesis, and ace inhibitors reduce the activity of the raas by inhibiting the production of angiotensin ii . Two benchmark randomized controlled trials, namely the cooperative north scandinavian enalapril survival study7 and the studies of left ventricular dysfunction treatment trial,8 demonstrated that ace inhibitors reduce mortality and improved new york heart association class, exercise capacity and cardiac function in patients with hf with reduced ef . Subsequently, the assessment of treatment with lisinopril and survival study5 investigated whether ace inhibitors had favourable effects on the outcome of patients with hf with reduced ef in a dosedependent manner . In this trial, an ace inhibitor, lisinopril, at high dose (32.535 mg daily) significantly decreased death or hospitalization for any cause by 12% (p = 0.002) compared with that at low dose (2.55 mg daily), but not significantly decreased death by 8% (p = 0.128). Show that benazepril at supramaximal dose results in prolonged survival by 41% compared with that at low dose [95% confidence interval (ci) 0.360.98, p = 0.042]. Further research should reveal whether ace inhibitors at supramaximal dose reduce the mortality rate in patients with hf compared with those at high dose . Common side effects of ace inhibitors are hyperkalaemia, hypotension, cough, and impaired renal function . According to the severity of these adverse events, it is crucial to manage when to stop uptitration, reduce dose, or discontinue treatment . Five ace inhibitors, namely ramipril, enalapril, lisinopril, captopril, and trandolapril, are listed for the treatment of hf with reduced ef, and the starting dose and the target dose are also described in the current guidelines.3 however, it remains unclear which ace inhibitor should be administered or if there are differences between the drugs, because headtohead comparisons are still missing . Angiotensin receptor blockers would be expected to exert beneficial effects on the treatment of hf through the stronger inhibition of angiotensin ii than ace inhibitors . The evaluation of losartan in the elderly study ii study9 showed that losartan was noninferior to captopril in reducing mortality in elderly patients with hf with reduced ef (hazard ratio 1.13, 95% ci 0.951.35, p = 0.16), and the candesartan in heart failureassessment of reduction in mortality and morbidityalternative trial10 showed that candesartan reduces the risk of cardiovascular death or hospital admission for hf in patients intolerant of ace inhibitors compared to placebo (hazard ratio 0.70, 95% ci 0.600.81, p <0.0001). Subsequently, the heart failure endpoint evaluation of angiotensin ii antagonist losartan study6 revealed that losartan at high dose (150 mg daily) reduced death or hospitalization for hf in patients with hf with reduced ef, compared with losartan at low dose (50 mg daily) (hazard ratio 0.90, 95% ci 0.820.99; p = 0.027), indicating the doserelated therapeutic effect of arbs on the outcome of hf as well as ace inhibitors . In the swedish heart failure registry,11 the use of losartan was associated with significant increase in mortality in patients with hf compared with that of candesartan (hazard ratio 1.43, 95% ci 1.231.65, p <0.001), suggesting the different clinical effects among arbs . Common side effects of arbs are well known and include hyperkalaemia, hypotension, and impaired renal function, similar to ace inhibitors; however, arbs are believed to be better tolerated than ace inhibitors . Reports that 29 patients (29%) at supramaximaldose benazepril withdrew from this study, as did 12 patients (12%) at supramaximaldose valsartan . Doserelated increase in the development of adverse events in patients receiving both benazepril and valsartan is observed, which is inconsistent with the findings of previous studies using very highdose arb.12, 13 in view of these findings, arbs are considered second choice in patients with hf with reduced ef who are not tolerated to ace inhibitors or mineralocorticoid receptor antagonists in the current guidelines . Because of the different mechanism of action of ace inhibitors and arbs in blocking the raas, combination therapy of an ace inhibitor and an arb was thought to be attractive for the treatment of hf as well as the monotherapy with an arb . In the valsartan heart failure trial,14 the addition of valsartan to conventional therapy for hf resulted in a significant decrease in cardiovascular events compared to placebo (relative risk 0.87, 97.5% ci 0.770.97, p = 0.009); however, a post hoc analysis indicated increased mortality and complications in the subgroup taking an ace inhibitor, valsartan, and a betablocker . Moreover, a recent metaanalysis15 suggested that combination therapy of an ace inhibitor with an arb should not be advocated in patients with hf, because the combination does not seem to reduce mortality or hospitalization and was associated with more adverse events . As described earlier, an ace inhibitor, lisinopril, or an arb, losartan, at high dose produced better clinical outcomes in patients with hf compared with each at low dose but did not decrease mortality significantly.5, 6 on the other hand, he et al . Reports that supramaximal dose of benazepril or valsartan improves not only cardiac function but also survival in patients with dcm compared with the low dose of each medication,16 which indicates that ace inhibitors or arbs at supramaximaldose might produce better outcomes in patients with ef compared with those at high dose (target dose). These unique findings are derived from a singlecentre prospective, randomized, and controlled trial . The sample size is small, but the followup period is long enough to detect the statistical difference in the outcomes of hf among the treatment groups . They should be confirmed with doubleblind multicentre randomized controlled studies . Recently, ivabradine17 and lcz696, a combination of the new neprilysin inhibitor sacubitril (ahu377) with valsartan,18 have attracted attention for the treatment of hf . If the efficacy and safety of supramaximaldose valsartan were well acknowledged in this field, it would be helpful to investigate the appropriate dose of lcz696 more precisely.
Indian population representing one - sixth of the world population has been the global melting pot of human diversity . It has all the world s major linguistic groups and the populations have been shaped by different waves of migrations and admixture (1, 2). Further, stringent mating patterns have led to the existence of several endogamous populations, which makes it an important resource for mapping genes (3). The indian genome variation consortium (igvc) project, an initiative of the council for scientific and industrial research (csir)was set up to develop a database of genomic variations in indian population for predictive marker discovery in complex diseases such as diabetes, asthma, neuropsychiatric, infectious and cardiovascular disorders, response to drugs, etc . The phase i of the project was conducted to determine the extent of genetic differentiation in india . Toward this genotype data of 405 snps from 75 genes and 4.2 mb contiguous chromosome 22 regions were studied in 55 contrasting populations (4, 5). These populations were identified from 4 major linguistic groups namely, austro - asiatic (aa), tibeto - burman (tb), indo - european (ie) and dravidian(dr) spanning 6 geographical regions of habitat (n, north; ne, north - east; w, west; e, east; s, south; c, central) and different ethnic groups (lp, large population, caste; ip, isolated population, tribes; sp, special population, religious groups). Five genetically distinct clusters were identified and a set of 24 populations that represent these clusters were selected for the phase ii of the project . In the phase ii, 3824 snps from 834 candidate gene as well as 50 000 (affy 50 k array) genome wide neutral markers this initiative lays the foundation for the integration of global genotype - to - phenotype data (6) with indian population data and development of a federated database . To address the need for an online comprehensive resource that enables users to visualize igvc data with integrated information about snps from different resources we have developed igvbrowser as shown in figure 1 . Distribution of markers in 2.41 mb region in human chromosome 1 from igvc data is displayed along with annotation data from different resources . Distribution of markers in 2.41 mb region in human chromosome 1 from igvc data is displayed along with annotation data from different resources . The database includes (i) final validated dataset from 1871 samples in phase i comprising of 405 autosomal snps spanning over 75 genes including 90 snps from 5.2 mb region of chromosome 22 from 55 diverse endogamous indian populations (3); (ii) phase ii dataset for 3824 snps spanning from 834 genes in 545 samples from 24 igvdb populations and (iii) 50 000 (affy 50k xbai array) neutral markers in 26 populations . The phase ii populations are a subset of the populations genotyped in the phase i. web - based tool snpper (http://snpper.chip.org/) was used to classify the 4229 markers in phase i and phase ii according to their location in genic regions (figure 2). Similarly, david (http://david.abcc.ncifcrf.gov/) was used to classify the genes containing these markers according to gene disease association class (figure 3) and their mapping in various kegg pathways (figure 4). We report that a large fraction of genes are implicated in cardiovascular, metabolic, cancer and immune system - related diseases . Thus, the igvc data provide a basal level variation data in indian population to study genetic diseases and pharmacology . More than 50% of the snps belong to intronic regions and 15% are in coding exons . Figure 3.bar graph shows the functional annotation of candidate genes in igvc according to gene disease association . Figure 4.bar graph shows the mapping of candidate genes in significant pathways (after bonferroni correction) of kegg pathway database . More than 50% of the snps belong to intronic regions and 15% are in coding exons . Bar graph shows the mapping of candidate genes in significant pathways (after bonferroni correction) of kegg pathway database . Igvbrowser also included hapmap snp genotype data from phases i + ii and iii of the hapmap project (http://hapmap.ncbi.nlm.nih.gov/downloads/gbrowse/2009-02_phaseii+iii/gff/) based on ncbi b36 assembly, dbsnp b126 from 4 populations: yoruba from ibadan, nigeria (yri); japanese in tokyo, japan (jpt); han chinese in beijing, china (chb); and ceph (utah residents with ancestry from northern and western europe) (ceu). Additional annotation information including cytogenetic positions, link to pathway annotations in the reactome knowledgebase and mrna sequences were retrieved from hapmap in generic feature finding (gff) format . Annotation data in tab - delimited format for non - coding rna genes and pseudogenes, omim - associated genes, mirbase and snornabase, simple repeats, database of genomic variants were downloaded from ucsc genome annotation database (http://hgdownload.cse.ucsc.edu/goldenpath/hg18/database) based on build hg18 . The browser implements one of the widely used platform - independent genome annotation viewer generic genome browser (gbrowse v1.69), developed by stein et al . (7) as a part of the generic model organism system database project (http://www.gmod.org). Gbrowse is a combination of database and interactive webpage for displaying genomic information along with providing data interoperability across systems running the same software . Integrated annotation data from primary sources like ncbi, ucsc and hapmap have been linked with variation data from different ethnic populations in india . Compiled data processed into gff format and complete human genome sequence as plain text files were loaded into mysql relational database management system using a script of gbrowse . A user can query chromosomal region of interest, reference snp i d, hgnc symbols, pathway name or any other unique feature recognized by database as a query . It allows researchers to upload their own data in gff format and view it along with data available in igvbrowser . In addition, the resource is facilitated with sequence analysis servers maintained by ncbi and ucsc . Online data analysis plugins allows text dumps of visible features using a number of standard formats and also facilitates the download of sequence corresponding to selected region . The browser implements one of the widely used platform - independent genome annotation viewer generic genome browser (gbrowse v1.69), developed by stein et al . (7) as a part of the generic model organism system database project (http://www.gmod.org). Gbrowse is a combination of database and interactive webpage for displaying genomic information along with providing data interoperability across systems running the same software . Integrated annotation data from primary sources like ncbi, ucsc and hapmap have been linked with variation data from different ethnic populations in india . Compiled data processed into gff format and complete human genome sequence as plain text files were loaded into mysql relational database management system using a script of gbrowse . A user can query chromosomal region of interest, reference snp i d, hgnc symbols, pathway name or any other unique feature recognized by database as a query . It allows researchers to upload their own data in gff format and view it along with data available in igvbrowser in addition, the resource is facilitated with sequence analysis servers maintained by ncbi and ucsc . Online data analysis plugins allows text dumps of visible features using a number of standard formats and also facilitates the download of sequence corresponding to selected region . Indian genome variation data would be enormously useful for the dissection of common complex diseases and in pharmacogenomics studies . Frequency profiles of markers on disease or drug - related genes that have been generated through the igvc are being used to identify at - risk chromosomes, founders, ld - based mapping, tracing history of diseases in pharmacogenetics as well as reference populations for mapping relatedness (3,4,5,819). The interactive web browser, igvbrowser, has been created as a central repository for the current and future dataset on indian populations and is being made accessible in the public domain . A possible integration of igvbrowser with hgvbaseg2p (20) can enable researchers for cross study comparison among different populations of the world for disease gene association study . Indian genome variation project was funded by the council for scientific and industrial research programme cmm0016 and sip0006 . Funding for igvbrowser and open access charge is provided by european community's seventh framework programme (fp7/2007 - 2013) under grant agreement number 200754the gen2phen project.
Coronary artery disease (cad) remains the principal cause of mortality and morbidity around the world [1, 2]. Although the mechanisms are still not entirely resolved, previous studies have demonstrated that an imbalance between t - helper 1- (th1-) mediated and th2-mediated immune functions contributes to the development of atherosclerosis . Recently, th17, which are a newly defined subset different from th1 and th2, reactive to autoantigens are involved in the pathogenesis of several autoimmune diseases [4, 5]. In addition to th1, th2, and th17 lineage, regulatory t cells (tregs), a special subset of cd4 t cells, inhibit atherosclerosis development by attenuating activated t cell responses [6, 7]. Inspiringly, previous clinical and experimental studies from our laboratory have found that th17/tregs functional imbalance exists during atherogenesis, indicating that the imbalance of th17/tregs plays a critical role in cad [8, 9]. Hence, these findings have showed that cd4 t cell subpopulations play an important role in the initiation and progression of atherosclerosis . It is well known that cd4 t cells are divided into different subsets depending on the cytokines they produce . Interferon- (ifn-), the principal cytokine of th1 cells, is proinflammatory and exacerbates atherosclerotic disease, while the th2 cytokine such as interleukin- (il-) 4 is considered to be mainly atheroprotective and can neutralize th1 cytokine activity [1012]. Moreover, hashmi and zeng have found that il-17 and il-17 induced cytokines (il-6 and il-8) were significantly increased in acs patients . Interestingly, tregs exert antiatherosclerosis partly by secretion of anti - inflammatory cytokine transforming growth factor (tgf-1), which can in turn induce the expression of forkhead box transcription factor p3 (foxp3) and promote differentiation of tregs derived from cd4cd25 t cells [15, 16]. Therefore, these findings suggest that cytokines as those mentioned above are also essential for the formation and progression of the atherosclerotic plaque . The galectin family is characterized by conserved carbohydrate recognition domains (crd) that can bind glycosylated proteins . Galectins are involved in a wide range of biologic processes such as cell adhesion and migration, proliferation and apoptosis, tumor biology, and regulation of the immune system . Galectin- (gal-) 9 is a member of the galectin family of carbohydrate - binding proteins comprising two carbohydrate recognition domains connected by a linker peptide . Gal-9 is mainly expressed by eosinophils, endothelial cells, macrophages, dcs, kupffer cells, vascular endothelial cells, intestinal epithelial cells, and in particular t lymphocytes . Furthermore, gal-9 is believed to function predominantly as a multifaceted player in adaptive and innate immunity, especially in t cell development and homeostasis [2023]. Accumulating evidence shows that gal-9 induces apoptosis in subsets of differentiated t cells, particularly in th1 and th17 cells, and stimulates the activity of tregs [19, 2428]. Indeed, treatment with recombinant gal-9 moderated the progression of experimental autoimmune encephalomyelitis (eae), arthritis, and diabetes in animal models [19, 2931], by reducing the number of th1 and th17 cells and downregulating circulating ifn- levels . T cell immunoglobulin mucin- (tim-) 3 has been identified as a receptor for gal-9 . Although gal-9 can function in a tim-3-independent fashion, the immune - regulatory effects of gal-9 are largely attributed to gal-9-tim-3 pathway [3235]. More recently, foks et al . Demonstrated that anti - tim-3-ab administration promoted atherosclerotic plaque formation, which indicated that tim-3-gal-9 pathway may be concerned with the development of atherosclerosis . Thus, it is reasonable to postulate that gal-9 may be involved in atherosclerosis based cad . In addition, several findings strongly support the original experimental observations that gal-3 plays an important role in the underlying heart failure (hf) processes and that elevation of gal-3 is associated with disease progression and poor outcome in hf [3739]. Herein, we investigate serum gal-9 levels in chinese patients with cad, and the severity of coronary arteries stenosis was evaluated by gensini score . Furthermore, ifn-, il-4, il-17, tgf-1, high - sensitivity c - reactive protein (hs - crp), and the classical atherosclerosis risk factors were evaluated . Additionally, th1, th2, th17, and cd4cd25foxp3 tregs differentiation were detected in pbmc cultures exposed to gal-9 . 232 patients presenting at the department of cardiology of huazhong university of science and technology affiliated union hospital between jan . 2013 with suspected or established cad were recruited, including 149 males and 83 females . They were divided into four groups: (1) the control group, which consisted of 50 subjects with nca (28 men and 22 women, mean age 56.7 11.7); (2) sap group (25 men and 15 women, mean age 62.4 10.0, inclusion criteria: typical chest discomfort that was associated with horizontal or downsloping st - segment depression> 1 mm in an exercise test); (3) nsteacs group (60 men and 30 women, mean age 60.6 9.9, which included unstable angina pectoris (uap) and non - st - segment elevation myocardial infarction (nstemi), inclusion criteria: chest pain at rest with definite ischemic electrocardiographic changes, t - wave inversions and/or st - segment changes, or typical ischemic chest pain which persists for more than 20 min, elevated serum myocardial necrosis markers concentration and dynamic evolution, but not the typical st - elevation electrocardiogram); (4) stemi group (36 men and 16 women, mean age 54.1 12.3, inclusion criteria: myocardial infarction that was confirmed by a significant increase of serum creatine kinase mb, troponin i levels, and elevation of st - segment). Furthermore, all participants underwent coronary angiography after admission and completed a standardized questionnaire about previous and present illness, medication history, and smoking status . Cardiovascular - interrelated factors, such as age, body mass index (bmi), gender, and ejection fraction (ef) were estimated via physical examination, ultrasonic cardiography (ucg), and electrocardiogram (ecg), respectively . Patients with the following characteristics were excluded from study enrollment: valvular heart disease, thromboembolism, autoimmune diseases (systemic lupus erythematosus, rheumatoid arthritis, etc . ), collagen disease, disseminated intravascular coagulation, severe liver and kidney disease, symptomatic heart failure, trauma or surgery, or malignant disease . The study was approved by the ethics committee of tongji medical college of huazhong university of science and technology, and all participants were provided written informed consent prior to study entry . Two experienced cardiologists, who were not aware of the patients' clinical and biochemical results, visually examined angiographic images to assess the extent and severity of cad . Cad diagnosis was made according to the presence of 50% stenosis in 1 main coronary artery . The stenosis severity of cad was assessed by gensini score as previously described . In the ami group, the blood was drawn with a 21-gauge needle for clean venipuncture of an antecubital vein and the samples were collected into sodium heparin vacutainers (becton - dickinson). After centrifugation, the serum obtained was stored at 80c until further use to measure the concentration of gal-9 . The level of gal-9 was measured by elisa according to the manufacturer's instructions (r&d systems). In addition, the levels of ifn-, il-17 (bolingkewei biotechnology, china), il-4, hs - crp (huijia biotechnology, china), and tgf-1 (fumeng gene biotechnology, china) were measured by elisa, following the manufacturer's instructions . The minimal detectable concentrations were 28 pg / ml for gal-9, 1.8 pg / ml for ifn-, 1.8 pg / ml for il-17, 0.4 pg / ml for il-4, 22 pg / ml for hs - crp, and 8 pg / ml for tgf-1 . The intra - assay and interassay coefficients of variation for all elisa were <5% and <10%, respectively . All the other biochemical measurements, including serum total cholesterol (tc), triglyceride (tg), low - density lipoprotein cholesterol (ldl - c), high - density lipoprotein cholesterol (hdl - c), apolipoprotein a - i (apoa - i), apolipoprotein b (apob), lipoprotein(a), creatinine, fasting plasma glucose (fpg), uric acid, and cardiac troponin i (ctni), were carried out by the biochemical laboratory of our cardiovascular institute using standard methods . Peripheral blood mononuclear cells (pbmcs) from nca (excluded from hypertension, diabetes, and dyslipidemia) were isolated by ficoll - hypaque density gradient centrifugation (pharmacia lkb technology, uppsala, sweden). Cells from the interphase were collected and washed twice with dulbecco's phosphate buffered saline (d - pbs). Remaining erythrocytes were removed using lysis buffer (4.14 g nh4cl, 0.5 g khco3, and 18.6 mg na2edta in 500 ml water, ph = 7.4 and sterilized) for 5 min on ice . Pbmcs were resuspended in rpmi1640 (lonza, verviers, belgium) supplemented with 2.5% fcs, penicillin (100 u / ml)/streptomycin (100 u / ml), and sodium pyruvate (1 mm, sigma). Pbmcs were stimulated with anti - human cd3 and anti - human cd28 antibodies (both 2 g / ml, sanquin) in the presence of recombinant gal-9 (m) (0.010.1 m, prospec - tany technogene ltd ., israel) for 24 h. cells were allocated into tubes and washed once in phosphate buffered saline (pbs). For tregs analysis, the cells were incubated with anti - cd4-fitc - hab and anti - cd25-apc - hab (bd pharmingen). After the surface staining, the cells were stained with anti - foxp3-pe - hab (bd pharmingen) after fixation and permeabilization according to the manufacturer's instructions . For analysis of th1, th2, and th17 cells, the cells were stimulated with phorbol myristate acetate (pma, 20 ng / ml, alexis biochemicals, san diego, ca) and ionomycin (1 g / ml, alexis biochemicals) for 4 h in the presence of 2 mol / ml monensin (alexis biochemicals). The incubator was set at 37c under a 5% co2 environment . After culture for 4 hours, the cells were collected for staining according to the instructions . Fixation and permeabilization were necessary before staining with anti - ifn--pe-, anti - il-4-pe-, or anti - il-17-pe- hab (bd pharmingen). Continuous variables are summarized as mean standard deviation (sd), and categorical data are presented as percentages . The shapiro - wilk test was used to assess the normality of distribution of continuous variables . In order to compare two groups, continuous variables were tested using the independent samples t - test for normally distributed data and the mann - whitney u test for nonnormally distributed data; the chi - square test was used for categorical variables . When three or more groups were compared, one - way anova was used . If significance was found, newman - keuls test was performed for post hoc analysis to detect the difference among groups . Multiple stepwise regression analysis was used to evaluate the influence of different variables on gal-9 and to adjust for covariates . Independent factors were sex, age, ctni, and the metabolic - related variables including bmi, fpg, lipid profiles, and hs - crp . To determine the independent predictors for the presence and severity of cad, all the conventional risk factors associated with cad were tested in multiple stepwise regression analysis . The prevalence of smoking and the levels of tg, lipoprotein(a), fpg, creatinine, hs - crp, and ctni were significantly higher in patients with cad compared to patients with nca group (all p <0.05). However, other biochemical results, including tc, hdl - c, ldl - c, and uric acid, were similar between nca and cad patients . Compared with stemi group, patients in sap and nsteacs groups showed markedly higher hdl - c levels and age and lower levels of lipoprotein(a), fpg, hs - crp, and ctni (all p <0.01). Compared to patients with sap, the use of aspirin, -blockers, and statins was rare in patients with acs (all p <0.05), whereas the levels of lipoprotein(a) and hs - crp were markedly higher in patients with acs (all p <0.01). A significant increase of creatinine levels was observed in patients with stemi compared with nsteacs group (p <0.05) and an obvious decrease of uric acid levels was found in patients with stemi compared to sap group (p <0.01). Unexpectedly, the distribution of hypertension, diabetes mellitus, dyslipidemia, and family history was similar among patients with acs and sap . Among the total 232 study participants, serum gal-9 levels ranged from 1733.86 to 5259.39 pg / ml . Compared with the nca group, patients with cad had significantly lower levels of gal-9 (3283.55 587.59 versus 3565.97 544.37 pg / ml, p <0.05; figure 1(a)). In addition, we found that serum gal-9 levels were significantly lower in the stemi (3126.36 637.7 pg / ml) than those in the sap group (3607.91 541.35 pg / ml) or the nca group (stemi versus sap and nsteacs versus sap, all p <0.01; stemi versus nca and nsteacs versus nca, all p <0.01; figure 1(b)). Interestingly, serum gal-9 levels did not differ significantly between patients with nsteacs and stemi (p> 0.05), nor was there a difference between the sap and nca groups (p> 0.05; figure 1(b)). As shown in figure 2, serum il-17, il-4, ifn-, and tgf-1 were detected in each group . The il-17 and ifn- concentrations in patients with stemi (il-17, 57.16 16.14 pg / ml; ifn-, 13.43 5.29 pg / ml) and nsteacs (il-17, 47.68 14.46 pg / ml; ifn-, 11.28 4.23 pg / ml) were significantly higher compared with those in patients with sap (il-17, 26.94 9.09 pg / ml; ifn-, 6.05 2.41 pg / ml) and nca groups (il-17, 24.00 10.14 pg / ml; ifn-, 5.83 2.12 pg / ml) (all p <0.01; figures 2(a) and 2(c)). Il-4 concentrations showed no difference in any of the groups (stemi, 7.25 3.82 pg / ml; nsteacs, 7.22 3.87 pg / ml; sap, 8.12 4.48 pg / ml; nca, 6.83 3.25 tgf-1 concentrations in patients with stemi (4.08 2.13 ng / ml) and nsteacs (4.73 2.71 ng / ml) were significantly lower than those in patients with sap (8.03 3.88 ng / ml) and nca (9.02 4.86 ng / ml) (all p <0.01; figure 2(d)). Importantly, gal-9 levels were negatively correlated with il-17 (r = 0.45, p <0.001; figure 3(a)) and ifn- (r = 0.53, p <0.001; figure 3(c)) but positively associated with tgf-1 (r = 0.58, p <0.001; figure 3(d)). However, gal-9 levels showed no correlation with il-4 concentrations (r = 0.04, p = 0.528; figure 3(b)). To detect the involvement of gal-9 in the induction of differentiation of human cd4 t cells gal-9 expanded the number of cd4cd25foxp3 tregs (control, 6.99 1.61%; gal-9 (0.01 m), 9.24 2.04%; gal-9 (0.03 m), 10.27 3.04%; gal-9 (0.1 m), 11.24 3.23%) and decreased the development of th17 (control, 0.628 0.239%; gal-9 (0.01 m), 0.418 0.171%; gal-9 (0.03 m), 0.214 0.064%; gal-9 (0.1 m), 0.059 0.015%) dose dependently (figures 4(c) and 4(d)), with resulting increase in secretion of tgf-1 (control, 165.3 48.6 pg / ml; gal-9 (0.01 m), 227.8 72.4 pg / ml; gal-9 (0.03 m), 312.9 81.7 pg / ml; gal-9 (0.1 m), 528.1 97.4 pg / ml) and suppressed il-17 production (control, 132.4 23.6 pg / ml; gal-9 (0.01 m), 110.3 25.7 pg / ml; gal-9 (0.03 m), 81.9 18.0 pg / ml; gal-9 (0.1 m), 56.6 13.8 however, the development of th1 and th2 cells and secretion of ifn- and il-4 in the supernatant were similar in the gal-9 untreated and treated pbmcs (figures 4(a), 4(b), and 4(e)). As we expected, gal-9 levels were found to be negatively correlated with hs - crp (r = 0.37, p <0.001; figure 5(a)), the gensini score (r = 0.23, p = 0.001; figure 5(b)), fpg (r = 0.15, p = 0.027; figure 5(c)), and lipoprotein(a) (r = 0.16, p = 0.012; figure 5(d)) but positively associated with creatinine (r = 0.21, p = 0.002; figure 5(e)) and age (r = 0.14, p = 0.028; figure 5(f)). To determine which variables were independently associated with gal-9, multiple stepwise regression analysis using gal-9 as the dependent variable identified sex, age, bmi, fpg, tc, tg, hdl - c, ldl - c, hs - crp, lipoprotein(a), creatinine, uric acid, ctni, and therapeutic use of statins as independent variables . After adjustments, only hs - crp (b = 72.158, p <0.001), lipoprotein(a) (b = 8.333, p = 0.045), creatinine (b = 5.228, p <0.001), and use of therapeutic statins (b = 362.29, p <0.001) retained significance for independently predicting gal-9 (table 3). In order to determine which variables gensini score was the dependent variable, and sex, age, bmi, fpg, lipid profiles, gal-9, hs - crp, uric acid, traditional risk factors, and statins were independent variables . After adjustments, only gal-9 (b = 0.145, p = 0.011), age (b = 0.276, p <0.001), and lipoprotein(a) (b = 0.280, p <in the present study, we found that participants with cad had lower serum gal-9 levels compared with nca . Furthermore, serum gal-9 levels in the acs group were significantly lower than those in the sap or nca group . Moreover, gal-9 was found to be independently associated with hs - crp, lipoprotein(a), and creatinine . Notably, gal-9 suppressed t - helper 17 (th17) and expanded regulatory t cells (tregs) as analyzed within the activated cd4 t cell population, resulting in decreased il-17 production and increased secretion of tgf-1 . To the best of our knowledge, this is the first study to investigate the relation of serum gal-9 levels with the presence and the severity of coronary arteries stenosis . Mor et al . Have reported that cd4cd25 tregs deficiency enhanced atherosclerotic lesion development in ldlr mice, and adoptive transfer of cd4cd25 tregs attenuated the initiation and progression of atherosclerosis in apoe mice [42, 43]. Moreover, our laboratory has detected that th17/treg functional imbalance exists in patients with acs, suggesting a potential role for th17/treg imbalance in plaque destabilization and the onset of acs [8, 9]. Of note, gal-9 is a multifunctional modulator of t cell immunity with apoptotic effects on th17 cells and stimulatory activity on tregs in autoimmune diseases . In line with this publication, we found that gal-9 was able to induce cd4cd25foxp3 tregs development and inhibit th17 differentiation in activated pbmcs of nca . Therefore, we speculated that gal-9 plays an immune - regulatory role in atherosclerosis via its effects on tregs and th17 cells . Acs patients have significantly higher il-17 and il-17 induced cytokines (il-6 and il-8), while the pleiotropic cytokine ifn- is a proinflammatory regulator that is expressed at high levels in atherosclerotic lesions . In addition, it has been demonstrated that increased expression of tgf-1 is a stabilizing factor in human atherosclerotic plaques . In this study, we also found that il-17 and ifn- concentrations in patients with stemi and nsteacs were significantly higher compared with those in patients with sap and nca, while tgf-1 concentrations in patients with stemi and nsteacs were significantly lower than those in patients with sap and nca . These results again confirmed that inflammatory cytokines mentioned above have differential effects in atherosclerosis based cad . Interestingly, gal-9 levels were showed to be negatively correlated with il-17 and ifn- but positively associated with tgf-1 . Furthermore, we found that gal-9 was able to increase secretion of tgf-1 and decrease il-17 secretion in activated pbmcs . Consequently, our data in vitro imply a protective role for gal-9 in atherosclerosis via expanding tgf-1 and suppressing il-17 secretion . However, the precise effect and mechanism of gal-9 in atherosclerosis are still to be elucidated in atherosclerosis animal models . Surprisingly, we found that the development of th1 cells and secretion of ifn- in the supernatant were similar in gal-9 untreated and treated pbmcs, since zhu et al . Reported that gal-9 induces apoptosis in th1 cells . This discrepancy may be ascribed to the diverse concentrations of gal-9 and different incubation periods . Additionally, il-4 concentrations in the supernatant showed no difference in gal-9 untreated and treated pbmcs . This result further confirms that the effect of il-4 is still controversial [45, 46], reflecting its dual pro- and anti - inflammatory character . Moreover, there are numerous publications showing that gal-9 has proinflammatory effects in addition to its immune - suppressive effects, especially through its actions on cells of the innate immune system like dc and nk cells [47, 48]. Thus, these results indicate that gal-9 has bidirectional effects like many immune - regulatory molecules . Our data showed that cad patients had lower serum gal-9 levels than those without cad, and gal-9 was independently associated with the gensini score in patients with cad . These results suggest that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis . Previous studies have elucidated that serum biomarkers are recognized as important tools for prediction, diagnosis, risk stratification, and therapeutic decision - marking for patients with cad . Accumulating studies demonstrated that atherosclerotic lesion instability and rupture induced by inflammation are the major mechanisms of acs [50, 51]. Recently, a large number of clinical experiments have reported that hs - crp is not only a biomarker of inflammations and atherosclerosis, but also a marker of atheromatous plaque vulnerability [5254]. In our study, patients with acs had higher serum hs - crp levels compared to patients with sap . Of note, gal-9 was found to be independently associated with hs - crp . Hence, low serum concentrations of gal-9 may take part in the onset of acs and may act as a predictor of atheromatous plaque vulnerability . However, whether a causal relationship exists between the two processes is still to be studied . Several lines of evidence indicate that lipoprotein(a) is an independent risk factor for patients with cad [55, 56]. All these findings together suggest that gal-9 might be a potential independent biomarker of cad . Interestingly, our data showed that serum gal-9 levels were higher in patients of cad complicated with t2 dm than those of cad without t2 dm (data not shown). 's study reporting that serum gal-9 levels are elevated in the patients with type 2 diabetes (t2 dm) and chronic kidney disease (ckd). However, serum gal-9 levels were decreased in cad and elevated in the patients with t2 dm . This paradox could be attributed to the fact that atherosclerosis is highly multifactorial, tregs and gal-9 being only some factors among many others . The exact mechanism is still to be elucidated but different diseases may have divergent immune states . Worth noting is the fact that the serum gal-9 level seems relatively higher in our study than in the previous studies [22, 57]. The differences among these studies might be explained, at least partly, by different experimental designs, different number and characteristics of participants recruited, ethnical differences, medical treatments, lack of standardization of methods for the determination of serum gal-9 levels, and different ways in which samples were handled . Nevertheless, several limitations of the present study should be considered . Firstly, the number of study participants (232) was relatively small . Secondly, given the cross - sectional design of the study, the causal relationship between gal-9 level and the severity of coronary artery stenosis cannot be determined . Thirdly, different methods of assessment of the coronary stenosis and the criterion for diagnosis of the diseased artery may lead to another result . Finally, although we accounted for confounding of traditional cardiovascular risk factors, a potential for uncontrolled or residual confounding that could influence the relationship between gal-9 and cad would be plausible . In conclusion, we have shown that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis . Importantly, gal-9 can expand cd4cd25foxp3 tregs and inhibit th17 development in activated pbmcs, leading to increased secretion of tgf-1 and decreased il-17 secretion . Our observations provide evidence of the role of gal-9 in cad and that gal-9 is an independent negative predictor of acs, which may also at least partially explain the protection of gal-9 against coronary atherosclerotic plaque vulnerability . Large - scale population - based clinical studies and further experimental studies are needed to elucidate the exact effect and potential biological mechanisms of gal-9 in the pathogenesis of cad and to evaluate whether gal-9 is a potential novel biomarker of cad.
Sedation is often required in young children in order to obtain good diagnostic cross - sectional imaging like computed tomography scan (ct scan) and magnetic resonance imaging (mri). Sometimes sedation is also needed for older children with complex neurodevelopmental disorders, such as autism and attention deficit hyperactivity disorder . Moderate sedation is unable to guarantee patient compliance; therefore, a deeper level of sedation is required [1, 2]. The success of sedation for mri has typically been measured by two factors: the safety of sedation procedure (lack of adverse events) and its effectiveness (completion of diagnostic examination). It can be challenging to obtain the deep sedation level required to prevent the patient's movement while maintaining respiratory and hemodynamic stability . Therefore, it is very important to select the appropriate drugs and dosage to achieve those objectives . Dexmedetomidine is a highly selective adrenoceptor agonist with a distribution half - life of six minutes and an elimination half - life of two hours . Five prospective trials have evaluated the efficacy of dexmedetomidine for sedation during noninvasive radiologic imaging [812]. Dexmedetomidine can be associated with side effects of hypotension, bradycardia, and transient hypertension with loading dose . Dexmedetomidine has been used at our institution since 2006 for mri and other noninvasive radiologic procedures . The purpose of this retrospective study is to present our institutional experience with dexmedetomidine in relation to efficacy and related side effects . Collection of quality assurance data includes patient demographics, adverse events, physiologic variables, drug dosages, the time required to sedate the patient, time needed to obtain the imaging study, and recovery time . After approval by the institutional review board, we conducted a retrospective analysis of all patients who received sedation for mri from july 2007 to december 2012 . Institutional sedation policies are based on guidelines by the joint commission on accreditation of health care organization and american academy of pediatrics and were closely followed [13, 14]. Vital signs including pulse oximetry, heart rate, noninvasive blood pressure monitoring, and nasal capnography (with concomitant oxygen delivery via the nasal cannula) are continuously monitored and documented every five minutes throughout sedation . At the start of sedation, intravenous dexmedetomidine was bolused at 2 g / kg over 10 minutes followed by a continuous infusion of 1 g / kg / h . Most of the patients were sedated by the end of the bolus; if not, a second bolus of 2 g / kg was repeated over another 10 minutes prior to the start of the maintenance infusion . Patients who continue to move after the second bolus or while the procedure is in progress, can compromise the quality of imaging and were given additional medications like midazolam or fentanyl as per physician discretion . Mg / kg to a max of 2 mg and for fentanyl was 0.5 mcg / kg1 mcg / kg to a max of 50 mcg . The goal was to achieve a minimum ramsay sedation score (rss) of 4 as assessed by a sedation nurse . Rss is a clinically derived sedation score generally accepted as a tool for assessing the depth of sedation . Usually, a score of 4 - 5 is targeted to ensure appropriate sedation for diagnostic imaging studies [11, 16]. Peak onset of the sedation was defined as the time from the start of the loading dose to achievement of a ramsay score of 4 . Procedure time was the time of achieving the required ramsay score to the end of the procedure (stoppage of drug administration). Discharge time was defined as time from the end of the procedure to actual time when the patient was discharged home . Normal ranges for heart rates, blood pressure, and respiratory rates for data analysis were based on the published normal of fleming et al . [17, 18]. A trained registered nurse provided continuous assessment and monitoring while the procedure was in progress under the direct supervision of a pediatric intensivist . Monitoring was continued until the patient was awake with a minimum aldrete score of 9 points, and the patient has tolerated clear liquids prior to discharge [19, 20]. All the parents were provided with written discharge instructions and a direct phone number for further assistance or to report any adverse effects . Database records were analyzed using dedicated statistical software sas v9.3 (sas institute, cary, nc). Changes in the vital signs associated with the use of dexmedetomidine from the baseline were evaluated and compared using student's t - test and mann - whitney rank - sum test, depending on the distribution of the data . The entire study cohort was divided into three groups depending on dexmedetomidine bolus and additional medications received . Dexmedetomidine group a received one bolus; dexmedetomidine group b received two boluses; dexmedetomidine group c received one or two boluses and additional medications . Three groups were compared with respect to age, weight, blood pressure, heart rate, procedure time, and discharge time using analysis of variance . Incidences of bradycardia and hypotension were analyzed with fisher's exact test, due to low cell counts . Discharge time of bradycardia and hypotensive patients was compared with normal cohorts using the t - test . The most common indications included seizure disorder, developmental delay and behavioral disorder, autism, and neoplasia . 382 (70.2%) of the total patients received one - time bolus, while two boluses were given to 162 patients (29.8%). In the population of 544 patients, additional medications (fentanyl or midazolam) were required in 117 (21.5%) for spontaneous movements to avoid motion artifacts that can compromise mri quality (54 patients with one - time bolus and 43 patients with two boluses). Dexmedetomidine - induced vital sign changes from baseline for the whole group are shown in figure 1 . Hypotension (systolic blood pressure 20% below the normal limits) was observed in 100 patients (18.4%) and transient hypertension (systolic blood pressure> 20% above age - specific high limits) was observed in 137 of the patients (25.2%). There were 210 (38.6%) patients who had a significant decrease in respiratory rate, that is,> 20% from the baseline, but no desaturation, upper airway obstruction, or apnea events were observed and the decrease was minimal in 403 patients (74%). Most of the patients received prophylactic supplemental oxygen to maintain oxygen saturation> 95% as per protocol, which makes it impossible to determine the actual incidence of desaturation at room air . Patients were divided into three groups based on numbers of dexmedetomidine boluses and additional medications received . Comparisons of blood pressure, heart rate, and other clinical measures between these groups of patients are shown in table 2 . Procedure time was significantly shorter in dexmedetomidine group a (40.31 19.40 min, p <0.0001) compared to the other two groups . Oxygen, dose, maximum dbp, maximum respiration rate, and initial spo2 had statistically significant (but clinically insignificant) differences between groups . The occurrence of hypotension among all three groups did not reach a statistically significant value (p = 0.82). Decrease in blood pressure from the baseline was frequently observed; however, medical intervention was not needed to correct it . The incidence of hypotension was usually noticed to be towards the completion of procedure or after stopping the infusion, with a mean procedure time of 48.73 25.46 minutes . Initial transient hypertension occurred in 137 patients approximately 20 minutes after the dexmedetomidine bolus and lasted for <15 minutes . In the population of 544 patients, 165 children (30.9%) had heart rates below the age - specific normal awake range during sedation (based on the published normal of fleming et al . ). Only in 21 children (3.9% of total cohort) did the lowest recorded heart rate fall> 20% below the given baseline average range . Bradycardia (hr <60/min), as defined according to pediatric advance life support (pals) guidelines, was observed in 10 children (4.5%) mostly in the age range of 13 years (figure 2). All the patients with bradycardia were continuously monitored and assessed by the supervising intensivist for normal blood pressure and oxygen saturation of 95% or above . No patient required treatment . The incidence of bradycardia was high in patients in the age range of 13 years compared with the rest of the cohort . The precise etiology of the bradycardia observed in this analysis cannot be delineated as electrocardiogram monitoring is known to be significantly distorted by the magnetohydrodynamic (mhd) effect and is nondiagnostic within the bore of any mri magnet . Time to discharge when dexmedetomidine was used alone was 92 25 versus 94 37 min when additional medications were used (p = 0.46). Average discharge time among 165 children with bradycardia was longer and statistically significant compared to the other 372 children in the study population without bradycardia (99.08 32.57 min versus 89.74 25.54 min, p = 0.0012, student's t - test). Gender was not associated with any change in discharge time (p = 0.30) (table 3). The ideal drug would allow optimal imaging, while maintaining hemodynamic and respiratory stability . As reported in previous studies, this occurred more frequently in hyperactive, uncooperative, and older children [4, 22]. Dexmedetomidine, a selective alpha 2 adrenoceptor agonist, has a very safe therapeutic window with respect to respiratory depression . This quality offers a distinct advantage in procedural sedations, where the patient is not immediately accessible to the medical team . There are various studies demonstrating that dexmedetomidine is a good option for procedural sedation [10, 12]. Interestingly, the dose used varies greatly indicating an open debate regarding the best dosage . Previous studies indicate that infusion of relatively low dose dexmedetomidine 0.10.7 /kg / h provides effective sedation [2327]. In a study conducted on eight healthy volunteers receiving dexmedetomidine infusion of 0.20.6 /kg / h, the visual analog sedation scores and bispectral index scores dropped by 3060% . However, this level of sedation will likely not be conducive to pediatric mri sedation, where higher doses of dexmedetomidine will be needed to accomplish the necessary sedation level . While dosing started low in initial reports, a recent one found that higher doses are required and have been well tolerated . A comparison of pentobarbital and chloral hydrate was performed by rooks et al . In 498 children for mri sedation . Several clinical trials have compared dexmedetomidine to propofol for pediatric procedural sedation . In a trial of 60 children undergoing mri, onset, recovery, and discharge time were all significantly shorter in the propofol group . Adverse effects such as lower blood pressure and heart rate and respiratory rate were also found more in the propofol group . Dexmedetomidine safety and efficacy for sedation during noninvasive radiologic procedures have been evaluated in four prospective trials [1, 810]. All of these studies reported that the use of high dose dexmedetomidine as a sole sedative resulted in high quality radiologic imaging with less use of additional rescue medicines . Our study also demonstrated that the use of high dose dexmedetomidine as sole agent is effective for mri sedation . We used fentanyl and midazolam as rescue medicines in 117 (21.5%) of our patients to avoid imaging artifacts, suggesting that these agents make a significant contribution to the sedation strategy for a sizeable minority of patients . In our study, the heart rate decreased significantly from baseline during sedation . This is an expected effect of anxiolysis and reflects higher baseline heart rate values from anxiety . Other studies reported a decrease in heart rate <20% from the baseline that was considered to be clinically insignificant in 86 (26%) patients . To have a true comparison with previously published data, we use the same definition of bradycardia; that is, the lowest recorded heart rate during sedation fell> 20% below the given baseline average range . Using that definition, the incidence of bradycardia in our study was 3.9% (21 children) in total cohort comparable to 4% as reported by mason et al . More importantly, during those periods of bradycardia, all patients maintained normal blood pressure and normal oxygen saturation (95% or higher). Treating bradycardia in normotensive children with glycopyrrolate has been reported to be associated with hypertensive episode, which could be beneficial to children who are hypotensive and bradycardic at the same time . Similar to our findings, previous investigators have also reported the occurrence of transient hypertension with bolus administration of dexmedetomidine . In our study, the occurrence of hypotension associated with high doses of dexmedetomidine was about 18%, observed immediately after stopping the infusion or towards the end of the procedure . None of the children needed any intervention, consistent with observation reported in other studies [12, 31]. Respiratory events make up a large proportion (5.5%) of sedation complications in children . In some studies, rapid administration of large loading doses has been described to cause respiratory complications [3234]. A loading dose of dexmedetomidine given over 2 minutes has been reported to cause irregular respiration, apnea, slight hypoxemia, and hypercapnia . However, similar to ours, several other studies have reported trivial effect of dexmedetomidine on respiration [12, 34, 36] which is consistent with the notion that the risk of respiratory depression is minimal with careful dexmedetomidine sedation . Despite these supporting lines of evidence, monitoring of respiratory function during the administration of dexmedetomidine in those receiving midazolam or fentanyl, which may depress respiratory function, appears warranted . Our reported discharge time of 92 minutes with dexmedetomidine only and 94 minutes when additional medicines were used is comparable to the 90 minutes' discharge time of heard et al . . Observed a recovery time of 47 minutes and mason et al . Reported recovery times ranging from 24.8 minutes to 35.2 minutes, depending on the dose of dexmedetomidine used . In some studies, recovery time is the time lapsed until the patient meets the discharge criteria, while in our study, like few others, discharged time is defined as the actual time of leaving the recovery room to go home . Discharge time of children experiencing bradycardia was longer than that for those who did not . In our study, 165 children with bradycardia had a mean discharge time which was statistically significant and longer than the other 372 children in the study without bradycardia (99.08 minutes versus 89.74 minutes; p = 0.0012). This probably could be due to the prolonged monitoring of these patients until the bradycardia resolved . Recovery time previously reported after sedation with propofol for mri has been 17 8 minutes, almost half of the recovery time when dexmedetomidine was used as a sole agent . In a second trial, forty children between the ages of 1 and 10 years were randomized to receive either a combination of midazolam + dexmedetomidine or propofol only for sedation during mri . The fast recovery times of propofol must be weighed against the fact that it induced deeper sedation with significant hypotension and oxygen desaturation . Taking the results of these comparison trials as a whole, dexmedetomidine appears to provide a useful alternative to propofol for procedural sedation in children, with a longer time of recovery but a lower incidence of adverse effects . This is a good alternative to propofol for patients with soy and egg white allergy and also in institutions where use of propofol is strictly limited to be used by anesthesiologists . It could be asserted that the reported efficacy is due to the use of an intensivist based specialized sedation team rather than to dexmedetomidine itself . This is reasonably true to some extent as specialization and experience should increase both success and efficiency . In spite of that, this can be stated with confidence; much of the reported success is specifically a function of dexmedetomidine . This is a descriptive study and few, if any, conclusions can be drawn about safety because the occurrence of serious sedation related side effects is fortunately rare . Additional prospective studies of the sedation for mri in children using a greater number of patients are warranted to provide a true idea of safety . In summary, high dose dexmedetomidine is an attractive and effective medication in children for mri sedation . When using high dose dexmedetomidine as the only agent for pediatric mri, it is not unusual to observe heart rate and blood pressure outside the established awake normal values . In our experience, these changes were pretty benign and were not associated with any adverse event . We conclude and recommend that, from hemodynamics and respiratory perspective, higher dose dexmedetomidine was well tolerated and is effective to use for successful completion of mri, in the majority of pediatric patients.
Exposure of ultraviolet (uv) irradiation to the skin causes acute and chronic detrimental cutaneous effects, which may result in photocarcinogenesis [17]. Native human melanin includes eumelanin and pheomelanin that contains sulfur, and eumelanin has been found in almost every type of human skin [8, 9]. In the skin, melanin synthesized in melanocytes located in the basal layer and hair bulbs transfers to keratinocytes . Melanin in keratinocytes acts as a photoprotector through body coloration and scavenging reactive oxygen species [1015]. In spite of photoprotective role of melanin, there are many cosmetics developed to prevent melanin formation in the skin because of aesthetic satisfaction by whitening ability . Of these, inhibitor of tyrosinase, which is a pivotal enzyme for melanin synthesis, has become major ingredient of cosmetics [1721]. Tyrosinase is an enzyme which catalyzes the biological conversion of tyrosine to dopaquinone with dioxygen at the dinuclear copper active site under physiological conditions [2224]. Other than oxidative catalysis of substrates by tyrosinase, a few studies on radical formation by tyrosinase have been reported . For instance, it was reported that tyrosinase - dependent activation of hydroxybenzenes formed reactive compounds including free radical, and some radicals were produced during the tyrosinase reaction and dopa - melanin formation . However, in these few studies, radical species have not been determined . We have studied the tyrosine - tyrosinase reaction in terms of melanin formation and ros scavenging ability of melanin . In a battery of studies, we found radical formation through the enzyme reaction, and discuss the formation mechanism in this paper . Reagents were purchased from the following sources: l - tyrosine, phosphate buffer solution (pb, ph 6.5) and catalase (from bovine liver) from wako pure chemicals (osaka, japan); tyrosinase (from mushroom), and superoxide dismutase (sod from bovine erythrocytes) from sigma - aldrich corp . (st . Louis, mo); 5,5-dimethyl-1-pyrroline - n - oxide (dmpo) from labotec (tokyo, japan); deuterium water (d2o) from tokyo chemical industry co., ltd (tokyo, japan). Then 1 mm tyrosine solution was prepared by mixing 5 l of 200 mm tyrosine solution with 5 l of 1 m naoh and 990 l of 0.2 m pb . Dmpo was dissolved in ultrapure water to be 4.5 m. the reaction mixture was prepared to contain different activity of tyrosinase, 20 l of 4.5 m dmpo, 60 l of 1 mm tyrosine and 0.2 m pb which was added to adjust a total volume of 200 l . Immediately after mixing the mixture was transferred to an esr spectrometry cell, and the esr measurement was started after 45 s. the measurement conditions of esr (jes - fa-100, jeol, tokyo, japan) were as follows: field sweep, 330.80340.80 mt; field modulation frequency, 100 khz; filed modulation width, 0.07 mt; amplitude, 400; sweep time, 1 min; time constant, 0.1 s; microwave frequency, 9.430 ghz; microwave power, 45 mw . Then 1 mm tyrosine solution was prepared by mixing 5 l of 200 mm tyrosine solution with 5 l of 1 m naoh and 990 l of ultrapure water or d2o . Tyrosinase was dissolved in ultrapure water to be 100 u/l . Immediately before the measurement tyrosinase was diluted to be 10 u/l with ultrapure water or d2o . Dmpo (8.9 m) was diluted to be 4.5 m with ultrapure water or d2o . The reaction mixture was prepared to contain 100 l of d2o, 20 l of 10 u/l tyrosinase (90% d2o solution), 20 l of 4.5 m dmpo (50% d2o solution), and 60 l of 1 mm tyrosine (99% d2o solution). Immediately after mixing the mixture was transferred to an esr spectrometry cell, and the esr measurement was started after 60 s. as a control, the reaction mixture was prepared to contain 100 l of h2o (ultrapure water), 20 l of 10 u/l tyrosinase dissolved in h2o, 20 l of 4.5 m dmpo dissolved in h2o, and 60 l of 1 mm tyrosine dissolved in h2o, and was similarly subjected to esr measurement . Reagents were purchased from the following sources: l - tyrosine, phosphate buffer solution (pb, ph 6.5) and catalase (from bovine liver) from wako pure chemicals (osaka, japan); tyrosinase (from mushroom), and superoxide dismutase (sod from bovine erythrocytes) from sigma - aldrich corp . (st . Louis, mo); 5,5-dimethyl-1-pyrroline - n - oxide (dmpo) from labotec (tokyo, japan); deuterium water (d2o) from tokyo chemical industry co., ltd (tokyo, japan). Then 1 mm tyrosine solution was prepared by mixing 5 l of 200 mm tyrosine solution with 5 l of 1 m naoh and 990 l of 0.2 m pb . Dmpo was dissolved in ultrapure water to be 4.5 m. the reaction mixture was prepared to contain different activity of tyrosinase, 20 l of 4.5 m dmpo, 60 l of 1 mm tyrosine and 0.2 m pb which was added to adjust a total volume of 200 l . Immediately after mixing the mixture was transferred to an esr spectrometry cell, and the esr measurement was started after 45 s. the measurement conditions of esr (jes - fa-100, jeol, tokyo, japan) were as follows: field sweep, 330.80340.80 mt; field modulation frequency, 100 khz; filed modulation width, 0.07 mt; amplitude, 400; sweep time, 1 min; time constant, 0.1 s; microwave frequency, 9.430 ghz; microwave power, 45 mw . Then 1 mm tyrosine solution was prepared by mixing 5 l of 200 mm tyrosine solution with 5 l of 1 m naoh and 990 l of ultrapure water or d2o . Tyrosinase was dissolved in ultrapure water to be 100 u/l . Immediately before the measurement tyrosinase was diluted to be 10 u/l with ultrapure water or d2o . Dmpo (8.9 m) was diluted to be 4.5 m with ultrapure water or d2o . The reaction mixture was prepared to contain 100 l of d2o, 20 l of 10 u/l tyrosinase (90% d2o solution), 20 l of 4.5 m dmpo (50% d2o solution), and 60 l of 1 mm tyrosine (99% d2o solution). Immediately after mixing the mixture was transferred to an esr spectrometry cell, and the esr measurement was started after 60 s. as a control, the reaction mixture was prepared to contain 100 l of h2o (ultrapure water), 20 l of 10 u/l tyrosinase dissolved in h2o, 20 l of 4.5 m dmpo dissolved in h2o, and 60 l of 1 mm tyrosine dissolved in h2o, and was similarly subjected to esr measurement . Representative esr spectra obtained from tyrosine - tyrosinase reaction with different activity of tyrosinase are summarized in fig . 1 . Each spectrum consists of quartet segments (intensity ratio, 1:2:2:1) and triplet of triplet segments (intensity ratio, 1:1:2:1:2:1:2:1:1). The former segment was assigned to dmpo - oh, a spin adduct derived from oh (hyperfine coupling constant, an = 1.49; ah = 1.49 mt). The latter segment was assigned to dmpo - h, a spin adduct derived from h (hyperfine coupling constant, an = 1.63; ah = 2.25 mt) as reported in a previous study . The ratio of dmpo - h and dmpo - oh generated in the tyrosine - tyrosinase reaction was approximately 1:2 . To examine if o2 and h2o2 are involved in a downstream of the generation process of h and oh, esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction were analyzed in the presence of sod, a scavenger for o2, and catalase, a cleaving enzyme for h2o2 . Neither sod (1 u/l) nor catalase (1 u/l) affected the amounts of dmpo - h and dmpo - oh (data not shown), suggesting that neither o2 nor h2o2 is involved in the generation process of h and oh . Then to further examine if h2o is a source of h and oh, esr signals of dmpo - h and dmpo - oh generated by the tyrosine - tyrosinase reaction where d2o was used as a major solvent (94% d2o solution) were compared with those where h2o was used as a solvent . As shown in fig . 2, the amount of dmpo - h was reduced by 70%, suggesting that approximately 70% of h is derived from h2o . Thus, we assume that the electron derived from the ligand of tyrosinase reacts with h2o or h to form h. that is, h2o + e oh + h or h + e h. on the other hand, the amount of dmpo - oh was not affected by the replacement of h2o to d2o, suggesting that oh was derived from the catalytic reaction of tyrosine to dopaquinone by tyrosinase . Since catalase had no effect on the generation of two radicals as described above, fenton - like reaction in which h2o2 reacts with transient metal copper in the structures of tyrosinase and its intermediates is least likely involved in the generation mechanism of oh . As reported in the previous study on dinuclear copper complexes, a rather stable dicopper - peroxide intermediate in aqueous solution decays through an internal electron transfer from the ligand to produce oh . Since tyrosinase is an enzyme which contains dinuclear copper ions at the active site, [2224], dicopper - peroxide intermediates formed during the catalytic process of tyrosine to dopaquinone possibly decay to produce oh through an internal electron transfer from the ligand . The proposed mechanism by which h and oh are generated in tyrosine - tyrosinase reaction is illustrated in fig . Although tyrosine - tyrosinase reaction is a key step of melanin formation as a photoprotection, it also produces not only h but oh which might be a causative factor of oxidative damage of the skin . Since there are many cosmetics developed for whitening ability by inhibiting tyrosine - tyrosinase reaction, our study revealed that they also might contribute to alleviate the oxidative damage of the skin by inhibiting oh generation via the enzyme reaction.
Bezoars are masses formed by the condensation of debris or stomach contents in the gastrointestinal tract . There are four major types of bezoars: a trichobezoar consists of hair, a phytobezoar of vegetable and fruit residues, a lactobezoar is formed from dairy products, a pharmacobezoar is caused by medications . A high - fiber diet, incomplete mastication, reduced gastric secretion, and decreased gastrointestinal motility from autonomic neuropathy can predispose patients to phytobezoar formation . It occurs more frequently in the small intestine, particularly in the terminal ileum and jejunum . The complications caused by bezoars are mechanical irritation, gastrointestinal obstruction, and, in severe cases, peritonitis caused by bowel perforation . Bezoars are responsible for 0.44% of cases of mechanical intestinal obstruction, which occurs mainly in the stomach and small intestine . Contrast - enhanced computed tomography (ct) is the method of choice to identify the location, severity, and etiology of intestinal obstruction . We describe our experience regarding a large - bowel obstruction caused by phytobezoar in a patient with a transplanted kidney . A 39-year - old man was admitted to our hospital for constipation, distension, and severe acute abdominal pain in the lower left quadrant . His medical history included alport syndrome resulting in progressive chronic kidney disease (ckd), hypertension, and iron deficiency anemia under medical treatment . Twelve years ago, he received a successful deceased - donor kidney transplant [figure 1]. At the time of admission, vitals were stable, and he was afebrile . Chest x - ray was normal and abdominal x - ray did not show signs of bowel obstruction . Coronal computed tomography scan shows transplanted kidney in left iliac fossa () non - contrast ct (ncct) of the abdomen was done (toshiba aquilion 64-tsx-101a / hc) as the patient had ckd (serum creatinine level = 7.71 mg / dl). Ct scan revealed a focal circumferential thickening of the sigmoid colon with an ovoid and encapsulated intraluminal mass measuring 4.7 3.6 cm [figure 2]. Dilatation of the proximal segment with abundant fecal material was seen [figure 2]. Based on the ct findings, particularly mottled gas pattern and encapsulated wall of the mass, we made the diagnosis of phytobezoar . Hence, because of acute intestinal obstruction and to avoid peritonitis, the patient underwent urgent exploratory laparotomy . (a) coronal computed tomography scan shows an ovoid intraluminal mass containing mottled gas pattern with encapsulating wall and associated wall thickening (arrows); note some dilated bowel loops (stars) and an abundant fecal material (arrowhead). (b) the mean attenuation value of the intraluminal mass is about 10.21 hu the obstructive intraluminal mass in the sigmoid colon was identified (approximately 4 4 cm in size) and manually broken down with evacuation of the obstructive material along with large amounts of liquid fecal material . The postoperative course was uneventful and the patient was discharged on the seventh postoperative day . At 3-month follow - up, phytobezoars are concretions of poorly digested fruit and vegetable fibres that are found in the alimentary track and are mostly composed of indigestible cellulose, tannin, and lignin from ingested vegetables and fruits . Normally, they are found in the stomach and may enter into the small bowel . The most important risk factors for phytobezoar formation are excessive consumption of fruits rich in fibers, poor dental health, insufficient mastication, diabetic gastroparesis, kidney failure, hypothyroidism, use of drugs which affect gastric motility, and previous gastrointestinal surgery . In our case, other symptoms include abdominal distension, vomiting, constipation, diarrhoea, anorexia, and weight loss . If treatment is delayed, colonic bezoars can be complicated by intestinal obstruction and perforation with consequent peritonitis . Plain abdominal radiography and ct scan are used for radiological evaluation of the patients presenting with intestinal obstruction . Simple abdominal x - ray may reveal air - fluid levels associated with mechanical obstruction, and occasionally it is possible to see the outline of bezoar, which is difficult to differentiate from abscess or feces within the colon . On abdominal ct, 89% of bezoars appear as spherically - shaped masses containing air - fluid levels . Ct is also helpful in discriminating bezoars from other causes of bowel obstruction such as gastrointestinal malignancies, gallstone ileus, etc . In our case indeed, many authors have highlighted some ct features to allow easier differential diagnosis between these two entities . Small bowel feces tends to be more amorphous and tubular in shape without evidence of an encapsulating wall, whereas phytobezoars have a well - defined ovoid shape and an encapsulating wall, such as in our case . Chen et al . Showed that a higher grade of obstruction is commonly associated with phytobezoar impaction without mesenteric fatty stranding and intraperitoneal fluid; moreover, they found that a combination of the food debris length <9.5 cm and the mean attenuation value <11.75 hu of the obstructed bowel at the transition point was suggestive for phytobezoar, such as in our patient . Although these features are typically found in the small bowel, they may also be considered very useful in the large bowel . Conservative management is considered in uncomplicated cases of colonic bezoars and consist of digital evacuation, enemas and manual disimpaction . If conservative approach fails, a colonoscopy can be attempted for bezoar removal with contextual mechanical fragmentation and extraction using several instruments . Although endoscopy has an acceptable success rate and fewer complications than surgery, surgical approach is mandatory in case of endoscopic treatment failure, sigmoid volvulus, hematochezia, intestinal obstructions, perforations, or peritonitis . When surgical approach is chosen, the first step is intestinal decompression and fluid - electrolyte replacement; later, open or laparoscopic abdominal exploration may be performed . In our case, the patient underwent laparotomic surgery and phytobezoar was manually broken down with evacuation . Bezoar is a rare disorder, however, it must be suspected in case of mechanical intestinal obstruction . Our case underlined the importance of early detection and removal of a bezoar to prevent complications such as obstruction, perforation, or peritonitis.
Mosquito samples were collected in vietnam in 2002 . Each species was separated and pooled, and each mosquito pool was then homogenized and centrifuged . The supernatant was filtrated through a 0.22-m filter and then inoculated onto a monolayer c6/36 mosquito cell culture and subsequently incubated at 28c for 7 days . Virus amplification in c6/36 cells was repeated twice, and the culture fluids were stored at 80c for further analysis . Rna was extracted from the second cell culture fluid by trizol ls reagent (invitrogen, carlsbad, ca, usa). Reverse transcription was performed by using superscript iii reverse transcriptase (invitrogen) and random hexamers, after rna denaturation at a temperature of 95c for 5 minutes in the presence of 15% dimethyl sulfoxide . Pcr was conducted by using takara la taq dna polymerase (takara bio, inc ., otsu, japan) with bav targeting primers (for segment 5, 5-cagctgcagtggttattgga-3 and 5accgtgcatcttaacccttg-3; for segment 8, 5-ttgcagtcgctgagctttta-3 and 5-cgcatttgatcgtatgcttg-3). These targeting primers were designed from sequences of bav strains from china and java (genbank accession nos . Af134519af134527, ay549307ay549309, af052024af052035, ay568287ay568290, nc_004211, nc_004217-nc_004221, nc_004200-nc_004204, nc_004198, and af052008af052013). The amplified cdnas were sequenced in the 3100-avant genetic analyzer (applied biosystems, foster city, ca, usa). The genomic sequences of all 12 genome segments for the 5 strains were determined (genbank accession nos . The sequences were translated to protein sequences by using the transeq program of the emboss package (8), version 5.00 . The protein sequences were aligned with the t - coffee program version 5.05 (9) with chinese and javanese bavs . Nucleotide sequences were then aligned, on the basis of protein alignment, with the tranalign program of the emboss package . Maximum likelihood phylogenetic trees were constructed from the protein - guided alignments of nucleotide sequences with dnaml software of the phylip package (10), version 3.67 . Five bav strains were isolated in ha tay and quang bing provinces (figure 1) in vietnam from culex annulus and cx . Phylogenetic analysis showed diversity within the phylogenetic clustering of each segment (table 2). Segments 7 and 9 formed 5 independent clusters, segment 12 formed 3 clusters, and the remaining segments formed 4 clusters . Phylogenetic trees for some representative segments (segments 2, 6, 9, and 12) are shown in figure 2 . Javanese strains formed a single cluster for segments 1, 2, 6, 8, 10, and 12 . For segments 7, 9, and 11, however, they diverged into 2 clusters, i and ii (figure 2, panel c). Segments 11 and 12 of the 2 vietnamese strains 02vn178b and 02vn018b were included in cluster iii with chinese bavs . Segments 6 and 9 of isolate 02vn178b belonged to cluster v, although segments 1, 2, 3, 4, and 8 belonged to cluster iv . Moreover, segments 5 and 7 of isolate 02vn178b and segments 8 and 11 of isolate 02vn009b showed a mixed electrogram pattern of clusters v and iv or iii . Mosquito collection sites in vietnam . * length, length of each segment in the reference sequence of banna virus (bav) from refseq (nc_004198, nc_004200nc_004204, nc_004211, nc_004217nc_004221); aligned area, the fragment used to make the alignment and phylogenetic tree . The numbers are set following the sequences of strain jht-6423, which is the reference strain in national center for biotechnology information reference sequences . I, the cluster including the javanese jkt-6423 strain; ii, the cluster specific to the jkt-6969 and jkt-7043 strains; iii, the cluster including chinese bavs; iv, the cluster including the vietnamese 02vn018b strain; v, the cluster including the vietnamese 02vn180b strain . We report isolation of bav in vietnam . The nucleotide sequences of the vietnamese isolates genomic rna segments diverged into 2 phylogenetically distant clusters . Our data indicate that 2 bav populations exist in the country and that both evolved independently . Two strains were clearly different from chinese and javanese bavs (02vn078b and 02vn180b). Two other strains (02vn018b and 02vn178b) were phylogenetically close to chinese bavs when 2 shorter segments, segments 11 and 12, were analyzed but not when the remaining 10, longer segments were considered . This finding implies that a recent reassortment event has occurred and that segments of chinese or vietnamese strains were replaced . In addition, although some vietnamese strains were phylogenetically distant, they could produce a viable progeny by reassortment of segments 5, 6, 7, 8, 9, and 11 . This finding is clearly illustrated by the diversity in the phylogenetic clustering pattern and the mixed electrogram pattern of these segments (table 2). Analysis of segment 12 showed that cluster iii (table 2) contained 2 vietnamese strains (02vn178b and 02vn018b) and the chinese strains af052030 and yn-6 (isolated in yunnan province) and bj95 - 75 (isolated in beijing). This finding suggests the possibility of wide circulation of bavs containing segment 12 type iii not only in southeast asia but also in east asia . A regionwide genotype shift of japanese encephalitis virus (jev), which is also carried by culex spp . Mosquitoes, from genotype iii to genotype i, was witnessed in east asia during the 1990s (11,12). These results and phylogenetic analysis of old jev strains in japan and southeast asia (11) strongly suggested that jev could be transferred frequently from southeast asia to east asia . Similarly, bavs with segment 12 belonging to cluster type iii were also distributed in both southeast and east asia . Therefore, we can speculate that bavs are actively circulating within the asian continent . To achieve a better understanding of the distribution dynamics of bavs in asia, more isolates are needed . In vietnam, a seasonal increase of viral encephalitis and meningitis cases is reported during the rainy season every year . Among these cases, 50%70% are diagnosed as japanese encephalitis, while the rest of them remain idiopathic (p.t . Nga et al ., unpub . Data). A clear association between bav and human diseases, as well as the prevalence of bav infection in humans, has not yet been established in vietnam . We also believe that bav is a good candidate for the differential diagnosis of viral encephalitis and meningitis cases of unknown origin in tropical and subtropical asia, where culex mosquitoes are abundant.
Clozapine remains an ultimate option for patients with treatment resistant schizophrenia (kane 2012). Efficacy of clozapine against positive symptoms of schizophrenia was confirmed in numerous studies and meta - analyses (chakos et al . 2001), including analysis published by the cochrane library (asenjo lobos et al . However, treatment with clozapine is associated with very severe metabolic side - effects (newcomer 2005). Clozapine - induced weight gain is very common (wetterling 2001), and so is impaired fasting plasma glucose levels . In patients with schizophrenia weight gain is associated with impaired physical functioning and negative body appraisal (bachmann et al . 2012), both of which affect quality of life . While treatment with clozapine may reduce mortality by reducing suicide rate, mortality due to clozapine - associated weight gain will diminish reduction in the suicide rate almost entirely over 10 years by the increased mortality associated with weight gain of 10 kg (fontaine et al . Also, obesity is linked with numerous secondary health problems (pressure overload on lungs, joints and bones) and is an important risk factor for life - threatening diseases, such as cardiovascular disease, type 2 diabetes and certain cancers . Several mechanisms are considered as taking role in clozapine - induced weight gain, including antagonism at histamine h1 receptors (kroeze et al . Various receptors, including aforementioned 5-ht1b and 5-ht1c, regulate activity of the hypothalamic nuclei (particularly the arcuate nucleus (arc), which plays the key role in appetite regulation). Activity of arc is also regulated by several hormones of anorexigenic properties: leptin, pancreatic polypeptide (pp), cholecystokinin (cck), glucagon - like peptide-1 (glp-1), oxyntomodulin (oxm), peptide yy (pyy) and the first discovered orexigenic substance - ghrelin (druce et al . 2004). Agouti - related peptide (agrp) is one of the hypothalamic hormones that works by increasing appetite and decreasing metabolism and energy expenditure, thus leading to weight gain . It was identified in 1997 based on its sequence similarity with agouti signaling peptide (asip), a protein synthesized in the skin that controls coat color . Compared with asip, agrp is physiologically expressed in the hypothalamus (shutter et al . Agrp is also expressed in the adrenal gland, testes, kidneys, and lungs . Agrp induces obesity by antagonism of the melanocortin receptors (mc3-r and mc4-r, subtypes implicated in weight regulation, via metabolism and appetite control) (ollmann et al . These neurons make peptides that potently stimulate food intake not only by increasing neuropeptide y (npy) signaling, but also by reducing melanocortin signaling via the release of agrp (morton and schwartz 2001). Activity of the agrp / npy neurons is modulated by leptin, released from the adipose tissue, and insulin . The adiposity signals (insulin and leptin) are secreted in proportion to body fat content and act in the hypothalamus to inhibit anabolic and stimulate catabolic, effector pathways (schwartz et al . The increase of food intake following a single central administration of agrp is sustained for up to a week (hagan et al . 2000), while the response to npy is sustained over hours, rather than days . This study was undertaken with the purpose to determine if patients with schizophrenia on clozapine monotherapy have higher fasting levels of agrp compared with healthy control . In order to provide more accurate measurements, biochemical and anthropometric measurements were combined with body composition determined using bioelectric impedance analysis (bia), which provides accurate measurements of body fat, lean mass and body water (bosy - westphal et al . This is the first study to investigate such combination of these parameters in subjects with schizophrenia treated with clozapine . Data for 24 european caucasian adult hospitalized patients with paranoid schizophrenia (295.30 according to dsm - iv, f20.0 according to icd-10) was included into the study . These subjects were on clozapine monotherapy for at least two months prior the assessments with a minimum dose of 100 mg / day . Most patients was in stable phase of the disease (i.e. No acute psychosis). Control group was 24 healthy subjects and was gender- and age - matched with patients in the clozapine group . Health status of the control subjects was determined on the basis of basic physical examination, including vital signs and an interview . All patients and volunteers included in the study expressed their written informed consent for participation in this study . The blood samples for the chemistry panel were collected between 7 am and 8 am, after ensuring at least 8 h of overnight fasting . Glucose, lipids, calcium and uric acid levels were measured using a dirui cs-400 analyzer (dirui, china). Homocysteine chemiluminescence assessments were performed using an immulite 2,000 analyzer (siemens, germany), insulin immunochemistry assessments were performed using a cobas e411 analyzer (roche diagnostics, switzerland) and albumin levels were assessed using a cobas integra 800 analyzer (roche diagnostics, switzerland). Levels of agrp were measured in blood serum using elisa (enzyme - linked immunosorbent assay) method . Prior to assays, serum samples were stored at 80 c for up to 6 months . Elisa assays were performed using commercial kits (intra - assay: cv <10%, inter - assay: cv <12%) manufactured by raybiotech (usa), according to protocol provided by its manufacturer (all samples were 2-fold diluted). Metabolic syndrome and abdominal obesity were defined according to international diabetes foundation (idf) criteria (alberti et al . Kg / m, 2530 kg / m and 30 kg / m were defined as normal weight, overweight and obesity, respectively . Raised triglycerides (tga) level 150 mg / dl and/or total cholesterol (tc) 200 mg / dl and/or reduced hdl cholesterol level <40 mg / dl for men and <50 mg / dl for women and/or raised ldl cholesterol level 135 mg / dl were interpreted as dyslipidemia . Corrected calcium was calculated using the formula: corrected calcium [mg / dl] = measured total calcium [mg / dl] + 0.8 (4.0 serum albumin [g / dl]). Insulin resistance was estimated from fasting glucose and insulin results by homeostasis model assessment and quicki index, using the formula: homa1-ir = (fasting plasma glucose [mg / dl] insulin [mu / l])/405 . Homa2-ir index was calculated using a calculator downloaded from http://www.dtu.ox.ac.uk . Quicki index (lower numbers reflect greater insulin resistance) was calculated using the formula: 1/(log (fasting insulin [mu / l]) + log (fasting plasma glucose [mg / dl])). Height was measured with a wall - mounted height measure to the nearest 0.5 cm . Weight was measured with a spring balance that was kept on a firm horizontal surface . Subjects wore light clothing, stood upright without shoes and weight was recorded to the nearest 0.5 kg . Body mass index (bmi) was calculated as body weight in kilogram divided by the height in meter squared (kg / m). Waist, abdominal and hip circumference was measured using a non - stretchable fiber measuring tape . Waist - to - hip ratio (whr) was calculated as waist circumference divided by hip circumference . Whr cut - off points were defined according to who recommendations (0.85 for women and 0.9 for men). Fat mass index (fmi) was calculated as total body fat in kilogram divided by the height in meter squared (kg / m). Excess body fat according to fmi classification ranges were defined as fmi> 6 for men and fmi> 9 for women body composition was measured using a maltron bf-906 body fat analyzer (maltron, uk), single frequency bioelectrical impedance analyzer to determine resistance and reactance at 50 hz . Standard operating conditions were observed by a trained operator including preparation of the participant, electrode placement and operation . The measurement using bia was taken immediately prior to anthropometry measurements with participants lying supine, in a rested state . Simple descriptive statistics (means, standard deviations, 95% confidence interval [ci]) were generated for all continuous variables . For discrete variables number of patients and percentages are given . Skewed variables were transformed to follow normal distribution using log, square root, inverse square root or square transformation . Means, standard deviations, and confidence intervals are reported for non - transformed variables, results of tests are reported for transformed or non - transformed variables . If transformation resulted in normal distribution, two - tailed t - test was used to assess inter - group differences, otherwise variables were analyzed using mann associations were tested by pearson s (for variables with normal distribution) or spearman's (for other variables) correlation coefficients . The significant level was set at p <0.05 . For group of patients treated with clozapine the mean age was 38.812.6 and 39.912.3 for the control group (p = 0.62). In both groups there were 12 men and 12 women . In the clozapine group 12 subjects smoked cigarettes and 8 in the control group (p = 0.38). The mean duration of monotherapy with clozapine was 60.579.4 [95% ci: 27.094.1] months and mean clozapine dose was 341.1148.6 [95% ci: 278.4403.8] mg / day . Detailed results for anthropometric measurements and laboratory tests are shown in table 1 . Table 1results of anthropometric measurements and laboratory testsclozapine n = 24control n = 24weight [kg]78.114.2(72.184.1)72.615.1(66.278.9) p = 0.08 t = 1.38bmi [kg / m]27.13.6(25.528.6)24.83.5(23.326.2) p = 0.01 t = 2.25fmi [kg / m]8.93.1(7.610.2)7.73.0(6.49.0) p = 0.09 t = 1.38abdominal circumference [cm]96.59.4(92.5100.4)85.511.6(80.690.3) p <0.001 z = 3.58waist circumference [cm]91.112.1(86.096.2)82.410.6(77.886.8) p = 0.005 z = 2.66hip circumference [cm]99.08.4(95.5102.5)95.97.1(92.998.9) p = 0.08 t = 1.39whr0.920.08(0.880.95)0.860.08(0.820.89) p = 0.005 z = 2.66sbp [mm hg]121.713.6(115.9127.4)136.717.9(129.1144.2) p = 0.001 z = 3.26dbp [mm hg]81.28.5(77.684.8)82.812.1(77.687.9) p = 0.30 t = 0.50uric acid [mg / dl]4.51.4(3.95.1)4.31.3(3.74.8) t = 0.57homocysteine [mol / l]14.5 4.4(12.616.4)13.6 5.0(11.515.7) p = 0.25 t = 0.67tc [mg / dl]194.2 53.2(171.7216.6)216.6 65.3(189.0244.1) p = 0.06 t = 1.54hdl [mg / dl]43.5 12.6(38.248.8)55.1 14.3(49.161.1) p 41.9(104.8140.3)128.3 39.7(111.5145.0) p = 0.29 t = 0.54tga [mg / dl]140.3 120.4(89.4191.1)104.3 81.4(69.9138.6) p = 0.07 t = 1.50fpg [mg / dl]103.5 31.7(90.1116.8)87.8 11.7(82.892.7) p = 0.03 t = 1.88insulin [u / ml]11.8 8.2(8.3 - 15.3)7.7 3.2(6.3 - 9.1) p = 0.02 t = 2.08homa1-ir3.3 3.4(1.84.7)1.7 0.8(1.32.0) p = 0.009 t = 2.46homa2-ir1.6 1.1(1.12.1)1.0 0.4(0.81.2) p = 0.01 t = 2.26quicki0.15 0.01(0.140.15)0.16 0.01(0.150.16) p = 0.007 t = 2.52corrected calcium [mg / dl]8.6 0.9(8.29.0)8.7 p = 0.37 t = 0.33data given as: mean standard deviation (95% ci)bmi = body mass index; fmi = fat mass index; whr = waist - to - hip ratio; sbp = systolic blood pressure; dbp = diastolic blood pressure; tc = total cholesterol; hdl = high density lipoproteins; ldl = low density lipoproteins; tga = triglycerides; fpg = fasting plasma glucose; homa1-ir = homoeostasis model assessment of insulin resistance 1; homa2-ir = homoeostasis model assessment of insulin resistance 2; quicki = quantitative insulin sensitivity check index results of anthropometric measurements and laboratory tests data given as: mean standard deviation (95% ci) bmi = body mass index; fmi = fat mass index; whr = waist - to - hip ratio; sbp = systolic blood pressure; dbp = diastolic blood pressure; tc = total cholesterol; hdl = high density lipoproteins; ldl = low density lipoproteins; tga = triglycerides; fpg = fasting plasma glucose; homa1-ir = homoeostasis model assessment of insulin resistance 1; homa2-ir = homoeostasis model assessment of insulin resistance 2; quicki = quantitative insulin sensitivity check index we have found no inter - group differences for body composition analysis . Detailed results for bia analysis are shown in table 2 . Lean body mass was higher in men in the whole study sample (60.16.4 [95% ci: 57.4 - 62.8] vs. 43.85.4 [95% ci: 41.546.1] kg, t = 9.56, p <0.001) and in the clozapine group (59.65.7 [95% ci: 56.063.3] vs. 45.37.0 [95% ci: 40.9 - 49.8] kg, t = 5.48, p <0.001). Similarly, basal metabolic rate was higher in men in the whole study sample (1,707.7182.3 [95% ci: 1,630.71,784.6] vs. 1,337.3138.4 [95% ci: 1,278.8 - 1,395.7] kcal / day, t = 7.92, p <0.001) and in the clozapine group (1,701.2138.2 [95% ci: 1,613.41,789.0] vs. 1,362.7173.0 [95% ci: 1,252.71,472.5] kcal / day, t = 5.29, p <0.001). N = 24control n = 24total body fat [kg]25.6 8.8(21.829.2)22.4 8.7(18.726.1) p = 0.10 t = 1.25total body fat [%] 32.6 8.4(29.136.2)28.9 7.1(25.831.8) p = 0.06 t = 1.67lean body mass [kg]52.5 9.6(48.456.5)51.4 10.8(46.855.9) p = 0.36 t = 0.36lean body weight [%] 67.6 8.1(64.271.0)71.1 7.1(68.174.1) p = 0.07 t = 1.61total body water [l]38.4 7.0(35.441.3)37.7 7.9(34.340.9)p = 0.36 t = 0.35total body water [%] 49.9 5.4(47.652.1)52.1 5.2(49.854.2) p = 0.09 t = 1.42basal metabolic rate [kcal / day]1,532.0 p = 0.39 t = 0.26data given as: mean standard deviation (95% ci) results of body composition analysis data given as: mean standard deviation (95% ci) there were no significant difference for fasting serum levels of agrp between the clozapine group and the control group (15.008.65 [95% ci: 11.3418.65] vs. 15.336.82 [95% ci: 12.4518.22] pg / ml, t = 0.32, p = 0.37), see fig . 1 . Women had significantly lower levels of agrp in the whole study group (12.535.65 [95% ci: 10.1414.92] vs. 17.808.66 [95% ci: 14.1421.45] pg / ml, t = 2.47, p = 0.009) and in the control group (12.905.77 [95% ci: 9.2216.57] vs. 17.777.14 [95% ci: 13.2322.31] pg / ml, t = 1.82, p = 0.04), but the difference was not significant for the clozapine group (12.175.75 [95% ci: 8.5115.83] vs. 17.8210.28 [95% ci: 11.28 - 24.36] pg / ml, t = 1.64, p = 0.06).fig . 1mean fasting agrp serum levels [pg / ml] in subjects on clozapine and in the control group (p = 0.37; horizontal bars indicate means). Mean fasting agrp serum levels [pg / ml] in subjects on clozapine and in the control group (p = 0.37; horizontal bars indicate means). For the whole study group significant correlations of agrp levels were found for total body fat [%] (r = 0.34, p = 0.02), lean body mass [kg] (r = 0.38, p = 0.006), lean body mass [%] (r = 0.34, p = 0.02), body water [l] (r = 0.38, p = 0.006), body water [%] (r = 0.34, p = 0.02) and homocysteine levels (r = 0.29, p = 0.04). For the clozapine group significant correlations of agrp levels were found for total body fat [%] (r = 0.48, p = 0.02), basal metabolic rate (r = 0.42, p = 0.04), lean body mass [%] (r = 0.49, p = 0.01), body water [%] (r = 0.49, p = 0.01). For the control group significant correlations of agrp levels were found only for basal metabolic rate (r = 0.42, p = 0.04). In the clozapine group there were no significant differences for agrp levels between subjects with or without excess body fat (based on fmi value) (p = 0.59), idf - defined metabolic syndrome (p = 0.33), smokers and non - smokers (p = 0.15), subjects with bmi <25 kg / m and with bmi 25 kg / m (p = 0.09), subjects with and without impaired fasting glucose (p = 0.16), subjects with and without abdominal obesity (p = 0.11), subjects with and without dyslipidemia (p = 0.09), and subjects with and without homa-1ir defined insulin resistance (p = 0.07). In the control group there were no significant differences for agrp levels between subjects with or without excess body fat (based on fmi value) (p = 0.20), idf - defined metabolic syndrome (p = 0.44), smokers and non - smokers (p = 0.35), subjects with bmi <25 kg / m and with bmi 25 kg / m (p = 0.12), subjects with and without impaired fasting glucose (p = 0.36), subjects with and without abdominal obesity (p = 0.10), subjects with and without dyslipidemia (p = 0.37), and subjects with and without homa-1ir defined insulin resistance (p = 0.13). In the whole study group there was no significant differences for agrp levels between subjects with or without excess body fat (based on fmi value) (p = 0.30), idf - defined metabolic syndrome (p = 0.43), smokers and non - smokers (p = 0.29), subjects with bmi <25 kg / m and with bmi 25 kg / m (p = 0.39), subjects with and without impaired fasting glucose (p = 0.29), subjects with and without dyslipidemia (p = 0.11), and subjects with and without homa-1ir defined insulin resistance (p = 0.33). There was a significant difference for agrp levels between subjects with or without abdominal obesity (13.466.50 [95% ci: 9.0917.83] vs. 16.926.93 [95% ci: 12.7321.11] pg / ml, t = 1.80, p = 0.04). The main objective of the present study was to find out whether there is a significant difference in fasting serum levels of agrp peptide between patients with schizophrenia on clozapine monotherapy and age- and sex - matched healthy controls . We have found the difference was not significant (clozapine: 15.008.65, control: 15.336.82 pg / ml, p = 0.37). We do not know pre - treatment values, so we cannot determine whether and how this parameter changed during therapy with clozapine . To our knowledge, no data are available on the effect of clozapine on blood agrp concentrations in humans . Limited data are available for olanzapine, which has clinical and pharmacological properties similar to clozapine . Results of these studies show that treatment with olanzapine does not affect agrp levels (basoglu et al . However, in animal study it was demonstrated that administration of olanzapine or clozapine upregulates npy and agrp and downregulates proopiomelanocortin in the arcuate nucleus of the hypothalamus (ferno et al . We do not know studies demonstrating correlation between blood levels of agrp and levels in the hypothalamus, but since it was showed recently that agrp neurons are unique among hypothalamic neurons by being the predominant neuronal subtype situated outside the blood brain barrier (olofsson et al . 2013), we may assume that changes in hypothalamic expression are reflected in changes in blood levels . The finding that women had significantly lower levels of agrp in the whole study group (12.535.65 vs. 17.808.66 pg / ml, p = 0.009) and in the control group (12.905.77 vs. 17.777.14 pg / ml, p = 0.04), but not in the clozapine group (12.175.75 vs. 17.8210.28 pg / ml, p = 0.057), might indicate that the testes are important source of agrp, as shown previously (shutter et al . However, several more recent studies showed no difference between men and (gavrila et al . 2001), therefore it may indicate that plasma agrp levels reflect central agrp concentrations . It was previously reported that total level of agrp is higher in obese subjects (katsuki et al . 2001), although in another study it has been demonstrated that agrp levels were lower in normal weight group compared with women with anorexia, probably due to increased leptin levels (moriya et al . We did not measure levels of leptin or acylated ghrelin, but we found in the same group of patients that there were no differences for desacyl ghrelin levels between patients taking clozapine and control group (wysokiski et al . While there was a significant difference in bmi values between the clozapine group and the control group, we found no differences between both groups for fmi values . Compared with bmi, fmi is a better indicator of central obesity since it is much more sensitive to body fat content (kelly et al . Moreover, there were no differences for any of the bia results between both groups . These indicate that there was a substantial similarity between both groups in terms of body fat content, which is important considering that agrp levels may affect or result more from the amount of adipose tissue than from body weight per se . On the other hand, we have found negative correlations between agrp levels and total body fat (r = 0.34 and 0.48 in the whole study group and clozapine group, respectively). These may indicate the effect of leptin on the expression of agrp, but require further studies . Additionally, there were positive correlations with lean body mass (r = 0.38 and 0.49 in the whole study group and clozapine group, respectively), body water (which amount is negatively correlated with the amount of body fat: r = 0.96, p <0.001) (r = 0.34 and 0.49 in the whole study group and clozapine group, respectively) and basal metabolic rate (r = 0.42 both in the clozapine group and control group), also being in line with the above - mentioned mechanism . In animal studies it has been found that high - fat diet leads to decreased expression of agrp in the hypothalamus, probably in the mechanism moderated by leptin (staszkiewicz et al . We have no detailed data on diet patterns of our study subjects, but reports are consistent that patients with schizophrenia consume higher amounts of high - fat foods compared with healthy population (dipasquale et al . . This might be one of the reasons why we have found no differences between our study groups . Also, level of physical activity (usually lower in hospitalized patients) might affect mechanisms regulating agrp levels . Finally, another important factor is the effect of treatment mediated by other anabolic or catabolic neuropeptides . Here, two most important peptides are leptin (which decreases agrp levels) and ghrelin (which has opposite effect) and have also been studied most extensively (sentissi et al . 2008). Since both leptin and ghrelin (andrews 2011) may regulate activity of agrp / npy neurons in the arcuate nucleus, effects of antipsychotics on these neuropeptides must be taken into consideration . Therefore, a study focused on interactions within this regulatory system would be very important for better understanding of this problem . Based on our results however, the low number of study subjects limited the probability of finding inter - group differences due to lack of statistical power . Due to the cross - sectional study design causal relationships cannot be established and the effect of previous antipsychotic treatment cannot be excluded . Dual - energy x - ray absorptiometry (dxa) should be used to measure body composition and adipose tissue mass more accurately . Due to complex structure of interactions between anabolic and catabolic neuropeptides, a longitudinal study comparing these interactions are crucial for understanding mechanisms of treatment - induced weight gain.
It is widely accepted that mutation is the main causal factor for diverse tumorigenesis [1, 2]. The recent development of next - generation sequencing (ngs) technologies has revolutionized the speed and throughput of dna sequencing, which facilitates the discovery of new driver mutations . For example, the arid1a gene is mutated in 83% of gastric cancers with microsatellite instability and the sf3b1 gene is somatically mutated in 9.7% of chronic lymphocytic leukemia patients [4, 5]. In colorectal cancers, although the majority of recurrent mutations have been previously known, some novel mutations, such as the mutations in the sox9 and fam123b genes, were also identified by ngs technology, which may have implications for colorectal tumorigenesis . In melanoma, approximately 40% was found to have causal mutations affecting b - raf function . Along with the well - known driver mutations, the novel mutations identified by ngs will be useful for predicting the prognosis and molecular characterization of cancers [8, 9]. Recently, a number of mutation calling tools, such as varscan, genome analysis toolkit, and mutect, have been developed to detect single - nucleotide variants (snvs) or indels from ngs data . However, snv calling from a single cancer case is not the final step for defining clinically meaningful mutations . To define the pathogenic snvs in human cancers, a commonly used approach is to identify the phenotype - associated recurrent mutations in the study group . Due to the data size burden of ngs output, profiling the mutations associated with a certain phenotype or prognosis in a study group by a researcher who does not have a bioinformatics background or facilities is very difficult . Currently, there is no user - friendly tool supporting methods for the detection of recurrent or phenotype - specific mutations . In this study, in order to support the definition of recurrent mutations or phenotype - specific mutations from ngs data of a group of cancers with diverse phenotypes, we aimed to develop a user - friendly tool, named mutation arranger for defining phenotype - related snv (map). Map software was designed as a standalone program, compatible in microsoft windows environments with a user - friendly graphic interface, and compiled codes of map can be easily installed . Both variant call format and its annotation files, such as annovar output, can be used as input files for map . When a user loads the input file, called sample descriptor file (gsf), map software displays the sample set information and filter options (fig . 1). Once data are loaded, the user can filter the data using the' analysis options' tab in map software based on annotation information . By using the sample set and analysis options summary metrics include the mutation status for each sample, total mutation frequency, p - values for differences between phenotype groups based on clinical information, genomic position, reference / observed sequence, and additional information, such as read depth by default . Map software also displays more detailed information in summary metrics based on the user - selected' analysis options .' In the' analysis result' tab, map software provides the frequency- or p - value - based filter function . This functionality supports detection of the recurrent mutations in study subjects and identification of the phenotype - specific mutation that occurs significantly in a certain phenotypic subclass . Then, map software represents the user - selected mutations graphically in the' visualization' tab . To sort the variants by genomic position, p - value, or frequency, users can use the sort function in the' analysis result' tab . Details of the program download, installation, and analysis procedures are available in the user manual . The major steps of map are as follows . - generating mutation summary metrics in study subjects- determination of recurrent mutations by frequency- determination of phenotype - specific mutations by association test- graphical illustration of user - selected mutations - generating mutation summary metrics in study subjects - determination of recurrent mutations by frequency - determination of phenotype - specific mutations by association test - graphical illustration of user - selected mutations mutation calling files from genome analysis toolkit can be used directly as input files for' sample descriptor .' Alternatively, manually prepared standard variant call format is also available . Annotation information files, such as annovar outputs, can be used as input files for sample descriptor . Gsf is a comma - separated values text file that describes the input files, such as gatk output files and annovar output files, and phenotype information . When users load the input file, map software displays the sample set information and filter options in the' source' tab . If any file is not prepared properly, map software informs the user in the' sample set files' tab . After data are loaded, the user can filter the data using the' analysis option' tab based on annotation information . Based on user - selected filters in the' analysis options' tab, summary metrics represent additional annotation information, such as gene name, function, known variant, and amino acid change . The additional annotations can be hidden using the snp filter option in the' analysis option' tab . These mutation metrics are exported into a comma - separated values text file for further investigation . In the' analysis result' tab, map software defines the recurrent mutations in study subjects by frequency . The frequency threshold can be determined arbitrarily by the user in the' frequency filter' function . Map also defines the phenotype - specific mutations using the mutation metrics and phenotype information . Phenotype - specific mutations that occur more frequently in one of two clinical conditions (e.g., drug - responder vs. non - responder) are determined by chi - square test under the user - defined significance level . The graphic user interface is implemented in the software for users to easily handle large - scale data or access the analysis result (fig . 2b and 2c). The recurrent or phenotype - specific mutations can also be demonstrated visually in heat - map style . For example, the left and right of the heat - map represent the sample name and mutational spectrum, respectively . The bottom of the heat - map represents the gene name by color for mutational spectrum . Frequency or p - value plots are represented on top of the heat - map (fig . 2b and 2c). Map is a user - friendly program with multiple functions that supports the determination of recurrent or phenotype - specific mutations and provides graphic illustration images to the users . Its operation environment, microsoft windows, enables more researchers who cannot operate linux to define clinically meaningful mutations with ngs data from cancer cohorts.
Echocardiographic measurements were performed in 19992001 (baseline) and 20072009 (follow - up) examinations of the hoorn study . The hoorn study is a population - based cohort study on glucose metabolism and cardiovascular diseases, previously described in detail (10). At baseline, 290 individuals with normal glucose metabolism (ngm), 187 with impaired glucose metabolism (igm), and 345 with type 2 diabetes participated . At follow - up, 167 (20%) individuals were excluded a priori because of incomplete baseline data (n = 26), mental incompetence to participate (n = 12), or death (n = 129). Of the remaining 655 individuals, 441 (67%) participated . Thirteen (3%) of those were excluded in the present analyses because of missing echocardiography data at follow - up . To restrict the study population to individuals at risk of developing lv systolic and/or diastolic dysfunction, we also excluded 34 individuals (8%) with an lv ejection fraction <50% or an la volume index> 40 ml / m at baseline . The local ethics committee approved the study, and written informed consent was obtained from all participants . Echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system (24 mhz transducer) according to a standardized protocol consisting of two - dimensional, m - mode, and pulsed wave doppler assessments as previously described (13). At follow - up, this protocol was expanded with tissue doppler assessments of mitral annular velocities . A set of three markers of lv diastolic dysfunction was determined: la volume index, lv mass indexed to height to the power of 2.7 (14), and the ratio of early (e) mitral valve flow to early (e) diastolic lengthening velocity (e / e) using tissue and pulsed wave doppler assessments . Incident heart failure was considered present if signs and symptoms were accompanied by lv systolic (lv ejection fraction <35%) and/or diastolic dysfunction (15). Presence of incident lv diastolic dysfunction (grade 2 or 3) was assessed according to international recommendations (16). In the present analyses, we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid, brachial, and femoral arteries to assess stiffness of the arterial wall (10). Additionally, systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure (17). Augmentation index was determined using applanation tonometry . In a subset of the study population, carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time, adjusted for height in the statistical analyses . Reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described (10). At follow - up, intraobserver intersession coefficients of variation in 10 individuals (aged 70.3 3.6 years) were comparable: 4.2, 13.1, 4.1, and 6.1% for lv ejection fraction, la volume, lv mass, and e / e, respectively . For assessment of glucose status, all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm, igm (impaired fasting glucose and/or impaired glucose tolerance), and type 2 diabetes groups according to the 1999 world health organization criteria (18). Health status, medical history, medication use, and smoking habits were assessed by questionnaires (10). Waist circumference, blood pressures (systolic, mean arterial, and pulse pressures), and levels of cholesterol, insulin, hba1c, and c - reactive protein were determined as previously described (10). Descriptive statistics are presented as means sd or, in the case of a skewed distribution, as median (interquartile range). The f test for anova in the case of continuous variables or tests in the case of proportions were performed to test for differences between groups of glucose status . In the case of skewed distributions, the above tests were done on log - transformed data . For every variable that had> 10% missing data, the number missing is presented in the legends of tables 13 . Linear regression analyses, adjusted for age and sex, were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up . Regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure . For investigation of changes in markers, lv systolic and diastolic dysfunction according to glucose status or arterial stiffness, we adjusted all models for baseline values of these markers (models assessing e / e adjusted for baseline la volume index). Lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors, like overweight, hypertension, insulin resistance, or previous cardiovascular events (19). Therefore, we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models (supplementary data). A secondary purpose of these analyses was to investigate potential residual confounding by these factors . Product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease . Associations of arterial distensibility coefficients, systemic arterial compliance, and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness . Additional analyses, adjusted for age and sex, were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function . P values <0.05, or <0.10 in case of interaction analyses, were considered statistically significant . Statistical analyses were performed with spss for windows (version 15.0; spss, chicago, il). Echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system (24 mhz transducer) according to a standardized protocol consisting of two - dimensional, m - mode, and pulsed wave doppler assessments as previously described (13). At follow - up, this protocol was expanded with tissue doppler assessments of mitral annular velocities . A set of three markers of lv diastolic dysfunction was determined: la volume index, lv mass indexed to height to the power of 2.7 (14), and the ratio of early (e) mitral valve flow to early (e) diastolic lengthening velocity (e / e) using tissue and pulsed wave doppler assessments . Incident heart failure was considered present if signs and symptoms were accompanied by lv systolic (lv ejection fraction <35%) and/or diastolic dysfunction (15). Presence of incident lv diastolic dysfunction (grade 2 or 3) was assessed according to international recommendations (16). In the present analyses, we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid, brachial, and femoral arteries to assess stiffness of the arterial wall (10). Additionally, systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure (17). Augmentation index was determined using applanation tonometry . In a subset of the study population, carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time, adjusted for height in the statistical analyses . Reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described (10). At follow - up, intraobserver intersession coefficients of variation in 10 individuals (aged 70.3 3.6 years) were comparable: 4.2, 13.1, 4.1, and 6.1% for lv ejection fraction, la volume, lv mass, and e / e, respectively . For assessment of glucose status, all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm, igm (impaired fasting glucose and/or impaired glucose tolerance), and type 2 diabetes groups according to the 1999 world health organization criteria (18). Health status, medical history, medication use, and smoking habits were assessed by questionnaires (10). Waist circumference, blood pressures (systolic, mean arterial, and pulse pressures), and levels of cholesterol, insulin, hba1c, and c - reactive protein were determined as previously described (10). Descriptive statistics are presented as means sd or, in the case of a skewed distribution, as median (interquartile range). The f test for anova in the case of continuous variables or tests in the case of proportions were performed to test for differences between groups of glucose status . In the case of skewed distributions, the above tests were done on log - transformed data . For every variable that had> 10% missing data, the number missing is presented in the legends of tables 13 . Linear regression analyses, adjusted for age and sex, were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up . Regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure . For investigation of changes in markers, lv systolic and diastolic dysfunction according to glucose status or arterial stiffness, we adjusted all models for baseline values of these markers (models assessing e / e adjusted for baseline la volume index). Lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors, like overweight, hypertension, insulin resistance, or previous cardiovascular events (19). Therefore, we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models (supplementary data). A secondary purpose of these analyses was to investigate potential residual confounding by these factors . Product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease . Associations of arterial distensibility coefficients, systemic arterial compliance, and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness . Additional analyses, adjusted for age and sex, were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function . P values <0.05, or <0.10 in case of interaction analyses, were considered statistically significant . Statistical analyses were performed with spss for windows (version 15.0; spss, chicago, il). A total of 394 individuals in the age range of 5087 years were included in the present analyses, 87 (22%) of whom had igm and 128 (32%) of whom had type 2 diabetes . Compared with individuals who were not included, these individuals were younger and less likely to have type 2 diabetes and had more favorable levels of arterial stiffness and lv systolic and diastolic dysfunction at baseline (data not shown). Individuals with type 2 diabetes were younger, had higher bmi and blood pressure, and had greater arterial stiffness and more severe lv systolic and diastolic dysfunction at baseline compared with individuals without type 2 diabetes (table 1). Heart failure incidence was not significantly different: 23 (21%) in type 2 diabetes versus 15 (19%) in igm and 24 (15%) in ngm . Incidence of lv diastolic dysfunction grade 2 or 3 was higher with deteriorating glucose status: 88 (72%) in type 2 diabetes versus 52 (61%) in igm and 55 (31%) in ngm . Characteristics according to glucose status the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction: lv ejection fraction, la volume index, lv mass index, and e / e (table 2). After adjustment for age and sex, lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm . Individuals with type 2 diabetes had a 3.71 ml / m higher la volume index, 5.86 g / m higher lv mass index, and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction, but type 2 diabetes was still significantly associated with a higher lv mass index (3.41 g / m) and e / e (1.43). Individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index, independent of baseline lv mass index . Markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status (ngm = reference) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure, lipid levels, renal function, c - reactive protein, and coronary artery disease (supplementary data table a). Adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index . Associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference . Interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd . A longer diabetes duration (per year) was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function . Greater stiffness of carotid, brachial, and femoral arteries, measured as lower distensibility coefficients, was associated with higher levels of la volume index, lv mass index, and e / e (table 3). After adjustment for age and sex, every 10 kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index . Every 10 kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10 kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index, a 0.91 g / m higher lv mass index, and a 0.09 higher e / e. these associations were independent of mean arterial pressure, except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction, 10 kpa lower distensibility coefficients of carotid, brachial, and femoral arteries remained significantly associated with a 0.29, 0.38, and 0.73 ml / m higher la volume index, respectively . Markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure (supplementary data table a). Lower compliance coefficients of brachial and femoral arteries, as well as a higher young elastic modulus or pulse pressure, were similarly associated with more severe lv diastolic dysfunction (supplementary data table b). A higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction: lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index . Carotid - femoral transit time was measured in a subset of 178 individuals, and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time, though not significantly (p = 0.13). There was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction (p values for interaction> 0.10). Figure 1 shows the associations of glucose status and sd scores of arterial stiffness (measured as femoral artery distensibility coefficients) with sd scores for all four markers of lv systolic and diastolic dysfunction, adjusted for age and sex . The right half of each histogram shows that values hardly changed if glucose status and arterial stiffness were combined in one model . For instance, the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex . After subsequent adjustment for arterial stiffness, this value remained similar, despite a loss of statistical significance: 0.25 (p = 0.08). Associations of glucose status with la volume index, lv mass index, and e / e hardly changed either after adjustment for arterial stiffness . Furthermore, each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex . After subsequent adjustment for glucose status, this value was 0.12 (p = 0.02). Associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status, despite a loss of statistical significance . Standardized associations of impaired glucose metabolism (black bars), type 2 diabetes (light gray bars), and lower femoral artery distensibility coefficients (dark gray bars) with markers of lv systolic and diastolic dysfunction, adjusted for age and sex (left half of each histogram) and, additionally, for each other (right half of each histogram). * there was no significant effect modification by sex, use of ace inhibitors, or lipid - lowering, glucose - lowering, or blood pressure lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction (p> 0.10, data not shown). The presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction: lv ejection fraction, la volume index, lv mass index, and e / e (table 2). After adjustment for age and sex, lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm . Individuals with type 2 diabetes had a 3.71 ml / m higher la volume index, 5.86 g / m higher lv mass index, and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction, but type 2 diabetes was still significantly associated with a higher lv mass index (3.41 g / m) and e / e (1.43). Individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index, independent of baseline lv mass index . Markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status (ngm = reference) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure, lipid levels, renal function, c - reactive protein, and coronary artery disease (supplementary data table a). Adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index . Associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference . Interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd . A longer diabetes duration (per year) was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function . Greater stiffness of carotid, brachial, and femoral arteries, measured as lower distensibility coefficients, was associated with higher levels of la volume index, lv mass index, and e / e (table 3). After adjustment for age and sex, every 10 kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index . Every 10 kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10 kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index, a 0.91 g / m higher lv mass index, and a 0.09 higher e / e. these associations were independent of mean arterial pressure, except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction, 10 kpa lower distensibility coefficients of carotid, brachial, and femoral arteries remained significantly associated with a 0.29, 0.38, and 0.73 ml / m higher la volume index, respectively . Markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure (supplementary data table a). Lower compliance coefficients of brachial and femoral arteries, as well as a higher young elastic modulus or pulse pressure, were similarly associated with more severe lv diastolic dysfunction (supplementary data table b). A higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction: lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index . Carotid - femoral transit time was measured in a subset of 178 individuals, and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time, though not significantly (p = 0.13). There was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction (p values for interaction> 0.10). Figure 1 shows the associations of glucose status and sd scores of arterial stiffness (measured as femoral artery distensibility coefficients) with sd scores for all four markers of lv systolic and diastolic dysfunction, adjusted for age and sex . The right half of each histogram shows that values hardly changed if glucose status and arterial stiffness were combined in one model . For instance, the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex . After subsequent adjustment for arterial stiffness, this value remained similar, despite a loss of statistical significance: 0.25 (p = 0.08). Associations of glucose status with la volume index, lv mass index, and e / e hardly changed either after adjustment for arterial stiffness . Furthermore, each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex . After subsequent adjustment for glucose status, this value was 0.12 (p = 0.02). Associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status, despite a loss of statistical significance . Standardized associations of impaired glucose metabolism (black bars), type 2 diabetes (light gray bars), and lower femoral artery distensibility coefficients (dark gray bars) with markers of lv systolic and diastolic dysfunction, adjusted for age and sex (left half of each histogram) and, additionally, for each other (right half of each histogram). There was no significant effect modification by sex, use of ace inhibitors, or lipid - lowering, glucose - lowering, or blood pressure lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction (p> 0.10, data not shown). This study shows that both glucose status and arterial distensibility coefficients were prospectively associated with more severe lv diastolic dysfunction . Furthermore, associations of glucose status and arterial stiffness with lv diastolic dysfunction were largely independent of each other and indicated a deterioration of lv diastolic dysfunction compared with baseline . Strengths include the population - based design and comprehensive assessment of glucose status, arterial stiffness, and lv systolic and diastolic dysfunction . These data provide a unique insight into many different aspects of arterial stiffness and their influence on lv systolic and diastolic dysfunction . Lv diastolic dysfunction at baseline was therefore based on la volume index only, and this might have been subject to some misclassification . Furthermore, differences between attendees and nonattendees suggest that a healthy cohort bias may have occurred in this study . We also had missing data for some echocardiographic markers, predominantly in individuals with high bmi . Our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction (20,21). The type 2 diabetes associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline . These results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes . There was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well, but this was not statistically significant . Individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up . Peripheral markers of arterial stiffness, like arterial distensibility and compliance coefficients, and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction . Associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure, since adjustment for mean arterial pressure only resulted in a small reduction of the associations . Associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index . This might imply that 1) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2) stiffening of arteries and lv walls occurs simultaneously . The first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion (6). The second might be due to coinciding structural changes in arterial and lv walls (9). In the current study, wave reflections seemed especially harmful to lv systolic function, since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction, followed by carotid - femoral transit time . Lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness . This might support the second theory: that stiffening of arteries and lv walls occurs simultaneously . Type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study . More severe lv systolic and diastolic dysfunction in type 2 diabetes therefore cannot be explained or can or just partly be explained by increased arterial stiffness . Other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension (11,2224). This might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c . Overweight and obesity are known to be associated with a higher lv mass index, and indeed, waist circumference seemed to play a role in type 2 diabetes related higher lv mass index (25). Lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . Since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline, the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining . To conclude, the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction . It is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways, since these associations were independent of each other . A better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure . Our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction (20,21). The type 2 diabetes associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline . These results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes . There was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well, but this was not statistically significant . Individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up . Peripheral markers of arterial stiffness, like arterial distensibility and compliance coefficients, and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction . Associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure, since adjustment for mean arterial pressure only resulted in a small reduction of the associations . Associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index . This might imply that 1) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2) stiffening of arteries and lv walls occurs simultaneously . The first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion (6). The second might be due to coinciding structural changes in arterial and lv walls (9). In the current study, wave reflections seemed especially harmful to lv systolic function, since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction, followed by carotid - femoral transit time . Lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness . This might support the second theory: that stiffening of arteries and lv walls occurs simultaneously . Type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study . More severe lv systolic and diastolic dysfunction in type 2 diabetes therefore cannot be explained or can or just partly be explained by increased arterial stiffness . Other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension (11,2224). This might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c . Overweight and obesity are known to be associated with a higher lv mass index, and indeed, waist circumference seemed to play a role in type 2 diabetes related higher lv mass index (25). Lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . Since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline, the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining . To conclude, the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction . It is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways, since these associations were independent of each other . A better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure.
Primitive neuroectodermal tumor (pnet), a highly aggressive tumor of the kidney is a rare malignancy with only few cases being reported in literature . Boys and men are more likely to suffer rpnet (renal pnet), and the sex ratio (male: female) is about 3:1 . It usually presents at an advanced stage, with distant metastasis and is associated with a poor prognosis . We report in this paper, a case of primary renal pnet in a young male . A 39-year - old male presented with complaints of hematuria and heaviness in the right flank since 15 days . Physical examination revealed a soft lump in the right hypochondrium . Ultrasound examination of the abdomen showed an irregular cystic structure in the right kidney at lower pole with few internal echoes . Ct scan of the abdomen demonstrated a large significantly enhancing right renal lower polar mass with necrosis with thrombosis in right renal vein . On the basis of these clinico - radiological findings, diagnosis of right side renal cell cancer gross examination revealed a friable, grayish - white, lobulated mass (7 5 cm) with multiple foci of hemorrhage and necrosis . The mass extended from mid to lower pole of the right kidney and infiltrated into the renal pelvis and was limited to the renal capsule the renal vein and ureter were free of the tumor . There was a thrombus inside the renal vein, which was not adherent to intima of renal vein . Microscopically, the tumor was composed of monotonous sheets of round cells with scant cytoplasm, hyperchromatic vesicular nuclei, coarse chromatin and few conspicuous nucleoli, transverse by thin fibrous bands infiltrating and entrapping the renal glomeruli and tubules [figure 1]. On immunohistochemistry, the tumor cells were positive for cd99 [figure 2a], fli-1 [figure 2b], nse [figure 2c], and synaptophysin [figure 2d], but negative for desmin, ck, ema, and lca . A diagnosis of pnet was made based on the above findings, and patient was started on vac (vincristine, adriamycin, and cyclophosphamide) alternating with ie (ifosfamide and etoposide)-based adjuvant chemotherapy regimen which he tolerated well . After 6 cycles of chemotherapy, a pet scan showed absence of any metabolically active focal uptake in whole body . Chemotherapy with vac (vincristine, actinomycin d, and cyclophosphamide) alternating with ie (ifosfamide and etoposide) for a year was planned . Section showing malignant round cells arranged in pattern sheets infiltrating and entrapping the renal glomeruli and tubules {figure 1a: h&e; 100, figure 1b: h&e; 200} (a) the tumor cells express membranous expression of cd99 {dab; 200} (b) the tumor cells express nuclear positivity for fli-1{dab; 200} (c) the tumor cells express cytoplasmic positivity for neuron - specific enolase (nse) {dab; 200} (d) the tumor cells expressing cytoplasmic positivity for synaptophysin {dab; 200} the primitive neuroectodermal tumor (pnet), was first recognized by arthur purdy stout in 1918, and is a member of the family of small round - cell tumors . Pnet arising in the kidney was first reported by mor in 1994 . Primitive neuroectodermal tumors occurs preferentially in the soft - tissues of the paravertebral region and chest wall, less frequently in extremities, with a slight male predominance . Rpnet appears to be an unique clinical entity that behaves more aggressively than pnet arising at other sites . The presenting symptoms and images of rpnet are non - specific and similar to other renal tumors . Rpnet show a male predominance with 85% cases being diagnosed during the second to fourth decade . It is an aggressive tumor that tends to recur locally and to metastasize to lymph nodes, lung, liver, bone, and bone marrow, which entail a worse prognosis . The differential diagnosis of rpnet includes other small round cell tumors like neuroblastoma, adult nephroblastoma, malignant lymphoma, rhabdoid tumor, small cell carcinoma and monophasic, poorly differentiated, round cell variant of synovial sarcoma, carcinoid, rhabdomyosarcoma, clear cell sarcoma of the kidney, the small cell variant of osteosarcoma, and desmoplastic small round cell tumor . Grossly, rpnet are typically very large tumors with about 65% measuring> 10 cm in diameter . They tend to be grayish in color, encapsulated, and contain focal areas of hemorrhage or necrosis . Microscopically, the tumor is composed of sheets of round cells; individual cells have a round nucleus with a distinct nuclear membrane, fine powdery chromatin, and 1 or 2 small nucleoli . The cytoplasm is ill - defined, scanty, usually pas - positive, and mitosis are variable . Perivascular pseudo rosettes and homer - wright rosettes are common . To better address the diagnosis, immunohistochemical markers to be used to confirm diagnosis are cytokeratin (for nephroblastoma, small cell carcinoma, and synovial sarcoma), lca (for lymphoma), nse / chromogranin a (for neuroblastoma), and cd-99 for pnet . Cd99 (the product of mic2 gene) greatly aids in recognizing the es / pnet family of tumors . Many pnet also stain for neural markers, including nse, leu-7, s-100 protein, synaptophysin, and pgp9.5 . The most frequent translocation in pnet is t(11;22)(q24;q12), detected in approximately 90% of cases, resulting in ews - fli fusion gene . The overall survival in patients who had localized rpent was longer than that in the patients who had rpent with regional nodes or distant metastases . The role of radiotherapy is not clear, but it may be advocated in locally advanced disease and involvement of gerota's fascia . Post - operative chemotherapy for rpent is usually used and can improve the prognosis of rpnet . The most commonly used chemotherapeutics are adriamycin, etoposide, dactinomycin, vincristine, cyclophosphamide, and ifosfamide . A multimodality management is recommended.
Female sex workers (fsws) are at high risk of hiv infection and transmission . A systematic review conducted in over 25 countries with medium and high hiv prevalence indicated that 36.9% of sex workers in sub - saharan africa were hiv positive with some countries having hiv prevalence as high as 70.7% . Despite this high burden of hiv infection, only 58% of sex workers have access to hiv prevention and care programmes and less than half of hiv positive sex workers are enrolled into hiv care . Unaids reported that, in 2013, art averted 6.3 million aids related deaths worldwide as a result of the increased number of people receiving art . In 2015, who released revised guidelines for hiv treatment and recommended initiation of art among all hiv infected individuals irrespective of their cd4 count, for improved treatment outcomes and prevention of hiv transmission . Sex workers are among the priority populations for expansion of hiv services including art . However, despite the existence of established targeted sex worker interventions, lots of challenges still exist that have hindered many from enrolling into care . Several studies have highlighted barriers to linkage to care among female sex workers including discrimination by hospital staff [3, 7], delays at the health facility [8, 9], lack of money for transport and food, fear of drugs, stigma, use of drugs and alcohol, fear to be seen by clients, lack of knowledge about the treatment center [10, 11], and delays in accessing treatment among those who felt they were still healthy . However, while these barriers have been reported, there is still limited information on linkage to hiv care among sex workers in sub - saharan africa, particularly on effective linkage models to inform scale up of hiv interventions among sex workers . In 2012, reach out mbuya hiv / aids initiative (rom), a faith - based community non - government organization in uganda, introduced mobile outreach services to promote hiv testing and treatment among fsws and their clients . Although the program has over 200 fsws enrolled in hiv care, some of the fsws who tested hiv positive did not enrol for treatment . We conducted a study to identify the facilitators and barriers to linkage to hiv care among the fsws who tested positive in order to design appropriate hiv interventions for this key population group . This was a cross - sectional qualitative study that involved in - depth and key informant interviews . This study was part of a larger study that explored the facilitators and barriers to linkage to hiv care among fsws in kampala and wakiso districts in central uganda . Rom promotes hiv prevention through peer educators and provides hiv counselling and testing (hct) services through established outreach clinics that provide care and treatment for hiv positive fsws . Mobile hct outreaches are conducted in locations surrounding mbuya parish in kampala and wakiso districts in central uganda . In - depth interview (idi) participants were selected from 301 hiv positive female sex workers who were registered in the hct registers at the different outreach points between may 2012 and december 2013 . Of the 301 registered clients, 144 were traced and interviewed as part of the larger study where 28 of these were purposively selected to participate in the in - depth interviews . These included 14 fsws who were in hiv care and 14 who had not yet enrolled into care . The selected fsws were contacted by counsellors by phone to explain the study and to ask them to participate in the study . Fsws with missing phone contact information or those who were unavailable after a minimum of three telephone attempts were tracked through their peer educators and friends . Key informant interview (kii) participants were selected from peer educators who were trained by rom and were involved in following up fsws to encourage them to access hiv care . In addition, five rom staffs who were involved in the implementation of the outreach program were interviewed as key informants . Rom provides static and bi - weekly outreach clinics for fsws using staff specifically trained on how to deal with key populations including fsws . At the time of the study, hiv testing and linkage to hiv care services were offered using bi - weekly mobile outreach clinics, brothel based testing, and nocturnal mobile vans at places where the fsws reside or conduct business . Hiv testing was conducted following the national rapid testing algorithm and posttest counselling was provided to hiv infected women who were advised on the available health facilities for follow - up care . Hiv infected women were advised to begin care immediately either at the static or mobile clinics operated by rom or other partners and were followed up with phone calls or by peer educators a week after testing and once every quarter thereafter, to ascertain linkage to care . Those who were enrolled at the static clinic were subjected to a home assessment by the community art and tb treatment supporters (catts) to enable adequate follow - up . The information collected using the home assessment tool included the names and number of household members, water and sanitation, economic status, property owned, distance to the facility, and acceptable mode for communication . Sex workers, who agreed to participate in the study, provided written informed consent and were interviewed in the language of their choice using unstructured, pretested questionnaires . The pretesting was conducted in the nearby project area two weeks before the commencement of the study . To ensure anonymity, data were collected on the facilitators and barriers to linkage to hiv care by trained research assistants with experience in handling fsws . Data were collected on age, education, and linkage status to aid proper categorization of the study participants (linked or not linked to care). We defined linkage to care as having registered within an hiv clinic . The interview themes (i.e., sociodemographic, socioeconomic, structural, and hiv - related factors) were informed by theoretical constructs from the health belief model and the socio - ecological model and further informed by literature on barriers and facilitators to hiv service access among fsws . Figure 1 presents a summary of the key variables and their interrelationships in informing linkage to hiv care among fsws . Interviews were conducted at selected brothels, fsws homes, and static and outreach clinics . Key informant interviews were conducted at the workplace or residence of the respondents, in english and luganda, using unstructured pretested questionnaires . The themes for the key informant interviews included hiv treatment seeking behaviour of the fsws, the challenges of enrolling into care, and the interventions to enhance linkage to hiv care in this population . Additional data, including age, education, job category, residence, and the time allocated to working with fsws, was collected . The review of transcripts was guided by a priori themes pertaining to linkage to care, loss to follow - up, stopping treatment, and facilitators and barriers to linkage . Issues that cut across the different themes which were captured by both authors were coded and categorized while those that were captured by one but not the other were subjected to further analysis until they were resolved and used to support the overriding themes or dropped . The study was approved by makerere university school of public health higher degrees research and ethics committee (irb #: irb00011353) and the uganda national council of science and technology . Of the 28 fsws interviewed, 14 (50%) were enrolled in hiv care (nine at rom), 8 had not yet enrolled, and 6 had previously enrolled but were either lost to follow - up or had stopped treatment . Of the 28 women interviewed, 24 (85%) were aged 2030 years while the rest where> 30 years . Half (14) had at least secondary or higher education and the other half (14) had at least primary education . The fsws who were lost to follow - up or had stopped treatment were aged between 15 and 36 years with their education level ranging from none to secondary . The respondents included fsws who operate in bars (6), on the streets (8), at home (3), and in brothels (11) (table 1). The findings have been grouped into two themes: (a) facilitators of linkage to hiv care and (b) barriers to linkage to hiv care among fsws, as presented below . The facilitators have been further grouped into two main themes: health system and social network factors (table 2). Majority of the participants associated their linkage to care with the good quality of the services including good attitude and friendliness exhibited by the counsellors towards them . They noted that the counsellors talked to them with ease and were not judgmental: the counsellors are good, they talk to us very well and are almost like our parents . When i was told my results i got so scared as though i would die there and then . I failed to breath for 30 minutes; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment . (fsw, 29 years, enrolled in care) the counsellors are good, they talk to us very well and are almost like our parents . When i was told my results i got so scared as though i would die there and then . I failed to breath for 30 minutes; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment . (fsw, 29 years, enrolled in care) participants reported that the counsellors provided detailed information and encouragement to help them start treatment . The counsellors told them that testing hiv positive was not the end of life and that with medication they would be helped to stay healthy and live longer . In addition, participants appreciated the good follow - up support for those who tested positive . Some said the counsellors called them and followed them up to their homes, which made them feel cared for: i started treatment due to counselor x's advice, she was so caring and always followed me up, she could visit me; otherwise on my own i had even disappeared but she was on my case . (fsw, 24 years, enrolled in care) i started treatment due to counselor x's advice, she was so caring and always followed me up, she could visit me; otherwise on my own i had even disappeared but she was on my case . (fsw, 24 years, enrolled in care) some participants acknowledged that it was easy for them to enrol in care because of the same - day results and immediate initiation of treatment . The peer educators and fellow sex workers enabled them to start treatment by encouraging them and helping to allay their fears regarding hiv treatment and occasionally escorting them to the clinics to start medication . Some respondents said they would forget the clinic days because the clinic did not run every day but the peer educators reminded them . During the key informant interviews the peer educators mentioned how tirelessly they worked to follow up the hiv positive fsws; they were sometimes abused by the sex workers but they never gave up on them: i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine . (peer educator, 29 years) they come and tell me that on such and such a date i will be going to the clinic so remind me, i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic . (peer educator, 25 years) i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine . (peer educator, 29 years) they come and tell me that on such and such a date i will be going to the clinic so remind me, i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic . (peer educator, 25 years) the proximity of the clinic to their residence was another enabling factor for linkage to care . Some fsws did not want to move long distances for hiv care and others did not have transport so they thought that enrolling at rom would help reduce such burdens . Across all interviews fsws who were enrolled in savings and they noted that the members of the support groups like village savings groups started by rom for sex workers only or mixed with other community members helped them to enrol into care . The members of the savings group reportedly advised them to start on treatment and live longer . Being part of a savings group also created hope that money would be available for food and they would have capital to start their own business and live longer: i thought i was alone but when i realized we were many, i stopped fearing and also started medicine . My group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more . (fsw, 25 years, enrolled in care) i thought i was alone but when i realized we were many, i stopped fearing and also started medicine . My group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more . (fsw, 25 years, enrolled in care) across all the interviews the participants cited the need to remain healthy as one of their motivators for linkage to care . In order to continue doing their work, they wanted to start medication to remain healthy and not show any signs of opportunistic infections, which would scare away their customers . Others wanted to remain healthy so as to live longer and take care of their children: it is because of my children; i have to live for them because i am a widow, if i don't take care of myself and die who will take care of them . (fsw with 3 children, 24 years, in care) it is because of my children; i have to live for them because i am a widow, if i don't take care of myself and die who will take care of them . (fsw with 3 children, 24 years, in care) several participants reported that seeing their colleagues improve after starting treatment motivated them to start on treatment while others did so because many of their friends were dying, which compelled them to start treatment so as to survive: i had a friend who stopped treatment and died, when we took her to the hospital the nurses said if only she continued with treatment she would be alive . When they (providers) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment . (fsw 20 years, enrolled in care) i had a friend who stopped treatment and died, when we took her to the hospital the nurses said if only she continued with treatment she would be alive . When they (providers) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment . (fsw 20 years, enrolled in care) barriers were similarly categorized into two themes: health systems and social network factors (table 3). The fsws enrolled at the government facilities mentioned discrimination from the health workers who openly exhibited a negative attitude towards sex workers . They said the health workers reacted differently after realizing that they were dealing with a sex worker and, upon telling their friends, those who had not yet registered for care feared to enrol: the counselor asked me what i do and i said sex work . She called four other counselors to ask me why i do that kind of work; i wanted to ask them, what do you expect me to do?' But the counselor stood up and asked me rudely; is that also work to mention among people?' That counselor annoyed me so much i walked off and never wanted to see her again . (peer educator who is a fsw, 29 years) the counselor asked me what i do and i said sex work . She called four other counselors to ask me why i do that kind of work; i wanted to ask them, what do you expect me to do?' But the counselor stood up and asked me rudely; is that also work to mention among people?' That counselor annoyed me so much i walked off and never wanted to see her again . (peer educator who is a fsw, 29 years) the four fsws who were enrolled at government facilities noted that they were discouraged by the requirement to present a treatment supporter coupled with the long procedures for enrolment that involved several visits to the facility: it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get . May be i do not want someone else to know that i am positive . (fsw, 29 years, enrolled in care) it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get . I felt this was not right because i must go for treatment by myself may be i do not want someone else to know that i am positive . (fsw, 29 years, enrolled in care) almost all the participants cited stigma as a major barrier to linkage to care . The majority feared to be seen by their clients at the clinic . Although the close proximity of the clinic was mentioned as a motivator, for some fsws it was a hindrance . Participants were afraid of being seen at the hiv clinic by customers, their friends, and other people in the village and were particularly concerned about rumours: i cannot go to that clinic because everyone here goes there and they will see me which will spoil my business . Right now the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick . (fsw 22 years, not enrolled) i cannot go to that clinic because everyone here goes there and they will see me which will spoil my business . Right now the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick . (fsw 22 years, not enrolled) due to lack of information, many participants had many myths about arvs (e.g., that they kill fast and need a lot of care). They avoided the arvs by not going to the clinic while some opted to wait until they got other jobs because of the perception that arvs could weaken them and disrupt their work . Some respondents feared drugs and could not imagine swallowing them for life so they opted out . On the other hand, some participants feared death after testing and lost hope for living; they did not see the need for starting on art: madam isn't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs (fsw, 20 years, not in care and never went to school) madam isn't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs (fsw, 20 years, not in care and never went to school) some fsws did not know what hiv is while others encountered difficulties in identifying an hiv clinic because they lacked proper information about the location of the clinics . This was mainly a challenge to the young girls (aged between 20 and 25 years) who lived in the brothels as indicated by the project staff: some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is . (key informant) some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is . (key informant) some fsws mentioned that they could not enrol in an hiv clinic since they did not believe their hiv results . Some thought it was just a prick performed hurriedly in the night and wondered how it translated into an hiv positive result so they waited and retested many times: when i reached home, i started doubting the results and decided to go for another test in a different place . I continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin . (fsw 29 yrs, not enrolled in care) when i reached home, i started doubting the results and decided to go for another test in a different place . I continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin . (fsw 29 yrs, not enrolled in care) a third of the fsws mentioned that they were using herbs as a substitute for arvs and therefore saw no need of being registered in an hiv clinic . Besides many of the fsws said they used local herbs from traditional healers to attract customers and did not have to line up for herbs like they did in the hiv clinic: no . I have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [meaning arvs]. Even with the herbs now am on medication; i do take the local herbs and am fine with the treatment, i don't want to be forced to take arvs . (fsw, 24 yrs, not enrolled) no . I have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [meaning arvs]. Even with the herbs now am on medication; i do take the local herbs and am fine with the treatment, i don't want to be forced to take arvs . (fsw, 24 yrs, not enrolled) some participants bought painkillers from general clinics instead of enrolling in an hiv clinic (self - medication). Even when the nurses in these clinics suspected that they were hiv infected, they did not tell them because they wanted business through treating their on and off sickness . Because they are always travelling in different places looking for clients, fsws cited lack of time as one of the barriers to linkage to care: i am still very busy right now looking for rent and food . I cannot lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries . (fsw 23 years, not enrolled) i am still very busy right now looking for rent and food . I cannot lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries . (fsw 23 years, not enrolled) the kis felt that the street based sex workers had more challenges with linkage to hiv care compared to those in brothels because they were difficult to trace given that they only relied on phone numbers which were off most of the time: those on the streets do not want to access care . (key informant) those on the streets do not want to access care . (key informant) across all interviews, the fsws mentioned lack of finances as a major hindrance to linkage to care . They were concerned about requirements such as good food and frequent transport to the health facility: it's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking . (fsw 22 years, not enrolled) it's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking . Our study of facilitators and barriers to linkage to hiv care among female sex workers in kampala and wakiso districts in uganda shows that health system and social network factors are the major facilitators and barriers to linkage to hiv care . Good quality health services (especially polite and caring providers, strong follow - up structures using peer educators, and provider telephone calls) and social network factors (encouragement from peers and membership of savings group and the need to maintain good health) were the primary facilitators of linkage to hiv care among fsws . On the other hand, perceived stigma, various forms of misinformation, and prohibitive clinic policies were major barriers to linkage to hiv care . Contrary to the experience of fsws at rom, those who were registered in the public health facilities encountered negative staff attitudes, a major barrier that has been documented in several studies [8, 16, 17]. These findings suggest that efforts to improve linkage and retention in hiv care among fsws should focus on quality of services and improving social support networks . Ensuring good quality services, particularly providers who can deliver services in a respectful and no - judgmental manner without undue delays and restrictions and adequate community education and mobilization as well as support structures for retention are key health system issues that can improve the quality of services . Using peers to educate communities and follow - up of those who need treatment reduce the burden on health workers and strengthens community engagement . As reported elsewhere, the requirement of a treatment supporter before initiating care and treatment was a barrier to treatment that should no longer be strictly enforced . The training of providers and peer educators prior to implementation of this program could have contributed to delivery of quality services at rom and are a key intervention area . Given the overriding concerns about being seen at hiv facilities, training for providers should emphasize the need for privacy and confidentiality . Further, providing an integrated package of services for sex workers could reduce stigma around accessing hiv and other services . Training should also emphasize flexibility with certain clinic policies that could hinder access to hiv services among sex workers . Social network issues were also strong facilitators and barriers to linkage and require interventions particularly targeted at enhancing support by peers and reducing misinformation and stigma . Enacted stigma, myths, and fears about arvs coupled with lack of correct information about hiv continue to be major barriers to linkage to hiv care . Financial constraints were a prominent barrier to linkage to care . The savings groups were a good avenue for social support and financial stability and could be integrated into health programs for low - income sex workers [20, 21]. Collectively, these findings suggest that programs targeting fsws should integrate continuous sensitization and provision of tailor made services that address the individual level, community, and health system needs of fsw . This study is based on interviews with 14 fsws who were in hiv care and 14 others who were not . By all counts, the numbers are too small to represent the views of all fsws who are enrolled or not enrolled in hiv care . However, our study findings provide good insights into the facilitators and barriers to linkage that can inform interventions to improving linkage for fsws . Future research is needed to further understand how the fsws can be maintained in care once they have been linked . Our study shows that fsws ability to be linked to hiv care is largely influenced by good quality friendly services and community support systems especially from their peers . Hiv programs for fsw should focus on enhancing these as well as dealing with the barriers that mainly stem from stigma, ignorance, and work - related challenges . These findings call for a need to design interventions that utilize a multichannel, multipronged approach to increase access to hiv services among fsws.
The acquired alexias may be categorized into posterior, anterior, central and deep alexias . Analagous to semantic paraphasias, semantic paralexias constitute the substitution of content related words during reading . Unlike the analogous subcortical aphasias such as thalamic aphasia, subcortical syndromes of alexia have rarely been described . This report depicts a patient with thalamic alexia with features of deep alexia (or paralexia), the latter which has not been described after review of the english based literature . A 57 year old white, right handed, english speaking woman with 13 years of education, presented to our stroke center with mild right sided numbness and weakness (graded + 4/5 medical research council grading scale) with the family reporting transient confused conversation and speech difficulty . The clinical evaluation at our center encompasses a cognitive screening evaluation in all patients followed by psychometric assessment in selected patients described in detail elsewhere . No dysnomia was documented . Specifically visual acuity and visual fields were normal and there was no object agnosia, dyschromatopsia, dyscalculia, finger agnosia, right left disorientation, or hemineglect syndrome . The substitution of semantically related words was documented with only mild slowing in her reading (family corroboration). Cerebrovascular risk factors included a significant smoking history and on investigation hyperhomocyteinemia (15.6 mmol / l). Within 4 days, the reading difficulty had normalized but she remained with mild speech dysfluency, right hand ideomotor apraxia and developed a dejerine roussey or post stroke thalamic pain syndrome . Multimodality magnetic resonance imaging investigations revealed a left lateral posterior thalamic infarct, well circumscribed on t2 weighted and diffusion weighted imaging without other brain parenchymal lesion (figure 1). Magnetic resonance angiography revealed a left internal carotid artery stenosis graded 5074% and basilar artery fenestration . Figure 1left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan (arrows). Left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan (arrows). In view of the isolated reading impairment, additional psychometric examination was performed on day 3 after the stroke onset, with the boston diagnostic aphasia examination (third edition - short form). In addition, the test for semantic paralexia prone words were tested with the standard version of the boston diagnostic aphasia examination (version iii) (table 1). In comparison to normative data, notable deficiencies were noted only in speech fluency, articulation, oral word and sentence reading, picture word matching and writing . Table 1boston diagnostic aphasia test - version iii.subtests%fluency phrase length30 melodic line60 grammatical form70conversation expository speech100auditory comprehension basic word discrimination100 commands100 complex ideational material100articulation (agility)70recitation (automatized sequences)100repetition words60 sentences100naming responsive naming100 boston naming test100 special categories100paraphasia100reading matching case and scripts100 number matching100 picture word matching40 oral word reading40 oral sentence reading80 oral sentence comprehension100 sentence / paragraph100 comprehension100wiriting form20 letter choice40 motor facility40 a 57 year old white, right handed, english speaking woman with 13 years of education, presented to our stroke center with mild right sided numbness and weakness (graded + 4/5 medical research council grading scale) with the family reporting transient confused conversation and speech difficulty . The clinical evaluation at our center encompasses a cognitive screening evaluation in all patients followed by psychometric assessment in selected patients described in detail elsewhere . No dysnomia was documented . Specifically visual acuity and visual fields were normal and there was no object agnosia, dyschromatopsia, dyscalculia, finger agnosia, right left disorientation, or hemineglect syndrome . The substitution of semantically related words was documented with only mild slowing in her reading (family corroboration). Cerebrovascular risk factors included a significant smoking history and on investigation hyperhomocyteinemia (15.6 mmol / l). Within 4 days, the reading difficulty had normalized but she remained with mild speech dysfluency, right hand ideomotor apraxia and developed a dejerine roussey or post stroke thalamic pain syndrome . Multimodality magnetic resonance imaging investigations revealed a left lateral posterior thalamic infarct, well circumscribed on t2 weighted and diffusion weighted imaging without other brain parenchymal lesion (figure 1). Magnetic resonance angiography revealed a left internal carotid artery stenosis graded 5074% and basilar artery fenestration . Figure 1left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan (arrows). Left thalamic infarct depicted on diffusion weighted and t2 weighted magnetic resonance scan (arrows). In view of the isolated reading impairment, additional psychometric examination was performed on day 3 after the stroke onset, with the boston diagnostic aphasia examination (third edition - short form). In addition, the test for semantic paralexia prone words were tested with the standard version of the boston diagnostic aphasia examination (version iii) (table 1). In comparison to normative data, notable deficiencies were noted only in speech fluency, articulation, oral word and sentence reading, picture word matching and writing . Table 1boston diagnostic aphasia test - version iii.subtests%fluency phrase length30 melodic line60 grammatical form70conversation expository speech100auditory comprehension basic word discrimination100 commands100 complex ideational material100articulation (agility)70recitation (automatized sequences)100repetition words60 sentences100naming responsive naming100 boston naming test100 special categories100paraphasia100reading matching case and scripts100 number matching100 picture word matching40 oral word reading40 oral sentence reading80 oral sentence comprehension100 sentence / paragraph100 comprehension100wiriting form20 letter choice40 motor facility40 the commonly referred to syndromic classification of benson and geschwind of posterior alexia (alexia without agraphia), central alexia (alexia with agraphia), anterior alexia (alexia in association with expressive dysphasia) and deep dyslexia (primarily semantic paralexia disorder) aptly describes most acquired reading deficiencies . Alexia secondary to isolated thalamic lesion has not been reported other than in combination with left occipital lesions, the latter lesions the usual focus . Semantic paralexia due to thalamic lesions has not been reported previously with neuroanatomical lesions mostly referred to as large perisylvian and even right hemisphere lesions implicated . Similar to subcortical aphasias, the subcortical (thalamic) alexia reported here is characterized by atypical alexia syndrome components . These include relatively mild deficit, transience, a mixture of syndromic components with rapid recovery . The lack of hemispheric lesions on magnetic resonance imaging using standard t2 weighted images diffusion weighted imaging for infarct exclusion, fluid attenuation inversion recovery sequences for covert white matter lesions and gradient echo sequences to exclude minor hemorrhagic lesions, implicated the thalamic lesion alone as the critical lesion in the syndrome described . Although not a routine stroke investigative tool, functional magnetic imaging (f - mri) would have been an important additional neuroimaging modality to assess whether the right hemisphere had or had not played a role . However, the rapidity of recovery precluded functional magnetic imaging, that may have helped ascertain whether right hemisphere linguistic processing was operative, the contemporary hypothesis of semantic paralexia.
Proliferative vitreoretinopathy (pvr) remains the major complication after retinal detachment surgery [13]. Pvr was identified as an independent clinical entity in 1983 by the retina society terminology committee and a classification was created, based on the condition formerly named massive vitreous traction or massive periretinal proliferation [57]. This classification divided pvr into four stages, a, b, c, and d, apparently by increasing its severity, from minimal to massive pvr (table 1). Nevertheless, this classification had numerous limitations . It did not consider the location of the vitreoretinal traction and the magnitude of the contraction . In addition, some of the stages provide a false idea of severity; for instance, d4 caused by a localized epiretinal membrane could be more easily treated by surgery than a c1 caused by intraretinal changes [8, 9]. In 1989, the silicone study group introduced a new classification including some characteristics, such as the location, anterior or posterior, and the type of contraction (table 2). Classification was then updated in 1991 according to modifications proposed by the silicone study group and also by other authors (table 3). This classification appears to be difficult to use in clinical practice and may not offer any special advantage for decision - making in relation to the treatment of the disease . Moreover, the classifications might have prevented advances in the understanding of the disease pathogenesis . For instance, because the retina society committee defined pvr as a proliferative disease, many treatments based on the inhibition of cell proliferation were developed for more than 20 years, none of which appears to have produced a significant clinical advance . Therefore, a review of both the classification and the pathogenesis of pvr appears to be appropriate to aid the development of new treatments . There is a clinical impression that over the last 15 years clinicians have abandoned current pvr classifications . Thus, the purpose of this study has been to evaluate the current use of pvr classifications in papers dealing with clinical practice and therapy as an indirect measure of the degree of usefulness of existing classifications . A search in pubmed for papers published between january 2000 and january 2014 was undertaken by two independent researchers . Inclusion criteria comprised human studies published in english, french, and spanish on pvr or retinal detachment with specific mention to pvr . Prospective randomized and nonrandomized clinical studies, retrospective clinical studies, short series, and pilot studies were included . A manual search of related articles was also performed through references reported in each article . We also analyzed how the different authors interpreted the classifications, comparing the results they provided with the original description . Therefore, we investigated if they used all the characteristics (grade [a, b, c], pvr localization, extension in hours, and type) of the updated retina society classification or if they were used only partially . Furthermore, it was also noted whether classifications were used both before and after an eventual pvr pharmacological or surgical treatment . Data were extracted using a custom - made data sheet performed with agreement between authors . Preliminary search identified a total of 219 publications, 81 of which were eliminated as they did not fulfill the inclusion criteria . 35 articles (25,4%) did not undergo detailed analysis because there was no reference to the type of pvr classification used by the authors or because no classification was used (generic terms such as initial or severe pvr or simply pvr were adopted, without using any grading system). 103 publications (74,6%) used standardized pvr classifications and they were analyzed in detail . The most used classification was the updated retina society classification (58 cites; 56,3%), followed by the first retina society classification (35 cites; 33,9%). In addition, 3 authors (2,9%) used modified or customized classifications [1315]. In 4 publications (3,8%), errors in the stated classification were identified [13, 1618]: in two papers [16, 18], the authors cite the updated retina society (91) but in fact they used the first retina society classification (83), koerner et al ., after citing the updated retina society classification used a customized one, and roldn - pallars et al . Cite the two retina society classifications at the same time . Among the papers using the updated retina society classification, the most documented was grade c pvr (48 articles, 83%), while grades b and a were less frequently documented (15 (26%) and 7 (12%) articles, resp . ). Of the publications documenting only c grade, the classification of subtypes was infrequently used . In 5 of 48 publications (10,4%), there was a full c grade description in terms of localization (anterior or posterior), extension (in clock hours), and type (focal, diffuse, subretinal, circumferential, or anterior displacement). A similar finding was observed when authors used the first retina society classification (35 papers): only c and d grades were frequently reported (27 and 10 papers, resp . ), while grades a and b were infrequently used (4 and 7 articles, resp . ). When the silicone study classification was used, only grade c was mentioned (in 2 out of 4 articles). Finally, there were 2 publications (1,9%) in which the standard classifications were used to assess changes in pvr status after any treatment, reporting pvr grade before and after it [19, 20]. The remaining authors used the classifications only to describe pvr status, rather than using them to analyze the improvement or worsening of the previous stage after the applied treatment . Classifications of pvr were developed to provide clinicians with a useful tool to compare results of treatments . They are, to date, purely descriptive and do not reflect the pathobiology of this complex vitreoretinal disease . The original classification of 1983, which was relatively simple to use, induces errors in the estimating severity in some cases . As mentioned, d grades were based on the ophthalmoscopic appearance of the detached retina, and experience demonstrated that some of these cases could be relatively easily solved by peeling localized epiretinal membranes although, in some cases, classified as c, intraretinal severe changes prevent reattachment, unless complicated surgical techniques were used . Subsequent classifications [10, 11] were more detailed and therefore more complicated to use on a routine basis . They incorporated the anterior pvr forms, which add severity to the case, but until now no one has incorporated the intraretinal changes, observed in many surgical retinal samples [8, 9], which may prevent the anatomical reattachment of the retina and require retinectomy . Another important limitation is that they do not provide information on the activity of the process, although it has been observed that pvr progresses through several stages [13]. This may be crucial in estimating the risk of reproliferation after surgery or when surgeons schedule removing silicone oil, and no data on the chronology of events is included in any classification . Furthermore, current classifications are not prognostic and do not correlate with visual prognosis or anatomical success after surgical treatment . Moreover, some of these classifications can be difficult to use in clinical practice, due to their complexity (grade, localization, extension, type, etc .) And because they may not provide useful information they have been largely abandoned by clinicians . Our findings in this study demonstrate that, in clinical research, investigators are using them inconsistently and reporting limited observations of stages (mostly grade c). Some authors simply do not use any, preferring generic terms, such as minimal, moderate, and severe pvr [21, 22]. Furthermore, early stages of pvr (named as grades a and b), which are common to all classifications, are not used, and most authors refer only to most advanced stages, basically grade c [20, 23]. Moreover, the presence of classification errors could indicate that there is confusion concerning their use [13, 1618]. An important relevant finding is that only a limited number of clinicians appropriately and fully use the current updated classification of 1991 [2427], while the majority of authors avoid using the added characteristics of localization, extension, and type, limiting the description to the grade . In addition, many colleagues still use the first reported classification, probably because although it presents many limitations, it is easier to use compared to the last version [23, 28]. Additionally, some authors decided to use alternative classification, probably to avoid problems evaluating pvr stages . The use of multiple classifications makes the efficient communication between clinicians and the comparison of different studies very problematic . As mentioned, one of the aims of this study was to evaluate the usefulness of these classifications for pvr management with surgery and other adjunctive treatments . Unfortunately, many ophthalmologists did not use the normalized classifications for comparing the outcomes using non - clearly defined terms such as pvr recurrence [29, 30]. Our findings showed the inconsistent and limited use of the current pvr classifications suggesting that it may be of benefit to produce a revised classification incorporating, if possible, the new knowledge on pvr which has been published since 1991, pointing out new potential targets for therapeutic agents distinct from those, mainly proliferative agents, targeted by the original description of this disease . Thus, it is possible that we could reduce its prevalence after retinal detachment surgery and to improve the anatomical and functional results of this disease which resists the attempts of both basic researchers and clinicians for more than 30 years.
The number of total joint arthroplasties (tjas) performed has increased steadily in recent years, with projected numbers for the coming years rising further . Consequently according to recent data, peri - prosthetic joint infection (pji) incidence constitutes between approximately 0.3% and 1.7% of all total hip arthroplasties (tha), and between 0.8% and 1.9% of all total knee arthroplasties (tka). Pji can be classified in intra - operative, early post - operative, acute haematogenous and chronic infections, according to both timing and cause of infection . The risk factors potentially correlated with acute pji infection can be divided into pre - operative (usually related to patient comorbidities), peri - operative and post - operative, which are mainly linked to the behaviours of the surgeon and the hospital staff . Conversely, chronic infections are less influenced by the conduct of the surgeon, as they are most often related to haematogenous diffusion of bacteria . The aim of this paper is to review the recent literature, summarising the most relevant risk factors that the surgeon can modify in order to reduce the incidence of peri - prosthetic joint infection . Several studies demonstrated that some comorbidity can be associated with an increased risk of pji . The american academy of orthopaedic surgeons (aaos) reported on the different risk factors for pji and developed a guideline for pji prevention and treatment . In 2013 consensus for definition, prevention and management of pji, as an update to the previous guidelines . Potential risk factors for development of surgical site infection or peri - prosthetic joint infection after elective total joint arthroplasty, according to the international consensus on periprosthetic joint infection bmi, body mass index; hba1c, glycated haemoglobin . Although the majority of these conditions must be considered as non - modifiable factors, many preventive measures may be adopted to reduce their impact on the development of pji . Table 2 summarises the modifiable and non - modifiable factors related to pji, including some preventive measures to manage reversible medical comorbidities, according to the existing literature . Pre - operative modifiable and non - modifiable risk factors; measures the surgeon can adopt to reduce impact of risk factors on development of pji pji, peri - prosthetic joint infections; bmi, body mass index; hba1c, glycated haemoglobin; mrsa, methicillin - resistant staphylococcus aureus; s. aureus, staphylococcus aureus other potential pre - operative risk factors are intra - articular corticosteroid injections and any infectious disease, particularly urinary tract infection (uti) and nasal colonisation with staphylococcus (s.) aureus . The relationship between steroid injections and post - operative pji was evaluated in several studies . Papavasiliou et al reported an incidence of only 2% of infections in a series of 114 tkas, but all of the infected tkas had previously been treated with an intra - articular corticosteroid injection within the 11-month period prior to surgery . Conversely, desai et al stated that the incidence of infection did not increase in patients with prior steroid injection treatment . The correlation between post - operative utis and pji has been demonstrated . However, the association between pre - operative bacteriuria and early deep infections remains uncertain . The presence of symptoms of a uti in association with urinary leukocyte counts greater than 1 10/ml and a bacterial count greater than 1 10/ml should be the only indication for surgical delay . Conversely, in asymptomatic patients it is still possible to proceed with tja by treating those patients with urine colony counts greater than 1 10/ml . Different authors confirmed that being a high - level nasal carrier of s. aureus is an important and significant independent risk factor for developing ssi with s. aureus . Nasal application of mupirocin is widely accepted as treatment for nasal carriers of s. aureus . In a recent randomised controlled trial (rct), mupirocin treatment resulted in a simple, safe and cost - effective intervention that can reduce the risk of ssi . Different intra - operative components may play an important role as risk factors for developing pji (table 3).the first six hours following surgery are the most important regarding infection, as during those hours the numbers of bacteria multiply exponentially . Maintaining a low blood level of bacteria in this period is critical, and for this reason prophylactic antibiotics are infused to decrease bacterial multiplication and to extend this golden period . The pre - operative dose of antibiotics should be administered within one hour before the surgical incision; this can be extended to two hours for vancomycin and fluoroquinolones . Most authors agree that a single pre - operative prophylactic infusion of cefazolin (1 gr if <80 kg; 2 gr if> 80 kg) is a good choice . However, recent studies have demonstrated that targeted use of vancomycin and cefazolin among patients undergoing revision tka significantly reduced the rate of overall infections, in particular of mrsa . Surgeons should consider additional antibiotic administration if the surgery time is twice the length of the half - life of the antibiotic, or whenever the blood loss exceeds 2000 ml and fluid resuscitation is over 2000 ml . To reduce pji infection rate, some authors advocate using antibiotic - loaded bone cement (albc) for the cementation . However, it was demonstrated that routine use of albc does not change the pji rate, though it may be useful in the reduction of the pji rate in high - risk patients (for example those with diabetes or immunosuppression). Intra - operative factors potentially associated with pjis pji, peri - prosthetic joint infections surgical site preparation also plays a role in reducing pji rate . Some authors have confirmed the reduction of pji in patients who underwent pre - admission surgical site preparation using chlorhexidine washing . Different studies have evaluated the best solutions for surgical site preparation to reduce pji . In a rct conducted by darouiche et al, it was demonstrated that a chlorhexidine alcohol solution was more protective than povidone iodine against both superficial and deep infections . This is probably due to the more rapid action, persistent activity despite exposure to bodily fluids, and residual effect of chlorexidine compared with povidone . The preparation of the surgical site often includes using plastic adhesive drapes and sterile stockinette . However, a recent cochrane review showed no evidence that adhesive drapes reduce surgical site infection rates . The bactericidal action of incision drapes containing iodine is inferior to conventional skin preparation solutions, so using incision drapes as a substitute for conventional skin preparation is not recommended . Furthermore, boekel et al concluded that the surgical field for tka can be contaminated by proximal microbial spread from the unprepared foot with the use of a sterile stockinette drape . The risk of pji is also directly correlated with the length of the surgery, which should be less than 2.5 hours as a reasonable cut - off point . Zhu et al, in their meta - analysis, concluded that increased operative time is associated with a higher risk of pji development (or = 2.18, ci 95% 1.39 - 3.42, p = 0.003). However, these data may also be correlated with the high complexity of long procedures . Furthermore, the surgeon s surgical volume may be directly associated with pji: surgeons with low volumes may have higher rates of infection . Hand care is another crucial point in reducing pji infection; hand surgical scrub recommendations were previously published by the centers for disease control and prevention . In particular, surgeons should remove debris from underneath the fingernails using a nail cleaner under running water, and either an antimicrobial soap or an alcohol - based hand rub should be used persistently for at least five minutes . Different studies evaluated the number of glove changes necessary to reduce the risk of pji . Of note, beldame et al recommended: renewing outer gloves after draping (before placing a cutaneous adhesive); opening the instrumentation secondarily, with a new glove change after handling instruments which may cause perforations; renewing outer gloves after each surgical stage . No strong evidence is available in the literature regarding the appropriate number of glove changes . Different authors recommended double gloving to reduce the risk of inner glove perforation, but no correlation with pji has been demonstrated . The role of the personal protection system (pps) in preventing pji is still debated . Kearns et al demonstrated that the external surface of the pps cannot be assumed to be sterile after removal from the original packaging, and they suggested the need to change gloves if the pps is touched or adjusted during the procedure . Other authors agree with the consideration of pps as more a personal protection than equipment specifically to reduce pji . Light handles can be a source of contamination, and surgeons must minimise their handling as far as possible . Furthermore a limited number of portable devices such as mobile telephones and tablet computers in the operating room is recommended, although no evidence in the literature is able to link their use to increased risk . As airborne pathogens are a potential source of infection, the location and length of time that surgical instruments remain exposed for this reason, all instruments should be opened in operating rooms with clean air systems . These basins are repeatedly used during a surgical procedure and should therefore be considered a potential source of contamination . Anto et al demonstrated that 23.8% of specimens from splash basins tested positive for bacterial contamination, and they suggested that surgeons should stop using them . A recent systematic review showed no conclusive results regarding the utility of laminar flow in reducing pji rate . Other authors agree that, despite the number of previous studies demonstrating the efficacy of laminar flow, more recent research has failed to prove this efficacy . Ultraviolet light seems to be more effective when compared with laminar flow in reducing pji; however it is characterised by potentially unacceptable health costs to operative personnel . A level iii study showed that the number of door openings had a role in increased infection rate . Surgeons may also play a role in reducing the rate of pji using power - pulsed lavage or wound lavage at surgery . In a level iv study, different post - operative variables may also play a role as risk factors for pji development . Antibiotic prophylaxis for other surgical procedures before and after tja, such as dental care or urological procedures, seems to play a role as a despite the lack of literature demonstrating the relationship between dental procedures and pji, the current recommendation of the aaos is to use antibiotic prophylaxis in patients with a tja who are undergoing dental procedures, as well as any other invasive procedure . Furthermore, patients should be aware that any infection is a potential source of haematogenous dissemination . As previously reported by different authors, patients with tja who have an active infection anywhere in the body are at risk of developing a pji . For this reason, a prompt diagnosis and management of those infections is a mandatory prevention mechanism . There is still debate regarding the association between blood transfusion and pji . In a level ii study, pulido et al demonstrated that transfusion with allogenic blood is an independent risk factor for pji . Patients receiving allogenic transfusions were 2.1 times more likely to develop pji compared with patients receiving no transfusion . In their study, innerhofer et al concluded that allogenic filtered transfusion is an independent variable for pji prediction (or 23.65; ci 95%, 1.3 - 422.1; p = 0.01). Furthermore, the centers for disease control and prevention guidelines defined peri - operative allogeneic transfusion as a potential risk factor for developing pji, but concluded that the interpretation of the existing literature is difficult due to variations in assessment criteria . However, different measures can be adopted to reduce the need for blood transfusion, such as pre - operative screening for anaemia and its treatment, intra - operative accurate haemostasis, minimisation of surgical time and use of tranexamic acid . Haematoma and persistent wound drainage were also related to an increased pji rate; these conditions should be treated promptly with antibiotic prophylaxis, a decrease in anticoagulation dose, surgical evacuation of the haematoma, irrigation and debridement and modular component exchange . Recently, more advanced surgical bandages such as hydrofibre absorbent dressings were proposed, with the aim of reducing the medication to allow for better wound healing and to prevent bacteria from entering the wound site from the external environment . In a level ii study, cai et al concluded that advanced surgical dressings such as hydrofibre may contribute to a reduction in the incidence of acute pji . Infection represents a major challenge in tja, and is costly and demanding to manage for both surgeons and patients . The main risk factors involved in pji development are divided into pre - operative, intra - operative and post - operative factors . The surgeon can act to reduce the impact of some reversible comorbidities, for example controlling glycaemia in diabetic patients or improving malnutrition . Various intra - operative risk factors such as operating theatre traffic, use of light handles, pulsed lavage or number of glove changes may also be related to infection . Post - operative risk factors include transient bacteraemia related to dental procedures or other infections, wound care and blood transfusion as well as haematoma, and wound drainage should be controlled with care . No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
The use of sodium hypochlorite as toilet detergent is related to a red - brown urine discolouration in patients taking mesalamine.it seems that the red - brown discolouration relates to a harmless reaction.polymerisation of mesalamine and/or metabolites could be a theoretical mechanism of this discolouration . The exact working mechanism of mesalamine is not fully understood, but it appears to have anti - inflammatory properties . The drug blocks interleukin-1 and tumour necrosis factor-. Mesalamine also inhibits the cyclo - oxygenase pathway, leading to inhibition of prostaglandin e2 in inflamed intestine . The most common adverse drug reactions (adrs), as described in the summary of product characteristics (smpc), are headache, rash and gastrointestinal symptoms, including diarrhoea, nausea, vomiting and abdominal pain . Mesalamine is also associated with the renal adrs of renal failure, interstitial nephritis and haematuria [1, 3]. In the period from august 2009 to april 2015, the netherlands pharmacovigilance centre lareb received two case reports of red - brown urine discolouration in association with mesalamine . Both cases were reported by patients and the urine discoloured after contact with the toilet bowl . This case concerned a 38-year - old male with a history of crohn s disease . The patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 (pentasa slow - release 500 mg tablet). The latest episode occurred in april 2015, 2 days after the use of mesalamine . The patient had a mild form of crohn s disease that had been stable for 68 years and only used mesalamine during episodes of exacerbations . Due to an exacerbation in september 2014, the daily dose of mesalamine was increased from 2 to 4 g. during this dose escalation, the patient recognised the same red - brown deposit that had occurred during previous use . In january 2015, the mesalamine was tapered over a period of 2 months to 2 g once daily and he remained stable . He observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl . Most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days, despite flushing the toilet . Of note, the deposit in the toilet bowl was more intense during the use of an increased dose of 4 g a day . The patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . In the evening a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . As the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent, he reported it to lareb . After the report had been assessed, the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test, to which he agreed (fig . 1). The urine (40 ml) was mixed with 20 ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . After shaking the mixture, 1 red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine (left, right). Agitation discoloured the urine completely (right) red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine (left, right). Agitation discoloured the urine completely (right) this case concerned a 36-year - old woman with ulcerative colitis, for which she is treated with 2 g of mesalamine (pentasa sachet 2 g prolonged - release granules) twice daily . Since she started taking mesalamine in june 2009, her urine appeared red - brown in the toilet bowl, but only when she had recently cleaned the toilet with sodium hypochlorite detergent . When the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet, the same red - brown discolouration occurred immediately . The patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet . This case concerned a 38-year - old male with a history of crohn s disease . The patient reported separate several episodes of a red - brown deposit in the toilet bowl after the start of mesalamine in 1999 (pentasa slow - release 500 mg tablet). The latest episode occurred in april 2015, 2 days after the use of mesalamine . The patient had a mild form of crohn s disease that had been stable for 68 years and only used mesalamine during episodes of exacerbations . Due to an exacerbation in september 2014, the daily dose of mesalamine was increased from 2 to 4 g. during this dose escalation, the patient recognised the same red - brown deposit that had occurred during previous use . In january 2015, the mesalamine was tapered over a period of 2 months to 2 g once daily and he remained stable . He observed that the urine initially had a normal colour and developed the red - brown colour after contact with the water in the toilet bowl . Most of the time the persistent deposit did not appear directly after contact with the toilet water but developed after a few days, despite flushing the toilet . Of note, the deposit in the toilet bowl was more intense during the use of an increased dose of 4 g a day . The patient wondered whether the use of detergents could have caused this kind of discolouration and therefore he cleaned his toilet in the morning with a chlorine - containing detergent . In the evening a red - brown deposit had developed in the toilet bowl and on the seat and the water was unaffected . As the patient wanted more information on the possibility of the discolouration being the result of a reaction with the sodium hypochlorite detergent, he reported it to lareb . After the report had been assessed, the pharmacovigilance centre lareb asked the patient to collect his urine and to add household bleach as a test, to which he agreed (fig . 1). The urine (40 ml) was mixed with 20 ml of sodium hypochlorite bleach and within a few minutes the discolouration developed . After shaking the mixture, 1 red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine (left, right). Agitation discoloured the urine completely (right) red brown discolouration after adding sodium hypochlorite bleach to normal - appearing urine (left, right). This case concerned a 36-year - old woman with ulcerative colitis, for which she is treated with 2 g of mesalamine (pentasa sachet 2 g prolonged - release granules) twice daily . Since she started taking mesalamine in june 2009, her urine appeared red - brown in the toilet bowl, but only when she had recently cleaned the toilet with sodium hypochlorite detergent . When the patient added a drop of sodium hypochlorite detergent to her normal - coloured urine in the toilet, the same red - brown discolouration occurred immediately . The patient is still taking mesalamine and the phenomenon is still present when cleaning the toilet . Likewise, the disordered haem production in patients with porphyria can result in red - brown discolouration . A red - brown urine discolouration could be perceived as a sign of possible renal impairment or other diseases; however, neither of the described patients are known to have an impaired renal function or physical abnormalities . Furthermore, various drugs are associated with red and/or brown discolouration, e.g. Rifampicin, doxorubicin, metronidazole, methyldopa, levodopa and sulfasalazine . A notable difference from the described mesalamine cases is that the urine of patients taking the above - mentioned drugs, such as sulfasalazine, is already discoloured at micturition . A pubmed search was conducted and it was found that two cases of red - brown urine associated with mesalamine have been reported previously by sacks and davis . Both of the male adolescents in these reports experienced the same red - brown urine discolouration after contact with sodium hypochlorite bleach . A similar experiment was performed to that performed by patient a, and after adding sodium hypochlorite bleach to the collected urine the discolouration developed spontaneously . To our knowledge, there are no other published reports of this association after a chemical reaction with drugs structurally related to mesalamine (sulfasalazine, olsalazine and 4-aminosalicylic acid). Notably, there has been discussion regarding this phenomenon in several patient forums and blogs . Case reports from altmann and mansell do, however, describe urine discolouration in patients using methyldopa or levodopa after contact with lavatory bleach and exposure to light . Normal biotransformation of methyldopa produces a variety of metabolites; presumably, these metabolites have properties that form melanin by spontaneous polymerisation, which results in darkening of the urine . Melanins are polymers of phenolic compounds (e.g. Dopaquinone, benzothiazole) that are formed by an extensive bio pathway . Melanin is an aggregate of smaller component molecules such as pheomelanin and eumelanin; both of these are initially formed by oxidation of tyrosine and then undergo cysteinylation and cyclisation, respectively . The end product is ultimately formed by polymerisation of several different derivatives . Altmann and mansell found that urine samples that were made alkaline in patients treated with methyldopa or levodopa turned black on exposure to sunlight . Mesalamine is acetylated by the enzyme n - acetyltransferase 1 (nat1) in the liver and gut mucosal wall into the metabolite n - acetyl-5-aminosalicylic acid (n - ac-5asa). Evidence from the literature indicates that nat1 is polymorphic, but that these polymorphisms are associated with relatively minor effects on acetylation function . It is unknown whether the patients in our reports have a nat1 polymorphism, but if so we would expect no significant effect on the propensity of discolouring . Furthermore, this acetylation is minimal when mesalamine and n - ac-5asa are both secreted back into the lumen by the drug efflux pump p - glycoprotein, excreted in the faeces, absorbed via the colon into the blood and, lastly, eliminated in the urine . The theory is that n - ac-5asa and/or mesalamine react with the sodium hypochlorite - containing bleach . Sodium hypochlorite usually has a ph of 1113 and could therefore initiate the reaction as described by altmann and mansell . According to the cases reported by altmann and mansell, it is also possible that a similar polymerisation of mesalamine and/or n - ac-5asa could cause the discolouration of the urine . Perhaps due to the alkaline environment, partial deprotonation of n - ac-5asa and mesalamine could also contribute to this reaction . Since the introduction of the national spontaneous reporting system in 2003 to the general public, consumers have now become the largest group of reporters in the netherlands . The quality of consumer reports are generally good and these reports highlight via first - hand information the real - life experience of an adr . Being unconstrained with regards to the probability of causality, consumers may report relevant adrs that healthcare professionals may not recognise initially or consider to be unlikely . Patient b s gastroenterologist recognised the phenomenon, but it is conceivable that not every gastroenterologist will establish a direct relationship between urine discolouration, mesalamine therapy and the use of a specific detergent . The two reports received provide important information about a harmless reaction as this phenomenon probably concerns a lot of other patients using mesalamine . Based on the described reports and the case reports in the literature, we suggest a causal relationship between red brown discolouration of urine after contact with sodium hypochlorite detergent and the use of mesalamine . The naranjo assessment scores for cases a and b were 9 and 6, indicating a certain and probable relationship, respectively . This seems to be a harmless reaction as the patients experienced no physical complaints and in both the lareb cases and those described by sacks and davis, the patients were under the care of a gastroenterologist . In the differential diagnosis it is important to be aware that the use of sodium hypochlorite as a toilet detergent could be the cause of coloured deposits in and on the toilet bowl . The presence of sodium hypochlorite does not exclude the possibility of renal impairment; therefore, it is important to distinguish already discoloured urine from normal - coloured urine that only discoloured after contacting the toilet bowl . Thus, to inform and reassure patients, it might be helpful to mention red - brown discolouration in the patient leaflet for mesalamine . No financial support was received for the conduct of this study or preparation of this manuscript.
Diabetic retinopathy (dr) is a leading cause of visual impairment and blindness in developed countries . The decrease in vision is due to diabetic macular edema (dme) and proliferative diabetic retinopathy (pdr) [1, 2]. The control of the blood glucose level, vitreous surgery, and photocoagulation are the major treatments used to prevent dr from progressing to the pdr stage . Currently, intravitreal injections of antivascular endothelium growth factor (vegf) or steroids have become the primary therapy for dme [35]. However, the use of a single therapeutic agent is not effective in all cases, and additional treatments or different agents are needed . The blood retinal barrier (brb) consists of two anatomical parts; the inner brb is located within the endothelial cells of the retinal capillaries, and the outer brb is located between the retinal pigment epithelial (rpe) cells . An intact brb is required for an efficient and regulated control of fluids in the subretinal space and for the maintenance of healthy rpe and retinal cells . A breakdown of the outer brb results in an increase in the paracellular permeability between the rpe cells, and the leakage can cause retinal edema . The tight junctions (tjs) of the rpe cells are intercellular junctions located at the apical ends of the rpe cells, and they are integral structural components of the brb . Tjs are made up of three tj related proteins, for example, the zonula occludens-1 (zo-1), occludin, and claudin . Recent studies have shown that interactions between inflammatory cells and retinal cells are critical for the development of intraocular neovascularization [1012]. The results of these studies also demonstrated that, in eyes with pdr, an elevation of vegf was significantly correlated with the levels of several cytokines including interleukin- (il-) 6, il-8, and the monocyte chemoattractant protein-1 (mcp-1). Another study showed that the levels of these cytokines were also strongly correlated with each other which suggest that there are common pathways involved in the inflammatory processes . In addition, various cytokines have been shown to be related to the maintenance of the conformation of the tj proteins . Lecithin - bound iodine (lbi, jolethin, daiichi pharmaceutical co., tokyo, japan) has been used clinically to reduce the antigen - induced immune responses in children with bronchial asthma . Lbi acts on the peripheral blood mononuclear cells and downregulates the il-4-induced ige synthesis, which suggests that lbi have anti - inflammatory properties [15, 16]. In patients with eye diseases, lbi has been used for the absorption of retinal or vitreous bleeding, vitreous opacities, and improvement of central serous choroidopathy . The purpose of this study was to determine whether lbi will alter the integrity of the tjs of arpe-19 cells in culture . Lbi (jolethin, daiichi pharmaceutical co., tokyo, japan) was dissolved in distilled water and diluted to the appropriate concentration for each experiment . An earlier study showed that lbi solutions contain 48.250.3% lecithin - iodine, approximately 10% free lecithin, and 40% phosphatidylinositol . Rabbit polyclonal anti - zo-1 antibody (sc-10804) was purchased from santa cruz biotechnology (santa cruz, ca), and alexa 594 anti - rabbit igg was purchased from invitrogen (molecular probs, eugene, or). Rabbit anti - mcp-1 antibody (#2029) was purchased from cell signaling technology (danvers, ma) and rabbit anti - eotaxin (ccl-11) antibody (ab133604) was purchased from abcam (cambridge, ma). Cells from a human retinal pigment epithelium cell line, arpe-19, were grown in dulbecco's modified eagle's medium / ham's f-12 supplemented with 10% fetal bovine serum (fbs, hyclone laboratories, inc ., logan, ut), 50 units / ml penicillin, and 50 g / ml streptomycin in an air-5% co2 atmosphere with constant humidity . Prior to the experiments, cells were placed in serum - free media and pretreated with 50 g / ml of lbi or 250500 pg / ml of antibody (anti - mcp-1 antibody and anti - chemokine (c - c motif) ligand-11 (ccl-11) antibody) for 24 h. to mimic hypoxic conditions, arpe-19 cells were incubated for 4 h with 100 m of cocl2 (sigma, st . Louis, mo), which is a chemical hypoxia - inducing agent . After the incubation, optimum concentration of lbi was defined as 50 g / ml because over 100 g / ml of lbi seems to be toxic (data not shown). For the immunofluorescence studies, cells were grown to a density of 2 10 cells / ml on 12 mm cover slips and fixed in 10% tricarboxylic acid for 10 min at 4c . They were then treated with 0.5% triton x-100 for 15 min . To detect the presence of zo-1, the cultured rpe cells were exposed to rabbit polyclonal anti - zo-1 antibody as the primary antibody and alexa 594 anti - rabbit igg as the secondary antibody . Samples were examined and photographed with a fluorescence microscope (bz-9000; keyence, osaka, japan, and eclipse 50i, nikon, tokyo, japan), and the intensity of fluorescence was quantified with a bz - ii analyzer (keyence). The levels of the bioactive molecules in the conditioned medium were determined by human il-6, il-8, and mcp-1 elisa kits (r&d systems, minneapolis, mn) and ccl-11 elisa kit (biosensis, thebarton, south australia, australia). All experiments were repeated at least three times and values are presented as the means standard deviations . Data were analyzed by two - way nonrepeated analysis of variance (anova) followed by bonferroni post hoc tests for the comparison of the means . We first determined whether the lbi pretreatment affected the changes in the conformation of the tj proteins caused by the hypoxic stress induced by cocl2 . Immunofluorescence microscopy showed that there appeared to be a disruption of tjs with cocl2 treatment (figures 1(a) and 1(b)). When the arpe-19 cells were pretreated with lbi before the addition of cocl2, the disruption of the tight junctions was not detected (figures 1(c) and 1(d)). The signal intensity measurements showed that there was a significant decrease with the cocl2 addition compared to the control . But there was no significant change in the lbi pretreated group (29244.6 2981.2 in controls; 5787.7 4126.4 with cocl2; and 27189.0 11231.1 with cocl2 after lbi pretreatment; p <0.05, nonrepeated anova, n = 5, figure 1(e)). Because disruption of tjs is detected as a decrease of signal intensity, these results indicate that lbi pretreatment can protect the tjs of the outer brb from hypoxic stress . Because lbi pretreatment protected the conformation of the tj proteins from hypoxic stress, we hypothesized that lbi will block different inflammatory molecules that are secreted from cells during hypoxia . The results of the elisa measurements of the conditioned culture media (n = 5) indicated that il-8 was not significantly changed after the addition of cocl2 or after pretreatment with lbi (control, 6.9 0.1; with cocl2, 6.8 0.1; and with cocl2 after lbi pretreatment, 6.9 0.1 pg / ml; figure 2(a)). Although the level of il-6 was increased after addition of cocl2 which was decreased by lbi pretreatment, these changes were not significant (control, 10.4 1.6; cocl2 addition, 15.0 6.7; and cocl2 addition after lbi pretreatment, 13.3 7.6 pg / ml; figure 2(b)). The level of mcp-1 increased significantly after the addition of cocl2, and it was significantly decreased after the addition of cocl2 after lbi pretreatment (control, 279.7 68.3; with cocl2, 340.8 43.3; and with cocl2 after lbi pretreatment, 182.6 23.8 pg / ml, p <0.05; nonrepeated anova; figure 2(c)). A similar tendency was observed for ccl-11 (control, 12.5 6.1; with cocl2, 15.2 12.9; and with cocl2 after lbi pretreatment, 5.46 1.9 pg / ml; p <0.05; nonrepeated anova; figure 2(d)). These results indicated that there is an increase of the mcp-1 and ccl-11 secretion under hypoxic stress, and all are suppressed by lbi pretreatment . To confirm that lbi effectiveness is related to suppression of mcp-1 or ccl-11, cells were pretreated with anti - mcp-1 or anti - ccl-11 antibody for 24 hrs . The tj disruption from hypoxic stress was reduced with pretreatment (figures 3(e) and 3(f)). These results indicated that both mcp-1 and ccl-11 were important to protect tjs from hypoxic stress and support the effectiveness of lbi . Vegf is a major antigen factor and is biogenic permeability factor which can increase the vascular permeability endothelial cell - cell junctions . Finally, to confirm that lbi pretreatment can protect tjs from direct vegf induced stress, after lbi pretreatment but after pretreatment of lbi, this disruption was decreased which indicates that lbi can also protect tjs from vegf induced damage . But there still remain patients who do not respond to the anti - vegf therapy and who had a reduction of their vision even after therapy . It must also be noted that the cost of anti - vegf therapy is quite high and continuous injections to control the dme can become an economic issue . Combination therapies have also been proposed that can enhance the efficacy and may minimize the cost of anti - vegf therapies . For example, photocoagulation can extend the interval of anti - vegf injections resulting in a reduction in the number of injections [21, 22]. If new drugs or treatments can be approved, this will make it easier and less expensive to use combination therapies . Moreover, adjunctive therapies that include oral kallidinogenase could also be used for combined therapy [23, 24]. Our results showed that the hypoxic stress induced by exposure of arpe-19 cells to cocl2 caused a disruption of the tight junctions and that lbi pretreatment can protect the tjs from the hypoxic stress . Hypoxic stress also enhanced the secretion of mcp-1 and ccl-11, and this enhancement can be suppressed by lbi pretreatment . Hypoxia has been reported to produce alterations in the tight junction proteins that are correlated with the vascular permeability increases . The integrity of the brb is dependent on tj - associated proteins such as occludin, claudin, and zo-1 . The function of zo-1 is to link the transmembrane protein, occludin, to the actin cytoskeleton . Under hypoxic conditions, the phosphorylation of zo-1 is enhanced which is related to the damage of the tight junctions . Mcp-1 is associated with a breakdown of the brb [3032], and it also induces a disruption of the tjs by the caveolae that are dedicated to the internalization of the tj proteins, such as zo-1 . Because mcp-1 is also increased in the vitreous of patients with dr, it probably plays an important role in the progression of dr and dme . In the photocoagulation - induced mouse model of retinal neovascularization, it was reported that an increase in mcp-1 contributes to the postischemic inflammation and dr progression [34, 35]. Mcp-1 has been recently postulated to have a direct effect on angiogenesis even though there is no link with macrophage recruitment . Ccl-11 also promotes the recruitment of vascular endothelial cells and relates the conformation of the tight junctions . Interestingly, there is also an increase of ccl-11 in the proliferative membranes obtained from eyes with pdr . So these cytokines relate to zo-1 and maintain tight junctions on diabetic retina . In the photocoagulation - induced retinal neovascularization or light - exposed mouse models, the levels of not only mcp-1 but also ccl-11 are increased in the rpe [3941]. These increases were related to the remodeling of f - actin that occurred after light damage and caused proinflammatory changes in the rpe cells . These findings support our results that showed that the hypoxia - induced stress affected the expression of cytokines especially mcp-1 and ccl-11 . The suppression of these cytokines by lbi leads to the protection of these cells from disruption of the tight junctions . Steroids are also commonly used for dme therapy because their anti - inflammatory property helps to protect the tissues from neovascularization or vascular permeability increases . Similarly, the effect of lbi on the suppression of the postischemic inflammation may be an additional contributor when used as either a primary or adjunctive dme therapy . Mainly in japan and china, lbi has been approved for the treatment of various retinal diseases including central serous chorioretinopathy, vitreous hemorrhage, or vitreous opacity for a long time . In addition, both lecithin and iodine are commonly used in food materials indicating the safety of lbi . Thus, the possibility exists that lbi can be even used as a safe adjunctive therapy with an anti - vegf drug to treat dme . Further investigations into the use of lbi in conjunction with anti - vegf therapy need to be undertaken to determine the effectiveness of lbi in preventing the development or progression of dme . Our findings showed that lbi pretreatment reduced the levels of mcp-1 and ccl-11 and blockage of them also results in protection of tjs . However, no significant changes were detected in the levels of il-6 and il-8, which are the major cytokines related to neovascularization . In ischemic retinas, il-8 causes increases in the vegf and mcp-1 levels but it is dependent on tnf- . Because il-8 has been shown to be secreted from either monocytes or macrophages and because our experiments were limited to arpe-19 cells in culture, there is the possibility that the results may not hold under in situ conditions . Though we observed that vascular permeability improved after oral lbi administration for diabetic mice model (data not shown), it is not clear whether this is the result of hypoxia improvement the same as we show here using arpe-19 . In addition, lbi pretreatment can also protect tjs from direct vegf induced damage which results in tj disruption . Further investigations will need to be undertaken to definitively explain these phenomena . In conclusion, our data demonstrate the therapeutic potential of lbi for protecting the integrity of the tight junctions from hypoxia - induced stress . These data suggest a new aspect on the clinical use of lbi in the treatment of dme.
A 21-year - old caucasian woman presented to an outside facility with altered mental status after being found lethargic by family . On arrival to the emergency department, plasma glucose was 20 mg / dl . The patient was hospitalized and started on dextrose infusion, including 10% dextrose in water but remained hypoglycemic with capillary glucose ranging between 50 and 60 mg / dl . She was transferred to our facility on suspicion of an insulinoma as the patient was unable to recall her prior diagnosis . The patient reported repeated episodes of seizures, syncope, dizziness, headaches, palpitations, and sweating around age 12; however, symptoms were never formally investigated as she did not seek expert care . These symptoms were also reported to have been present since birth but had been intermittent and of varying severity . She remained seizure - free and without syncopal episodes until 17 when she experienced another syncopal episode . At that time, she underwent extensive inpatient hypoglycemia evaluation with the following results: blood glucose of 47 mg / dl after 2 h of fasting, proinsulin levels varying from 10.4 to 84.1 pmol / l (normal range 010 pmol / l), c - peptide level 3.1 ng / ml (normal range 1.14.4 ng / ml), negative insulin antibodies and sulfonylurea screen . Magnetic resonance imaging of the abdomen showed no insulinoma, but genetic studies revealed val452 leu activating mutation of the gck gene . She was successfully treated with diazoxide and discharged home on oral diazoxide 250 mg daily, which she discontinued due to side effects of hirsutism and fluid retention . On current admission, her blood glucose level remained low in spite of a continuous infusion of 5% dextrose necessitating transfer to an intensive care unit . As review of her medication list did not show any implicating drugs, no additional work - up was pursued . She was then placed on octreotide 200 g subcutaneously twice daily and diazoxide suspension 100 mg three times a day on consultation with the pediatric endocrinologist . The patient's hypoglycemia improved with capillary glucose of 55110 mg / dl by the time of discharge . Importance of compliance with the treatment and follow - up with an endocrinologist was emphasized . When encountered in the inpatient setting, it is easily recognized as patients already have risk factors such as diabetes mellitus (dm) on treatment, sepsis, and end - organ failure . However, for patients presenting emergently with hypoglycemia alone, appropriate investigations are warranted to treat and prevent recurrence . After establishing a diagnosis of hypoglycemia by whipple's triad (low plasma glucose, hypoglycemic symptoms, and resolution of adrenergic / neuroglycopenic symptoms with correction of the blood sugar), interim treatment and search for risk / causative factors should ensue (table 1). Ideally, treatment is only given after obtaining a plasma sample to ensure accuracy of diagnosis . Medical history of ethanol use, surreptitious insulin ingestion, medication use and interaction, autoimmune disease, gastric bypass (4), and subtle risk factors such as undiagnosed psychiatric disorder and polypharmacy should be excluded (5). In seemingly well patients, the timing of hypoglycemic spells (fasting / postabsorptive or postprandial / reactive) is required to determine diagnostic studies . A mixed meal tolerance test under supervision is done for postprandial hypoglycemia while a 72-h fast should be done for fasting hypoglycemia . Hepatic or renal disease, endocrine disorders of growth hormone, cortisol, adrenal insufficiency, and hypopituitarism should be excluded . Plasma insulin, proinsulin, c - peptide, and beta - hydroxybutyrate levels should be assessed to exclude surreptitious use of insulin or insulin receptor antibodies . Intravenous glucagon can be given to distinguish disorders of gluconeogenesis from glycogenolysis (5). A 48-h supervised fast should be done to exclude insulinoma, with plasma insulin and proinsulin levels . Pro - igf ii to igf ii ratio can be measured to look for non - beta - cell tumors (5). Negative findings suggest non - insulinoma pancreatogenous hypoglycemia syndrome (niphs), which encompasses chi (4, 6). A selective pancreatic arterial calcium stimulation test (spaci) done at this time can detect hyperfunctioning of b - cells and confirm the diagnosis of niphs . Causes of hypoglycemia in adults chi is uncommon in the pediatric population with an estimated incidence of 1 in 40,000 to 50,000 live births annually in europe (7). Gck mutations account for 7% of all causes of chi (8, 9); gck facilitates phosphorylation of glucose to glucose-6-phosphate and is the main glucose sensor of the pancreatic beta cell and hepatocytes - controlling glucose - stimulated insulin secretion and glycogenesis (10). Activating mutations of the gck gene are typically inherited in an autosomal dominant pattern, showing variable phenotypic penetrance and expressivity ranging from asymptomatic to marked hypoglycemia (11, 12). Inactivating forms of the mutation cause varying severity of persistent hyperglycemia depending on zygosity (13). De novo gck mutations are very rare and exhibit marked variation in phenotypic expression (14). Over 600 heterozygous inactivating mutations have been reported, causing mild persistent hyperglycemia known as maturity onset diabetes of the young (mody) as opposed to only 13 known activating mutations of the gene (15, 16). Activating mutations are usually clustered around the allosteric activator site of the enzyme and led to hyperinsulinemic hypoglycemia by lowering the glucose set point and threshold for glucose stimulated insulin release . The sheer numbers of existing mutations of the gck gene show it to be a highly mutable gene with varied disease outcomes depending on the type of activation . It is key to blood glucose homeostasis and has been identified as a potential target for anti - diabetic therapy . Trials manipulating gck regulatory protein an allosteric switch for gck, which indirectly restores gck activity, are currently underway to evaluate its effect on blood glucose in patients with type 2 dm (17, 18). Treatment of niphs involves lifelong dietary modification consisting of frequent, low carbohydrate meals (5), which prevent surges in plasma glucose level, insulin level, and the consequent hypoglycemic spells . Medications such as diazoxide, octreotide, glucagon, verapamil, and somatostatin, whose actions all involve inhibition of calcium channels, have been employed with varying degrees of success (7). Diazoxide binds to and activates the sulfonylurea receptor 1 subunit of the potassium adenosine triphosphate [k(atp)] channel, causing calcium channel closure . Response to diazoxide therefore varies with genetic mutations [ineffective in mutations of the k(atp) channel genes] (8) and with pattern of inheritance (7). Octreotide, a somatostatin analogue (secretin and insulin inhibitor), has been used in diazoxide unresponsive chi with improved blood glucose in these patients; however, side effects such as gastrointestinal symptoms, dilated gallbladder, necrotizing enterocolitis, and qt prolongation sometimes limit its use (7). Long - term use of glucagon is effective in diazoxide - unresponsive cases or severe / diffuse chi but gastrointestinal side effects and crystallization in infusion tubing have been reported (19). Glucagon - like protein 1 receptor agonists such as exendin-4 are newer drugs that improve hypoglycemia in diazoxide - unresponsive patients by inhibiting insulin release (7). Partial pancreatectomy is indicated for medical treatment failure [suggesting a k(atp) defect] and is only curative for focal lesions while extensive resection is reserved for diffuse chi to limit pancreatic burden (7, 8, 11, 20). Affected patients can present in adulthood; de novo mutations should be considered given the following:- positive family history of unremitting hypoglycemia- lifelong symptoms of low plasma glucose, even after eating- increased bmi- negative sulfonylurea and autoimmune work up- positive glycemic response to octreotide - positive family history of unremitting hypoglycemia - lifelong symptoms of low plasma glucose, even after eating - negative sulfonylurea and autoimmune work up - positive glycemic response to octreotide to our knowledge, only three families with affected adults have been reported . Our case is unique because it expounds on genetic causes of hypoglycemia in adults . In addition, this case highlights the pediatric - to - adulthood transition for rare diseases and the key role that coordination and transition of care plays in timely management . After establishing a diagnosis of hypoglycemia by whipple's triad (low plasma glucose, hypoglycemic symptoms, and resolution of adrenergic / neuroglycopenic symptoms with correction of the blood sugar), interim treatment and search for risk / causative factors should ensue (table 1). Ideally, treatment is only given after obtaining a plasma sample to ensure accuracy of diagnosis . Medical history of ethanol use, surreptitious insulin ingestion, medication use and interaction, autoimmune disease, gastric bypass (4), and subtle risk factors such as undiagnosed psychiatric disorder and polypharmacy should be excluded (5). In seemingly well patients, the timing of hypoglycemic spells (fasting / postabsorptive or postprandial / reactive) is required to determine diagnostic studies . A mixed meal tolerance test under supervision is done for postprandial hypoglycemia while a 72-h fast should be done for fasting hypoglycemia . Hepatic or renal disease, endocrine disorders of growth hormone, cortisol, adrenal insufficiency, and hypopituitarism should be excluded . Plasma insulin, proinsulin, c - peptide, and beta - hydroxybutyrate levels should be assessed to exclude surreptitious use of insulin or insulin receptor antibodies . Intravenous glucagon can be given to distinguish disorders of gluconeogenesis from glycogenolysis (5). A 48-h supervised fast should be done to exclude insulinoma, with plasma insulin and proinsulin levels . Pro - igf ii to igf ii ratio can be measured to look for non - beta - cell tumors (5). Negative findings suggest non - insulinoma pancreatogenous hypoglycemia syndrome (niphs), which encompasses chi (4, 6). A selective pancreatic arterial calcium stimulation test (spaci) done at this time can detect hyperfunctioning of b - cells and confirm the diagnosis of niphs . Chi is uncommon in the pediatric population with an estimated incidence of 1 in 40,000 to 50,000 live births annually in europe (7). Gck mutations account for 7% of all causes of chi (8, 9); gck facilitates phosphorylation of glucose to glucose-6-phosphate and is the main glucose sensor of the pancreatic beta cell and hepatocytes - controlling glucose - stimulated insulin secretion and glycogenesis (10). Activating mutations of the gck gene are typically inherited in an autosomal dominant pattern, showing variable phenotypic penetrance and expressivity ranging from asymptomatic to marked hypoglycemia (11, 12). Inactivating forms of the mutation cause varying severity of persistent hyperglycemia depending on zygosity (13). De novo gck mutations are very rare and exhibit marked variation in phenotypic expression (14). Over 600 heterozygous inactivating mutations have been reported, causing mild persistent hyperglycemia known as maturity onset diabetes of the young (mody) as opposed to only 13 known activating mutations of the gene (15, 16). Activating mutations are usually clustered around the allosteric activator site of the enzyme and led to hyperinsulinemic hypoglycemia by lowering the glucose set point and threshold for glucose stimulated insulin release . The sheer numbers of existing mutations of the gck gene show it to be a highly mutable gene with varied disease outcomes depending on the type of activation . It is key to blood glucose homeostasis and has been identified as a potential target for anti - diabetic therapy . Trials manipulating gck regulatory protein an allosteric switch for gck, which indirectly restores gck activity, are currently underway to evaluate its effect on blood glucose in patients with type 2 dm (17, 18). Treatment of niphs involves lifelong dietary modification consisting of frequent, low carbohydrate meals (5), which prevent surges in plasma glucose level, insulin level, and the consequent hypoglycemic spells . Medications such as diazoxide, octreotide, glucagon, verapamil, and somatostatin, whose actions all involve inhibition of calcium channels, have been employed with varying degrees of success (7). Diazoxide binds to and activates the sulfonylurea receptor 1 subunit of the potassium adenosine triphosphate [k(atp)] channel, causing calcium channel closure . Response to diazoxide therefore varies with genetic mutations [ineffective in mutations of the k(atp) channel genes] (8) and with pattern of inheritance (7). Octreotide, a somatostatin analogue (secretin and insulin inhibitor), has been used in diazoxide unresponsive chi with improved blood glucose in these patients; however, side effects such as gastrointestinal symptoms, dilated gallbladder, necrotizing enterocolitis, and qt prolongation sometimes limit its use (7). Long - term use of glucagon is effective in diazoxide - unresponsive cases or severe / diffuse chi but gastrointestinal side effects and crystallization in infusion tubing have been reported (19). Glucagon - like protein 1 receptor agonists such as exendin-4 are newer drugs that improve hypoglycemia in diazoxide - unresponsive patients by inhibiting insulin release (7). Partial pancreatectomy is indicated for medical treatment failure [suggesting a k(atp) defect] and is only curative for focal lesions while extensive resection is reserved for diffuse chi to limit pancreatic burden (7, 8, 11, 20). Affected patients can present in adulthood; de novo mutations should be considered given the following:- positive family history of unremitting hypoglycemia- lifelong symptoms of low plasma glucose, even after eating- increased bmi- negative sulfonylurea and autoimmune work up- positive glycemic response to octreotide - positive family history of unremitting hypoglycemia - lifelong symptoms of low plasma glucose, even after eating - negative sulfonylurea and autoimmune work up - positive glycemic response to octreotide to our knowledge, only three families with affected adults have been reported . In addition, this case highlights the pediatric - to - adulthood transition for rare diseases and the key role that coordination and transition of care plays in timely management . Despite the congenital nature of gck mutations, some patients can go undiagnosed till early adulthood . Future research into a cost - effective and prompt scheme of patient information dissemination is warranted for uncommon diseases . The authors have not received any funding or benefits from industry or elsewhere to conduct this study.
Obstructive sleep apnea (osa) is a common public health problem worldwide and it has been shown to be associated with an increased incidence of cardiovascular complications such as death, stroke, or myocardial infarction . The 2014 american heart association / american stroke association guideline recommends that patients with stroke / transient ischemic disorder (tia) showed be worked up for osa . In this study, patients who had osa and stroke had an improved outcome with continuous positive airway pressure . Extrapolating data from western population and implementing in the asian populations may not be possible as there are differences in genetic and anatomical factors . For example, the cutoff point for obesity based on body mass index (bmi) criterion for western population is 30 kg / m meanwhile the bmi for asian population is 25 kg / m . It is also evident that there are differences in anatomical factors between asian and caucasian population such as retrognathia and oropharyngeal width which are main risk factors for osa . Alternatively, the stop - bang questionnaire developed by chung et al . Has been widely used as a sensitive screening tool for osa . The stop - bang acronym stands for: snoring history, tired during the day, observed stop breathing while sleep, high blood pressure, bmi more than 35 kg / m, age more than 50 years, neck circumference more than 40 cm and male gender . The authors recommended that if a patient had 3 or more criteria mentioned above, it is strongly suggestive for osa . However, the cut - off values of this questionnaire are based on the data from caucasians, therefore we hypothesized that to use it in asian population, some adjustments are needed . We compared the clinical features in the stop - bang questionnaire between osa - induced hypertension patients and healthy control subjects . The study was conducted in the faculty of medicine, khon kaen university (thailand). The study protocol was approved by the ethics committee for human research, khon kaen university . Osa induced - hypertension patients were diagnosed by: i) met the criteria of hypertension; ii) having average apnea - hypopnea index (ahi) by polysomnography more than or equal 5 times / hours and iii) no evidence of other secondary hypertension . The control subjects filled up the modified berlin questionnaire and epworth sleepiness scale less than 10 and defined as low risk for osa with low scores . Demographic data and clinical features of both osa - induced hypertension patients and healthy subjects were recorded and compared . The clinical features included age, gender, bmi, neck circumference, mallampati classification, torus palatinus, and torus mandibularis . The mallampati classification was defined by asking the subjects to protrude their tongue as much as possible and classified as class 1 to 4 . The sample size of the study population was calculated by using the proportion comparison between osa and healthy subjects using winpepi program . The proportion of osa was 0.35 and the healthy control was 0.07 with deviation of 5%, power of 90%, and missing data of 10% . The sample size was calculated to be 102 subjects (osa 34 subjects and healthy control 68 subjects). Due to the incompleteness of the database of medical students, 120 healthy controls were selected by systematic sampling from the database (total of 1174 medical students). Baseline and clinical characteristics of the participants in both groups were compared using descriptive statistics . Univariate logistic regression analyses were applied to calculate the crude odds ratios of individual variables for having osa . All clinically significant variables or p<0.20 by the univariate analyses were included in subsequent multivariate logistic regression analyses . Analytical results were presented as crude odds ratios (or), adjusted or, and 95% confidence intervals (ci). Significant risk factors were calculated for the best cut - off points by the receiver operator characteristic curve (roc curve). Data analyses were performed with stata software (college station, tx, usa) and spss software (chicago, il, usa). In this study, 42 osa - induced hypertension patients and 82 control subjects who had a complete set of clinical data were included . All clinical features of both groups the osa - induced hypertension patients were significantly older (59.5 vs 21.0 years), with higher proportion of males (64.3 vs 59.8%), more obese (78.6 vs 6.1%), higher incidence of mallampati class 3 or more (54.8 vs 24.4%), larger neck circumference (41.3 vs 32.0 cm), higher incidence of torus palatinus (26.6 vs 0%) and of torus mandibularis (9.5 vs 0%). By multiple logistic regression analysis, only two factors bmi and neck circumference, the adjusted odds ratios for both factors were 1.49 (95% ci: 1.06, 2.09) and 1.67 (95% ci: 1.11, 2.51), respectively . By the roc curve analyses, the best cut - off points for the bmi and the neck circumference were 24.5 kg / m (figure 1a) and 36 cm (figure 1b). The sensitivity and the specificity for bmi cut - off point were 97.2% and 91.4%, whereas those for the neck circumference were 94.7% and 82.9% . The present results showed that the stop - bang questionnaire needed to adjust the cutoff values of the bmi and neck circumference suitable for thai population . Subjects should referred for polysomnography if positive at least 3 questions: s, snoring history; t, tired during the day; o, observed stop breathing while sleep; h, high blood pressure; b, bmi more than 25 kg / m; a, age more than 50 years; n, neck circumference more than 36 cm; g, male gender (modified from chung et al . ). For the criteria of obesity for asians, the cut - off of the bmi should lower from 35 kg / m to 24.5 or round up to 25 kg / m . The cut - off of the neck circumference also should be lowered from 40 cm to 36 cm for asians . Proposed the cutoff points of 24.1 kg / m for the bmi and 35.5 cm for the neck circumference for japanese population . Using stop - bang questionnaire is very suitable for thailand and other developing countries due to limited availability of polysomnography, the standard diagnostic tool for osa . This low cost tool can select appropriate patients for referral to sleep center for further polysomnography . Healthy control subjects were not performed polysomnography to exclude osa . However, both the berlin questionnaire and the epworth sleepiness scale were used to exclude osa, which has a sensitivity and specificity of 0.86, 0.95 and 0.49, 0.80, respectively . Even though the age is a significant factor by univariate logistic regression, it was not included in the subsequent multivariate regression . This is because the difference of age group composition between the osa and the healthy control subjects . Further studies, therefore, are needed to confirm the results of this study and to identify the appropriate cut - off point using the age- and sex - matched controls . Also, to increase their sensitivity and specificity, osa patients with other complications than hypertension should be included and compared . Whether simple measurement of the neck circumference and the bmi calculation are sufficient to identify osa patients in routine practice by primary health care personnel including physicians, nurses and volunteers should be examined in further study . Osa has been proved to be a contributing factor for major cardiovascular diseases; stroke, hypertension, sudden death, and also coronary artery disease . Treatment of osa may reduce large economic burden from prevention of the morbidity and mortality from stroke and acute coronary syndrome . In conclusion, the appropriate cut - off points for the bmi and the neck circumference for stop - bang questionnaire were 25 kg / m and 36 cm for asian people . All hypertensive patients should have their bmi and neck circumference measured to detect the risk factors for osa . Public health campaign for osa screening is also needed to reduce morbidity and mortality from osa complications.
Triple - negative breast cancer (tnbc) is characterized by absence of expression of the estrogen (er) and progesterone receptors (pr) and no human epidermal growth factor receptor 2 (her2)/neu gene amplification [1, 2]. Unlike hormone receptor - positive and her2-overexpressing breast cancers, tnbc is unresponsive to endocrine therapy and her2-targeted agents, respectively, thereby limiting available systemic treatment options to conventional cytotoxic chemotherapy [1, 2]. Patients with tnbc are more likely to have visceral or brain metastases and short relapse - free survival compared with those with other breast cancer subtypes [3, 4, 5, 6]. While chemotherapies have been effective for treating early - stage disease, with pathologic complete response (cr) rates exceeding those of hormone receptor - positive subtypes, patients with metastatic disease have shorter disease - free survival progressing rapidly through several lines of chemotherapy . Thus, response to chemotherapy has not translated to improvements in progression - free (pfs) or overall survival (os) in the metastatic setting, and the overall prognosis for tnbc remains poor [3, 7]. The median os of patients with metastatic disease is relatively short, with reports between 9 and 13.3 months [3, 4, 5, 6]. An unmet clinical need exists for more effective therapies for tnbc, particularly in the metastatic setting . Taxane - based chemotherapy has demonstrated activity in patients with tnbc [1, 2]. In the phase iii eastern cooperative oncology group 2100 trial in which 91% of the patients had her2-negative disease, 51% of the patients were pr negative and 37% were er negative, the addition of bevacizumab to solvent - based paclitaxel demonstrated significantly improved median pfs compared with solvent - based paclitaxel alone (11.8 vs. 5.9 months, respectively); however, this did not translate into a significant improvement in median os for the combination arm . Nab - paclitaxel, a novel formulation albumin - bound paclitaxel with a mean particle size of 130 nm, was initially designed with the intent to improve the therapeutic index of solvent - based taxanes [9, 10]. Currently, nab - paclitaxel is indicated for the treatment of breast cancer after failure of combination chemotherapy for metastatic disease or relapse within 6 months of adjuvant chemotherapy . A phase ii study examined the combination of nab - paclitaxel, bevacizumab, and gemcitabine as first - line therapy for patients with her2-negative metastatic breast cancer . In this small cohort of patients with stage iv tnbc, the clinical benefit rate as defined by cr, partial response, and stabilization of disease was 85% . Additionally, in this study, there were no significant differences in pfs or os between triple - negative and hormone receptor - positive patients . This work reports on a patient with metastatic tnbc enrolled in this phase ii study of nab - paclitaxel, bevacizumab, and gemcitabine who experienced a long - term complete remission lasting just under 2 years and a pfs from diagnosis of nearly 3 years . In june 2007, a 52-year - old woman was diagnosed with invasive ductal carcinoma . The patient initially presented with chest pain, and a subsequent breast examination revealed a mass in the left breast and a palpable sternal mass . A lumpectomy and axillary lymph node dissection were performed at an outside institution and results revealed a moderately differentiated, infiltrating ductal carcinoma, high nuclear grade, measuring 4 cm in maximal diameter, with metastases to 2 of 24 axillary nodes . Further staging also revealed sternal and mediastinal metastases, and a computed tomography (ct) scan revealed bilateral pulmonary nodules (fig . Tumor samples were negative for er and pr, and positive (+ +) for her2 by immunohistochemistry . However, fluorescent in situ hybridization results were negative for her2 gene amplification, and the patient was diagnosed with triple - negative primary metastatic / stage iv breast cancer . The patient was initially seen at the university of miami sylvester comprehensive cancer center and enrolled in the phase ii study of nab - paclitaxel, bevacizumab, and gemcitabine for her2-negative metastatic breast cancer . She was treated on study with gemcitabine 1,500 mg / m by 30-min infusion, followed by nab - paclitaxel 150 mg / m by 30-min infusion, then bevacizumab 10 mg / kg by 30-min infusion once every 2 weeks in a 4-week cycle . The patient experienced pain relief after 5 weeks on the study (2 infusions). Serial ct scans, performed every 2 months, demonstrated progressive reduction in the size of all target lesions (fig . She showed a complete clearance of lesions in all imaging studies, which was confirmed on at least 2 separate occasions approximately 2 months apart according to response evaluation criteria in solid tumors . One dose reduction, as required by the study protocol, occurred in november 2008 on cycle 12, due to treatment - related fatigue . The dose of gemcitabine was reduced to 1,250 mg / m, nab - paclitaxel to 125 mg / m, with no modification of the bevacizumab dose, and treatment was continued once every 2 weeks on a 4 week cycle . Throughout her treatment, she experienced no major side effects and continued to work uninterrupted except for visits for chemotherapy and evaluation, and her treatment - related fatigue was successfully managed with the dose reduction . The patient maintained her response and continued to receive treatment for just over 2 years . After 12 months on study, follow - up was performed every 3 months . In may 2010, the patient had documented disease progression by ct scan . Metastases were found in the lung, bone, and lymph nodes . As specified in the study protocol, the patient discontinued study treatment due to disease progression . She received her last dose of nab - paclitaxel, bevacizumab, and gemcitabine on may 28, 2010 . Three weeks after progressing, the patient received capecitabine at a dose of 2,000 mg / m orally twice a day for 2 weeks in 3-week cycles and showed minor response . However, therapy was stopped due to unacceptable toxicity, including diarrhea, dehydration, and severe palmar plantar erythrodysesthesia . After 2 months, she initiated therapy with ixabepilone, with a minor radiographic response . From august 2010 to december 2010, she received 8 cycles of treatment with ixabepilone (30 mg / m) administered intravenously every 3 weeks . In december 2010, the patient requested a chemotherapy holiday . Beginning in november 2011, the patient was treated with eribulin 1.1 mg / m intravenously on days 1 and 8 of a 3-week cycle . Treatment was discontinued in march 2012 due to rapid disease progression with development of central nervous system metastases . She was transferred to hospice care where she died a month later . Throughout the course of her disease, she received zoledronic acid at 4 mg intravenously every 4 weeks beginning in november 2007 when she began the phase ii study . In may 2010, she continued to receive denosumab throughout her chemotherapy holiday from december 2010 to november 2011 until discontinuing therapy in march 2012 after being transitioned to hospice care . Patients with metastatic tnbc have a very poor prognosis . In a large analysis of 255 patients diagnosed with tnbc at the md anderson cancer center between 1985 and 2004, the median os of patients with recurrent disease was only 1.0 year (95% ci, 0.81.2 years) for tnbc patients, significantly shorter than the median os of 2.3 years (95% ci, 1.92.7 years) for those with other breast cancer subtypes (hr, 2.5; 95% ci, 1.83.5; p <0.001). Other studies have reported median os times for patients with recurrent or metastatic tnbc between 9 and13.3 months [4, 5, 6]. While median os has not been specifically assessed in a large patient population initially presenting with metastatic tnbc, it is likely to be similarly poor . Additionally, patients with metastatic tnbc exhibit progressively shorter response durations to successive lines of therapy . In a retrospective analysis of 111 patients with tnbc (14% of whom presented with metastatic disease at diagnosis), the median duration of response was only 12 weeks (range, 073.1 weeks) to first - line therapy, 9 weeks (range, 0120.9 weeks) to second - line chemotherapy, and 4 weeks (range, 059 weeks) to third - line chemotherapy . There is an unmet clinical need for effective therapies that prolong survival for patients with metastatic tnbc . In the absence of curative therapies for metastatic disease, the goal of therapy is primarily palliative in nature, leveraging systemic treatment with minimal toxicity to both prolong os and at the same time enhance quality of life by delaying the onset of cancer - related symptoms . Numerous studies have shown that the combination of gemcitabine and a taxane is an effective regimen that is well tolerated with good response rates . The case described here is of a patient with metastatic tnbc treated with first - line combination chemotherapy consisting of nab - paclitaxel, bevacizumab, and gemcitabine every other week . After only 5 weeks and 2 infusions, the patient experienced significant symptom relief from her pain from bone metastases, including the sternum . Following 7 months of treatment, she achieved a complete remission that, with continuous therapy, persisted for nearly 2 years for an overall progression - free period from initial diagnosis of 2 years and 7 months . Following progression, subsequent lines of therapy were able to produce only limited efficacy in disease control . Following her initial progression - free interval of almost 2 years in cr with nab - paclitaxel, bevacizumab, and gemcitabine therapy, subsequent treatment with capecitabine then ixabepilone resulted in short progression - free intervals of only 2 months and 4 months, respectively . While ixabepilone did provide a pfs benefit, it was very modest . From her initial diagnosis in june 2007, the patient survived nearly 5 years, which is greatly improved compared to reported median survival of 913.3 months for stage 4 tnbc [3, 4, 5, 6]. With the aim to enhance quality of life, systemic treatments with minimal toxicity while the patient did experience treatment - related fatigue while on nab - paclitaxel, bevacizumab, and gemcitabine combination therapy, this was easily managed with a dose reduction . In addition to a lengthy complete remission, the patient was able to work uninterrupted throughout her treatment period, a factor that may significantly elevate quality of life for many patients . This patient initially received treatment as a participant in a phase ii study of nab - paclitaxel, bevacizumab, and gemcitabine as first - line therapy for her2-negative metastatic breast cancer . A large proportion (13 of 29) of the patients enrolled in this study had triple - negative disease . Overall, this subset of patients demonstrated promising outcomes, with 38% achieving a complete radiographic response and 85% experiencing clinical benefit . Most notably, survival outcomes were almost identical between tnbc patients and those with hormone receptor - positive disease . Additionally, severe adverse events appeared to be less frequent in the overall study population relative to results in other published chemotherapy studies in patients with metastatic breast cancer . This case study demonstrates the potential for triplet chemotherapy with nab - paclitaxel, bevacizumab, and gemcitabine to elicit prolonged responses, potentially prolong os, and improve quality of life for patients with metastatic tnbc . This combination shows promise for the treatment of tnbc and should be the subject of further investigation in larger randomized trials.
In humans, type i interferons are encoded by ~16 genes and individual genes encoding,, and . Their gene products are highly structurally related and all bind to a single receptor (ifnar) consisting of heterodimeric r1 and r2 subunits . In contrast, ifn- consists of 3 genes, ifn-1, 2 and 3 (formally, il-29, il-28a and il-28b) that are more related to the il-10 family than to interferon . Ifn- binds a receptor (ifnlr) composed of a unique ifnlr1 and a shared il-10r2 subunit . While stats 1 and 3 are promiscuously activated by a variety of other cytokines and growth factor receptors, stat2 is selectively recruited to the ifnar and ifnlr . Stat2 recruitment and activation by both receptors involves tyrosine phosphorylation of stat2 by jak kinases and subsequent oligomerization with stat1 and irf-9 . Although a fraction of stat1:stat2 heterodimers can translocate to the nucleus and bind atypical gamma activated sequence (gas)-like elements, the canonical interferon - stimulated gene factor-3 (isgf3) complex of stat2:stat1:irf-9 regulates a large fraction of the interferon pathway genes . Thus, stat2 is central to the induction of these genes in response to both ifn-/ and ifn-. In some cases, type ii interferon, ifn-, can mobilize the antiviral response in a stat2-dependent manner . Although there is no evidence for direct recruitment of stat2 to the ifn-r, ifn- signaling can drive the formation of an isgf3-like complex containing stat2, inhibit viral replication and induce expression of ifn-/ target genes, perhaps through the pairing of phosphorylated stat1 with latent stat2 . Regardless of its mode and route of activation, stat2 acts as the gatekeeper to the antiviral response, which is underscored by the severe susceptibility of stat2 knockout mice to viruses ranging from influenza to dengue . Type i and iii interferon, while produced in different amounts by distinct cell populations, regulate the expression of an overlapping set of interferon stimulated genes (isgs). Some examples include 25-oligoadenylate synthase (oas), which decorates viral rnas with branched polyadenosine, and rna endonuclease l, which promptly degrades rnas containing these polyadenosine modifications . Most isgs, however, have not been well characterized, and some of these genes are so enigmatic that they do not contain any canonical secondary structures that could aid in predicting their function . Over 300 isgs have been identified by microarray and genomic analysis, which is curious given the magnitude of antiviral activity exhibited by select individual isgs . For example, mxa potently inhibits the replication of a wide range of viruses and can do so in the absence of other isgs or interferon signaling if ectopically expressed . Not all isgs exhibit such robust effects individually; nonetheless, when combined with other isgs, the host can mount an impressive arsenal of anti - viral proteins that could conceptually block every step in the viral life cycle . However, like oas and mxa, most isg expression requires interferon signaling through stat2 . Thus, while there are three major nodes to drive interferon secretion (tlrs, rig - i and nlrs) and two pathways available to drive stat2 activation (ifn-/ and ifn-), there is only one stat2 . In addition to its role in interferon mediated antiviral effect, stat2 also confers anti - proliferative and apoptotic activities on a variety of cell types in response to both type i and iii interferon . This activity is perhaps one of the most fundamental actions of interferon to inhibit viral spread by blocking the replication of virally infected cells . However, much attention has been focused on the use of ifn-/ to inhibit cancer outgrowth and metastasis given its unique ability to inhibit proliferation of a variety of tumor cell types . Stat2 is a critical signaling intermediate that regulates much of the apoptotic and anti - proliferative effects of ifn-/ and ifn-. Further, mutations within stat2 that protract its ligand - mediated phosphorylation can drive cells down the apoptotic pathway . A variety of genetic mutations have been identified in humans that affect ifn-/ and ifn- production, including mutations in nemo, tyk2 and tlr3 . Further, functional defects in the human unc93b1 gene, which controls signaling via tlr3, 7, 8 and 9, results in dramatic reductions in ifn-/ and ifn- secretion in response to ligands that activate these receptors . However, these subjects are susceptible to only a few types of viral infections, mainly encephalitis caused by herpes simplex virus . This restriction in viral susceptibility may reflect the inherent redundancy in driving ifn-/ and ifn- production through alternative pathways such as rig - i and nlrs . In contrast, to date, no mutations have been found that block responsiveness to type i or iii interferons in humans, particularly within either the ifn-/ receptors or stat2 . Yet, defects in a variety of genes that affect both ifn- production and responsiveness have been characterized including ifngr1, ifngr2, stat1 and il-12rb1 . Thus, it is interesting to speculate that mutations that affect ifn- responsiveness, while debilitating, can be tolerated; yet, mutations that cripple ifn-/ or ifn- responsiveness such as stat2 are perhaps lethal . A cascade of cytokines regulate processes of inflammation that drive both local and systemic activation of immune cells, and this process can be initiated by both pathogen sensing as well as sterile tissue injury . When discussing the role of stats in inflammation, it is perhaps more accurate to discuss their function in the context of distinct cytokine receptors, as most of the stat family members can be activated by multiple cytokines . For example, stat3 can be activated by both il-6 and il-10, yet their biological activities are diametrically opposed and counterregulatory with il-6 contributing to inflammation and il-10 blocking inflammation . While ifn-/ can induce local inflammation under certain circumstances, it does not mediate the intense forms of inflammation seen with tnf- or il-1. Initially, ifn-/ was thought to act similarly to il-12 in driving th1 development through the recruitment and activation of stat4, thereby driving th1-mediated inflammation . Indeed, stat2 was shown to be involved in recruiting stat4 to the human ifnar . However, our recent findings demonstrated that although stat4 is transiently activated by the human ifnar, it is not sufficient to promote th1 development . Further, distinct ifn- subtypes can drive stat4 activation to the murine ifnar, which can occur in mouse cells even in the absence of stat2, yet still does not promote either th1 or tc1 development compared with the effects of il-12 . Nonetheless, ifn-/ may regulate some aspects of inflammation that play a more supportive rather than direct role . Ifn-/ (as well as ifn-) is a potent regulator of the chemokine cxcl10 (ip-10), which recruits activated effector t and nk cells to sites of infection . Thus, direct pathogen sensing by epithelial and endothelial cells can lead to recruitment of inflammatory cells via autocrine responses to ifn-/. Ifn-/ also activates nk cells directly by driving lytic activity and ifn- secretion . Further, recent studies by tracey and colleagues demonstrated that the ifn-/-induced kinase, pkr, regulates an alternative pathway for inflammasome activation . As a result, the induction of pkr by ifn-/ can lead to potent secretion of hmgb1 and other leaderless cytokines that are dependent upon inflammasome - mediated cleavage . Although ifn-/ regulates these processes, a formal test for the direct role stat2 in driving these responses has not been performed in stat2 knockout mice . However, a recent study by gamero and colleagues demonstrated reduced skin and colonic chemokine and cytokine expression in stat2 knockout mice in two models of chemically induced carcinogenesis . These results suggest that stat2 may directly contribute to carcinogenesis by promoting expression of proinflammatory cytokines and chemokines . In summary, stat2 participates in some aspects of inflammation by autocrine responses to ifn-/ and downstream induction of inflammatory chemokines and cytokines . Given the critical nature of the interferon pathway in viral defense, it is not surprising that viruses have evolved to antagonize the antiviral state induced by type i and type iii interferons . The different antagonistic mechanisms include inhibition of interferon production, interferon signaling and inhibition of interferon induced antiviral proteins . Stat2 in particular is a target of many viral proteins, which in some cases determines viral host range . Here we discuss the viruses that have particularly acquired the ability to subvert the interferon signaling pathway by targeting stat2 (summarized in table 1). Viruses from a broad array of genera have developed unique ways to subvert the interferon response, and such examples include herpes simplex virus 2 (hsv-2), mouse cytomegalovirus (mcmv) and lymphocytic choreomeningitis virus (lcmv). However, the paramyxoviridae family of viruses contains perhaps the most replete examples of how viruses have subverted the interferon response pathway by targeting stat2 . The henipavirus (nipah and hendra virus) are newly emerging zoonotic pathogens causing acute respiratory disease in both human and other animal populations . Each of these viruses along with other members of the paramyxoviridae family express a v protein, which is encoded by a polycistronic gene segment (p gene) that utilizes unique overlapping reading frames . The v protein from measles, nipah and hendra viruses can bind to stat2 and prevent the interferon response via sequestration of stat2 in the cytoplasm . However, the modes of interaction of the v protein from measles mediate this interference by distinct mechanisms from that of henipahviruses . While the v proteins of these viruses contain a conserved c - terminal domain (ctd), only the measles virus requires the ctd for binding and targeting stat2 as means of interference with interferon signaling . In contrast, the nipah virus v protein can also bind stat2 in a stat1 dependent manner through distinct regions of the v protein mapping outside the boundary of the ctd . These complexes are unable to translocate to the nucleus, thus sequestering stat2 to the cytoplasm and inhibiting efficient interferon signaling . The hendravirus v protein also operates in a similar manner to nipah virus, although the distinct interaction with stats have not been mapped . In addition, the nipah virus v protein has been shown to contain a nuclear export signal (nes) that enables the v protein to shuttle between the cytoplasm and nucleus . Nonetheless, binding of stat1:stat2 complexes by nipah v protein is sufficient to prevent ifn-/ signaling even in the absence of the nes . Therefore viruses belonging to the same family have evolved to target stat2 using diverse mechanisms in order to evade interferon response in the host . Respiratory syncytial virus (rsv) is a member of the paramyxovirus family and causes severe flu and cold like symptoms in humans, particularly in children . Rsv antagonizes the interferon - signaling pathway through two virally encoded nonstructural proteins, ns1 and ns2 . Together, ns1/2 form a complex with stat2 and drive ubiquitin - mediated proteasomal degradation . Binding of stat2 with this complex requires the c - terminus of ns2, while ns1 acts as an e3 ligase that targets stat2 for degradation . In a similar manner to rsv, the rodent pathogen sendai virus (sev) encodes a c protein that antagonizes ifn-/ signaling at all phases of the infection process by interacting with stat1 and blocking activation of stat2 . Finally, the human parainfluenza virus 2 (hpiv2) is an rna virus of the paramyxoviridae family that causes upper and lower respiratory infections . In human, hpiv2 blocks type i interferon signaling by selectively inducing degradation of stat2 but not stat1 . The degradation of stat2 is independent of ifn-/ signaling, and like other members of this family of viruses, the v protein mediates the degradation of stat2 . Post - translational degradation of stat2 protein by hpiv2 v protein was blocked by proteasome inhibitors indicating a role for proteasomal degradation . In contrast, the v protein of parainfluenza virus 5 [piv5, previously known as simian virus v (sv5)], another member of the same family of viruses, preferentially induces stat1 degradation in a stat2-dependent manner in human . The c - terminal domain of this v protein is highly conserved across the members of rubulavirus genus of the paramyxoviridae family that includes both hpiv2 and piv5 . Although piv5 can target human stat1 via stat2, mouse stat2 does not interact with the piv5 v protein . Thus, mouse stat2 restricts piv5 v protein interaction leaving the interferon response pathway intact . As such, piv5 is unable to efficiently replicate in mice due to an intact interferon response . Transgenic mice expressing human stat2 restores the ability of piv5 v protein to mediated degradation of stat1 . Likewise, expression of human stat2 in mouse embryonic fibroblasts permits antagonism of endogenous murine ifn-/ signaling by both piv5 and hpiv2 v proteins . Thus, the requirement for stat2 interaction for antagonism of interferon response limits the host range of piv5 . Dengue virus (denv) is another example of how host - pathogen co - evolution has become a host range - determining factor . Dengue virus is a mosquito - borne rna virus that causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome in humans . Denv belongs to the genus flavivirus and has been shown to replicate efficiently in a variety of human immune cells including monocytes, macrophages, b cells and dendritic cells (reviewed in ref . Initial studies with denv replicons expressing all of the non - structural proteins demonstrated reduced ifn- stimulated genes concomitant with a reduction in endogenous stat2 protein levels . In later studies, denv ns5 was found to directly bind to and promote ubiquitin - mediated degradation of human, but not mouse stat2 . Mouse stat2 expression in human cell lines restored the ifn - dependent block in virus production . Ns5 interaction with human stat2 was mapped to the coiled - coil domain of human stat2 containing a species - specific sequence between residues 181301 . As ns5 failed to bind mouse stat2, replacement of this region of mouse sequence with the human counterpart restored ns5 binding to the chimerized mouse molecule . In parallel studies, denv ns5 was found to specifically antagonize ifn-, but not ifn- signaling by binding to and inhibiting stat2 phosphorylation . Recently, schoggins et al . Have demonstrated that overexpression of stat2 in stat1-deficient fibroblast can limit denv replication without any significant isg expression . Collectively these studies demonstrate that denv is able to establish infection in human due to its ability to surmount human antiviral response via stat2 degradation and blocking phosphorylation that may be circumvented in humans through augmented stat2 expression . However, murine stat2 has evolved to disengage this mechanism of stat2 antagonism by denv and thus restricting denv host range to humans . Given the central role of stat2 in the induction of isgs, it is perhaps surprising that only a restricted subset of viruses have adapted their mechanism of innate subversion to target stat2 . For some viruses, stat2 determines the host range across species, and this is particularly evident with dengue virus, discussed above . However, closer examination of the stat2 gene and protein product reveals some important insights into how viruses shape the evolution of their hosts . In this section, we will explore how stat2 has emerged as a highly divergent member of the stat family while retaining its ability to exclusively regulate the interferon response in each species . The stat family can be traced as far back to rudimentary stat - like genes that exist in slime molds (d. discoideum), thus suggesting that the current members that exist in mammals (stats 1, 2, 3, 4 5a, 5b and 6) arose from a common ancestral gene through duplication . In mammals, the seven stat family members are located as clusters in 3 genomic locations: in humans, stats 1 and 4 on chr . 17 and stats 2 and 6 on chr . 12 . Considering their syntenic arrangement across species, how conserved are the various stat members in mammals? Within the ensemble database, there are seven species, including human, for which the full - length sequences are available for six of the seven stat family members . An alignment of these sequences reveals remarkable similarity (> 95% for most pair - wise comparisons) for each of the stat genes except stat2 (fig . 1). Stat3 is the most highly conserved member, retaining> 98% sequence similarity comparing human with both mouse and rat . However, human stat2, while being relatively well conserved with macaque and chimpanzee, diverges significantly when paired with mouse, rat, horse and elephant . A phylogenetic analysis further reveals the degree to which stat2 has diverged even within the non - human primate species when compared with the human counterpart (fig . 2). Moreover, stat2 s dimerization partner stat1 displays roughly 5-fold greater sequence similarity than stat2 within primates (based on sequence identity within the phylogenetic analysis in fig . The full length protein sequences of stats 1, 2, 3, 4, 5a and 6 were aligned against the human sequence for each of the species shown in the radar plot . The clustal alignment algorithm within macvector was used for the alignment, and the percent sequence similarity to human in pair - wise comparisons is plotted in the graph . The full length sequences of stat1 and stat2 for each species shown were aligned with the clustal algorithm within macvector . Phylogenetic analysis was performed with this alignment, and the scale indicates the absolute numbers of sequence differences across the length of the dendrogram . Within each stat family member, the sh2 domain is the most highly conserved segment of the gene across species, and stat2 is no exception to this (fig . However, just beyond the sh2 domain, the c - terminal transactivation domain of stat2 diverges so significantly that only small segments of conservation are retained at the distal end of the protein . Most striking is the disruption of the structure and insertion of a mini - satellite segment within the c - terminus of mouse stat2 that contains a repetitive element papqvlle . This element has no significant similarity to any other protein in the database, and mouse is the only species that harbors this sequence . The sequences of stat2 from the indicated species were aligned beginning with residue 651 of human stat2 . The sh2 and c - terminus of stat2 is indicated along with the conserved tyrosine residue (y690), which is phosphorylated in responses to ifn-/ signaling . The divergence in stat2 was first appreciated when mouse stat2 was cloned and sequenced over 12 years ago . At that time, it was known that stat2 interacted with other transcription factors to regulate expression of various isgs, and one of those factors was cbp . Demonstrated that while the c - terminus of mouse was significantly different than human, cbp could still interact with the c - terminus of both mouse and human stat2 and functionally cooperate to regulate transcription . In addition, stat2 was also found to be required for recruitment of stat4 to the human, but not mouse ifnar . Further, the human stat2 c - terminus was found to be required for this recruitment in human cells, and the significant divergence in this region partially explained the difference in signaling between the two species . However, the interaction was perhaps indirect as replacement of the mouse c - terminus with the human counterpart via knock - in failed to restore significant ifn-/-dependent stat4 activation in mouse t cells . Nonetheless, these animals still responded normally to ifn-/ by activating isgf3 and by driving the expression of isgs . These types of domain - swapping experiments reveal some very interesting aspects of stat2 biology . Despite drastic differences within the c - terminus, this domain still maintains a physical interaction with cbp and perhaps many other transcription factors whose evolution has remained relatively constant compared with stat2 . Indeed, the c - terminus of stat2 is relatively unstructured compared with other domains within the protein . However, when bound to the transcriptional activator zinc binding (taz) domain of cbp, the human stat2 c - terminus undergoes dynamic folding to create a stable tertiary structure, thereby stabilizing its bound conformation to the taz domain . Unfortunately, the structure of the c - terminus from other species such as mouse with the cbp taz domain has not been solved . Nonetheless, stat2 displays remarkable plasticity in both its proximal receptor activation and downstream gene transactivation capabilities regardless of the remarkable structural differences that exist, particularly within the c - terminus . Thus, even though stat2 has diverged at a much greater rate than other stat family members, the interactions that stat2 must maintain to mediate its function are still preserved and presumably contain enough inherent flexibility to accommodate large changes in stat2 s structure . Considering the central role that stat2 plays in regulating the intracellular anti - viral response, it represents an important target for viral interference . While speculative, it is likely that lethal pandemic viruses drive stat2 evolution, in some cases, by targeting stat2 directly . Perhaps the unique haplotypes that have been identified in stat2 among east asians and shared with neanderthal point to recent selection events in human evolution . Regardless, the survival of each species, including humans, to viral assaults thus depends upon the flexibility of stat2 to maintain distinct physical interactions, which preserve its function in driving the interferon response.
For patients with operable breast cancer, the major prognostic determinant is whether there has or has not been spread to the axilla and the number of involved axillary nodes . Initially it was suggested that breast cancer first spreads locoregionally via lymphatics to the axillary lymph nodes and then metastasises more distantly . In accordance with this concept, subsequently fisher postulated that the extent of micrometastases at diagnosis of breast cancer is an indicator of outcome, with biological behaviour of cancer predetermining the likelihood of progression of the disease . Nowadays gene expression profiling arrays can delineate tumour types with different prognoses . The surgical approach for breast cancer treatment evolved from the extensive radical mastectomy and the patey modified radical mastectomy to breast conserving and minimally invasive techniques . Traditionally the surgical management of breast cancer comprised wide local resection of the primary tumour and axillary lymph node dissection (alnd). Axillary status is the most important prognostic factor in breast cancer providing staging information and therefore largely defining treatment strategy . Diagnostic imaging modalities such as ultrasound, magnetic resonance mammography, positron emission tomography, and 99 m technetium (tc) sestamibi scintimammography are not reliable for staging the axilla, particularly with lymph node metastases <0.5 cm [8, 9]. Clinically palpable lymph nodes prove to be false positive in 2530% of patients and about 40% have positive results after ultrasound with or without fine - needle aspiration node negativity . Studying 24,740 women with invasive breast cancer, carter et al . Showed that approximately 80% with tumour size <1 cm, 50% with up to 5 cm, and 30% with> 5% had negative axilla, a fact suggesting that metastases do not occur exclusively via the axillary lymph nodes, but rather lymph node status serves as an indicator of the tumour's ability to spread . Additionally, it has been recently shown that the molecular profile of the primary tumour is a more significant prognostic indicator in terms of disease - free survival (dfs) and overall survival (os) than lymph node metastases . The combination of the introduction of population - based mammographic screening for breast cancer, modern imaging methods, and increased public awareness resulted in patients being diagnosed more often with smaller - size tumours and less likelihood of axillary lymph node metastases . It is evident now that 6070% of patients with early breast cancer are node negative at the time of diagnosis and alnd puts them at significant risk of short- and long - term morbidities without benefit . Alnd is associated with acute complication rates of 2030% including seroma formation, local swelling, numbness, impaired shoulder movement, neuropathy, infection, and chronic lymphoedema rates of 737% . In a prospective study by petrek et al . Evaluating a cohort of 923 women with 20 years follow up, it was shown that breast - cancer - related lymphoedema following alnd occurred maximally in the first 3 years following surgery; however, up to 23% of patients may still develop arm swelling during the rest of their lives . Several randomised studies have established that sentinel node biopsy (snb) is a safe and accurate procedure for detecting tumour cells in sln and predicting the status of the other axillary nodes (non - sln). Although accuracy and appropriateness of snb were disputed by the finding of 510% false - negative cases when snb was followed by axillary dissection at high - risk patients for axillary nodal disease [13, 18], false - negative snb results seem to have decreased with the increasing experience of surgeons, and it is expected that the utilisation of snb in the future will be increased . A meta - analysis of seven prospective randomised controlled trials by kell et al . Demonstrated that snb is equivalent to alnd for the detection of lymph node metastasis with the additional advantage of reduction of up to 75% in morbidity in patients with early stage breast cancer . Furthermore, a trend towards an improved detection of ln metastases was shown when snb is used . Patients undergoing snb have a 22% higher odds ratio of having a positive sln, due to the more intensive pathological examination which utilises multiple sections and immunohistochemistry (ihc). In contrast, the false - negative cases seen after axillary dissection are probably due to the inability of the pathologist to perform serial sections and ihc on the 2030 lymph nodes found in a complete axillary clearance specimen . Studies have shown that sln is the only positive lymph node in 3867% of patients when alnc followed . Interestingly, it has been reported that only 48% of patients with negative alns have internal mammary lymph node involvement (imn) whereas 2550% of patients with affected alns have also imn metastases [23, 24]. Dissection of imn is not recommended because of the high morbidity and the uncertain benefit on survival . The recently published outcomes of nsabp b-32 trial established the efficacy of sln biopsy alone with no further alnd in 5611 breast cancer patients with clinically negative lymph nodes . Women with invasive breast cancer who were randomly assigned to either sln resection plus alnd (group 1) or to sln resection alone with alnd only if the slns were positive (group 2), after 8 years of followup, showed statistically equivalent overall survival, disease - free survival, and regional control . Patient followup is still continuing for longer - term assessment of survival and regional control . Moreover, a closer look into mature studies focused on axillary relapses and overall survival is in agreement with current findings favouring slb . The national surgical adjuvant breast and bowel project (nsabp) b04 randomised study compared breast cancer patients with clinically negative alns managed either by radical mastectomy, total mastectomy with axillary radiation, or total mastectomy alone . The results clearly defined that alnd decreases the risk of locoregional relaps; however, no significant differences in survival were found among the treatment groups . This study has been criticised because of the variability of numbers of lymph nodes resected in the total mastectomy alone arm and the lack of statistical power to detect a small difference in outcome . Indeed, a meta - analysis has suggested that inadequate axillary treatment may lead to not only an increased risk of local relapse but also a 5% reduction in survival . As a result of increasing detection of early breast cancer and the high rate of micrometastases and itcs (itcs) found in the detailed pathological examination of sln, a new debate has opened about the consequent necessity of alnd in these patients . This has arisen because of better understanding of breast cancer behaviour and improved efficacy of combined therapeutic modalities . In this paper we report the current guidelines concerning the management of the axilla after slnb and review the different aspects arising from recent studies on the role of micrometastases and itc clusters in sln on decision making . The american society of clinical oncology (asco) expert panel conducted a systematic review of the literature available through february 2004 on the use of snb in early - stage breast cancer in order to develop guidelines for the management of the axilla (http://jop.ascopubs.org/content/1/4/134), and these are similar to those of the national institute of health and clinical excellence (nice) recommendations in uk (http://www.nice.org.uk/nicemedia/live/12132/43413/43413.pdf). Alnd is the standard of care in those with a macrometastatic or micrometastatic positive sln to maximise local control . If the sln is negative, a calnd (calnd) is not necessary . Itcs detected by ihc are of unknown clinical significance, and when identified, the sln is regarded as negative and no further alnd is required . Although ihc is often used, it is not included in routine sln evaluation for breast cancer at this time . Asco and nice recommendations for slnb, alnd alone and managing of the axilla after slnb are summarised in table 1 . In contrast the german guidelines do not recommend axillary clearance for 1 - 2 sln positive in patients with t1 and t2 tumours (http://www.ago-online.de). It has been suggested that slnb should be carried out by an experienced team in order to minimise false negativity and improve the predictive value of the procedure . The american joint committee on cancer (ajcc) in the sixth edition of the cancer staging manual defined a lymph node metastatic tumour with maximum diameter> 2 mm as macrometastasis (pn1), when the diameter of deposit is 0.22 mm as micrometastasis (pnmi), and a lesion of single tumour cells or small cell clusters with diameter <0.2 mm as itcs [pn0(i+)]. Itcs are not distinguishable by h&e staining but detected only with immunohistochemistry (ihc) or molecular methods . Moore et al . Suggested that the presence of itcs was unrelated to known prognostic variables and partly the result of instrumentation and manipulation of the tumour . The management of patients with minimal sln involvement is problematic . In a meta - analysis of 25 studies of patients with sln micrometastases, in approximately 20% there was nonsentinel node disease falling to 9% when the sln involvement was detected by ihc . Furthermore, the consequent effect on dfs and os remains controversial, so the biological relevance and clinical significance is a matter of debate . Amaros investigates the benefit of a calnd in comparison to treatment with axillary radiotherapy (art) in patients with sln - positive breast cancer . A recently published substudy evaluated the identification rate and the nodal involvement of the first 2,000 patients between 2001 and 2005 who entered from 26 european institutions . The sentinel node identification rate was 97% which is high considering the relatively early days of this procedure . 34% were sln positive of whom 63% had macrometastases, 25% had micrometastases, and 12% had itcs . In the calnd arm non - sln involvement was identified in 41% of patients with macrometastases and in 18% of patients with either micrometastases or itcs . Several studies have investigated the significance of occult metastases, such as micrometastases or small clusters of tumour cells in association with non - sln involvement and the impact of calnd on disease - free survival and overall survival, and the larger ones are summarised in table 2 [3743]. Although the majority show no prognostic impact of itcs in the sentinel node, a large dutch investigation with 5-year followup indicated that women with itcs who received adjuvant chemotherapy had a significantly better event - free survival compared with untreated cases with itcs . Furthermore, a finnish study showed a worse 5-year breast - cancer - specific survival for those with itc compared with node negative cases . In the largest published multicenter retrospective study of 187 sln - itcs patients undergoing calnd, houvenaeghel et al . Reported an incidence of 16% non - sln involvement . The difference in the risk of non - sln involvement between sentinel nodes with itcs (16%) and those with micrometastases (14%) was not statistically significant . However it was not apparent whether the presence of non - sln metastases should affect the therapeutic decision in these patients . The authors proposed that calnd could be avoided in patients with tubular, colloid, or medullary small primary tumours (pt1) with a risk of non - sln involvement approximately 5% . Mirror is a large dutch cohort retrospective study which assesses the impact of sln - itcs and micrometastases on 5-year disease - free survival in patients with favourable primary tumour characteristics . According to recent published data, both patients with snb micrometastases and those with itcs who did not undergo calnd experienced a far higher 5-year axillary recurrence rate, 6% in comparison to 1% of snb - negative patients who did not undergo calnd . Additionally, both patients with sln micrometastases and itcs had approximately 5-year disease - free survival improved by 10% with adjuvant systemic therapy . Mirror findings support an aggressive treatment approach in patients with either sln micrometastases or itcs . Many investigators have studied the incidence of non - sln involvement in patients with sln micrometastases to define which patients may need further axillary treatment . Wada and imoto . Collected 22 studies from 1999 until 2006 referring to the frequency of sln micrometastases in patients with breast cancer and the prevalence of non - sln involvement in those patients after alnd . The frequency of sln micrometastases was 38% with non - sln micrometastases ranging from 0 to 57% . Additionally, a wide range of non - sln macrometastases was found (018%). Because the prevalence of non - sln micrometastases was low, the prognostic impact was unclear . The wide range of results arose from the different numbers of patients involved, variations in number of pathological sections examined, and differences in tumour stage and grade . Results of studies in which patients with micrometastases in snb and who were not treated by completion axillary node clearance are summarised in table 3 [38, 4854]. Most of the studies had small numbers and relatively short followup and tended to conclude that there was no benefit from completion axillary node clearance . The largest study; however, found a significantly worse disease - free survival for women with micrometastases who did not undergo calnd . . De boer et al . Conducted a systematic review of 58 studies conducted from 1977 to 2008 included 297,533 patients, aiming to define the prognostic relevance of micrometastases and itcs in patients with breast cancer . Using random - effect meta - analysis they showed that the presence of aln metastases <2 mm in diameter detected on single - section examination was associated with poorer overall survival . Moreover the presence of occult metastases on retrospective examination of aln - negative patients by step sectioning and/or immunohistochemistry (n = 7740 patients) was associated with poorer 5-year disease - free and overall survival . Outcomes from sentinel lymph node biopsy studies were not assessable due to small patient groups and short followup . The international breast cancer study group trial ibcsg-23 - 01 is a randomised multicentre study designed to determine the significance of minimal ln metastasis in patients with breast cancer . The trial was initiated in april 2001, and it compares survival between patients with sln micrometastases who undergo slnb alone with those who receive calnd . It is known that the extent of macrometastases in sln is strongly correlated with non - sln involvement . The long - term effect of the residual axillary disease in the sentinel - lymph - node - positive patient on local and systemic recurrence has not been clearly defined for patients receiving modern radiotherapy and chemotherapy . Older studies of patients with symptomatic breast cancers have shown that inadequate axillary surgery does lead to reduced overall survival [5760]. The study set out to randomise 1900 women with breast cancer and 15 involved snln to either calnd or observation . All had a lumpectomy and tangential breast irradiation, but systemic therapy was at the discretion of the treating centre . After a median followup of 6.3 years, the relapse - free survival for the alnd group was 82% compared with 84% for the observation group, and the overall survival was 92% in both groups . Unfortunately the trial stopped accrual after 891 cases had been entered which makes it underpowered to detect a 5% difference in outcome . An additional aspect to be considered is that the guy's wide excision studies showed no difference between the wide excision group and the radical mastectomy group at 10 years whereas after 25 years there was a significantly worse relapse - free and overall survival in the wide excision group with inadequately treated axillae . In a retrospective study, takei et al . Confirmed the importance of calnd in sln - positive patients with high nuclear grade and hormone - negative breast cancer . It was noticed that of 459 patients with macrometastatic disease treated with calnd, after a median follow - up period of 34 months, the axillary recurrence rate was only 0.6% . Bilimoria et al . Studied a cohort of 403,167 patients with clinically node - negative breast cancer that underwent slnb from the us national cancer data base (19982005). Of the 97,314 (24%) patients identified with nodal metastases, 28% had no further surgical intervention in the axilla and 72% underwent calnd . After a median followup of 63 months, it was found that in all patients and separately in those with macroscopic and microscopic nodal disease, the unadjusted axillary recurrence rate and overall survival were comparable . After adjustment for clinicopathological differences, there was a trend towards a lower risk of axillary recurrence and death in patients with macroscopic nodal involvement undergoing calnd . For those with micrometastases, recurrence rates were similar in those undergoing either slnb alone or calnd . Of 26,986 patients with slnb - positive breast cancer from the seer database (surveillance, epidemiology, and end results), 16% had no further axillary treatment and 84% had calnd . After a median followup of 50 months, although a higher rate of ipsilateral regional recurrence was noticed in patients who underwent slnb alone, no statistically significant differences in overall survival (os) between patients who underwent slnb alone versus complete alnd were found . The investigators suggested that in patients with small, low - grade primary tumours, positive er status, older age and who have received segmental mastectomy, calnd may be omitted . Hwang et al . Reviewed the outcome of 3,366 patients with invasive breast cancer who underwent slnb from 1993 to 2005 . Of 750 sln - positive patients, 65%, 45.9%, and 34.2% were pn1, pn1mi, and pn0 (i+), respectively . Of these patients, 196 had no further axillary surgery due to clinician and patient preference . According to clinicopathological variables, adjuvant treatment was applied and locoregional and distant recurrence and survival were studied . After a median followup of 29.5 months, no patient had an axillary recurrence, one had supraclavicular lymph node recurrence, and three patients developed metastatic disease to the lung or bone . The median time to recurrence was 32 months . Notably the patients with distant metastases had t3 grade iii invasive carcinoma . Despite the low axillary recurrence rate, authors suggested that it is not possible from these results to conclude definitively that calnd should be abandoned for these patients . Several factors correlated with the likelihood of additional non - sln metastasis have been investigated in an effort to distinguish which patients could avoid extensive axillary surgery . Characteristics of the primary tumour, such as size [40, 66], grade, hormone receptor and her2 profile, tumour type, multifocality, mean proliferative fraction, and lymphovascular invasion [68, 69], have all been studied . Additional features of the involved slns, such as size of metastases, number of positive slns, ratio of positive to resected slns, and the extracapsular spread have been also examined [21, 61, 7072]. Particularly patients with minimal sln metastases are at a significantly lower risk to have further non - sln invasion than those with sln macrometastases (1324% versus 4579%). However, none of these characteristics individually can determine a subset of patients for whom alnd is unnecessary . Molecular profiling of metastatic foci different from the primary tumour could be used as indicator for the selection of patients who might benefit of completion axillary dissection . The most important prognostic factors for the presence of non - sln metastases in patients with minimal sln involvement are presented in table 4 . Several mathematical models have been developed to predict the risk of non - sln involvement in patients with sln - positive breast cancer . These include four nomograms: the memorial sloan - kettering cancer center (mskcc), (https://www.mskcc.org/mskcc/html/15938.cfm), mayo (https://www.mayoclinic.org/breast-cancer/sentinelbiopsy.html), cambridge, and stanford (https://www3-hrpdcc.stanford.edu/nsln-calculator/). There are three scoring systems, the tenon, mda, and saidi, and two recursive partitioning (rp) tools developed by kohrt et al . . The institut curie studied 588 consecutive patients with positive slns who underwent alnd to compare the actual rate of non - sln metastases with those predicted by breast cancer nomogram of memorial sloan - kettering cancer center (mskcc). While the predicted rate in non - sln macrometastases was relatively accurate, when the nomogram was applied to the 213 slns that contained only micrometastases, the predicted rate 59% was far away from the actual rate 44% of non - sln micrometastases detected by ihc . Consequently, the authors concluded that a different predictive model should be created for patients with micrometastases . Molecular tests based on technology such as oncotype dx (genomic health, redwood city, calif, usa) or other multigene arrays developed prognostic and predictive markers aiming to personalize surgical and adjuvant treatment of early breast cancer . An accurate, intraoperative sentinel lymph node test probably could help in avoidance of delayed axillary dissections . Molecular tests may be proved more sensitive than current intraoperative tests but have not yet been validated . Osna is an automated assay for the detection of cytokeratin message, ck19 mrna, present in approximately 98% of breast cancers . It provides an opportunity to make an intraoperative diagnosis of sentinel node involvement within 30 minutes, avoiding frozen section and allowing a one - stage procedure . Larger verification studies in which half of the bisected sentinel node was sent for histology and the other half homogenised for osna are shown in table 5 [71, 72, 7981]. The usual reason for discordance between histopathology and osna was an uneven distribution of nodal metastases (tissue allocation bias). The studies indicate a good concordance between results of histopathology and osna . Indeed in a study conducted by osaka, comparing osna with frozen section, it is likely that in time osna will replace histopathological examination of sentinel lymph nodes because of its ease, accuracy, and potential for enabling almost all patients to have a one - stage operation for early breast cancer . Recent studies aiming to determine if calnd is beneficial in patients with sln - positive breast cancer and even more in patients with minimal sln involvement have reached contrary conclusions . Limitations in the published studies include the methods of pathological evaluation of lymph nodes and that the number of additional non - sln - positive nodes is usually not known thus lymph node step sectioning and ihc lead to increased identification of minimal metastases upstaging 9% to 25% of patients who initially were considered node negative [83, 84] and did not have further axillary surgery . The different rates of recurrence may be due to the molecular type of cancers and so that patients with sln metastases may also have different risks of metastatic involvement . Finally it is possible that not all minor tumour foci in axillary lymph nodes progress to local recurrences . According to al - hajj et al ., only a minority of cancer cells potentially give metastases and most itcs are not viable and do not have the ability to form new tumours . There may be two different breast cancer cell populations, true stem cells that have the capacity to develop metastases and the nonstem cells that never grow and are finally destroyed . At the level of everyday clinical practice, with both promising and disappointing results of the published studies, most breast surgeons will hardly ever take the risk of avoiding completion axillary dissection in breast cancer patients even with minimal sentinel lymph node metastases . Many are seeking to find a balance between the needs of the majority to have minimal axillary surgery with minimal postoperative morbidity against the possibility that a minority will suffer relapse, morbidity, and possible increased mortality from undertreatment . Results from ongoing phase iii trials will perhaps provide new guidelines for the treatment of patients with micrometastases or itcs . A predictive model which estimates accurately the likelihood of additional disease in the axilla might help tailor surgical therapy to the needs of the individual patient and identify those most likely to benefit from completion or alnd . Genetic assays defining prognostic markers and new intraoperative tests detecting accurately sln involvement will help in early therapeutic decision making in the future . It is important that premature decisions to restrict axillary surgery are not made on a basis of early results from underpowered clinical trials.
Labor wellbeing refers to all efforts of employers, trade unions, voluntary organizations, and governmental agencies, which help employees feel better and perform (work) better . It is a comprehensive term that refers to the physical, mental, moral, and emotional wellbeing of an individual including intra and extra mural facilities . Although wellbeing includes a wide range of programs and services in organizations, the focus of this study is on the employee assistance programs and employee group services, which are provided by employers according to the real needs of the employees . Hence, it is necessary for organizations to identify the real needs, as a base to provide a wellbeing package for their employees through wellbeing need assessment techniques . Wellbeing need assessment is a systematic process of gathering information to determine and address the needs or gaps between the current conditions and the desired conditions . A need can be a desire to improve the current performance or to correct a deficiency . The wellbeing needs assessment includes two main targets; exploring the effectiveness of the delivered services and exploring those wellbeing needs that are not fulfilled by an organization . Davis and gibson emphasize on the importance of a comprehensive wellbeing needs assessment, both for obtaining the required information needed to design the appropriate interventions and also for providing the basic required information to evaluate the effectiveness of the wellbeing program . Wellbeing assessments are vital for the determination of the economic and social needs of employees and for the best decisions to be taken in response to such needs . Reviewing the human resources management literature showed an absence of attention given to the employee's benefits . Moreover, various expectations of employees in the field of wellbeing services caused organizations, especially the human resources (hr) offices, to face many challenges (chen et al . ). Therefore, for keeping employees satisfied with the wellbeing services, all challenges had to be truly and effectively met (hyde et al . ). Research indicated that satisfaction of the employees was not positively correlated with the benefits of the welfare package (e.g. Dreher, ash, and bretz). Dencker, joshi, and martocchio argued that individual differences in benefit preferences emerged from particular employment relationships, with individuals having greater experience . The tehran university of medical sciences (tums), including 69 research centers, 25 hospitals, 11 educational and health care centers, three health networks, and 10 faculties with more than 2020 faculty members and 19,000 students (tums 2012), requires an improved comprehensive system for delivering the wellbeing services (financial, insurance, healthcare services, educational and training services, etc . ). Taking a look at the functions of the university's wellbeing services system; it uncovers a gap between the employees real needs and what is delivered to meet their needs . It is because of the lack of a participatory strategic wellbeing delivery plan that is based on prioritized wellbeing needs (requirements). As the first step to make a strategic plan is identifying the needs, the main purpose of this study is to assess the wellbeing services of the staff working in tums first to see if they are satisfactory enough or they need to be changed, and second, to help decision - makers develop services that are essential to improve the well - being of employees, as also the quality and effectiveness of the tums's wellbeing delivery system . What are the services used and to what extent they have been satisfactory? This question aims to explore the reasons why they are not delivered . The second question is, what type of services are essential to be provided by the university? With respect to the main purpose of this research, the wellbeing delivery system of tums is assessed with a thorough answering of the questions mentioned above . The research design was a cross - sectional survey (2008 - 2009) using a structured non - disguised questionnaire . The sample size was 98 with a 95% level of confidence and absolute error of 0/01 . Cluster sampling based on the occupational scope as the main variable, was used as the sampling method, with regard to the existence of different occupational scopes . Data was collected from 98 officers working at different departments of central organizations of tums (tehran university of medical sciences) including logistics, education, research and technology, student and cultural affairs departments . The research participants were assured of the confidentiality of their personal information at the beginning of the questionnaire . The questionnaire included 45 questions (43 close - ended questions and 2 open - ended questions1) organized into two separate parts (cranach alpha = 0.85): according to table 1, within the first part, the respondents were supposed to mark the wellbeing services if they had ever used any of them . Those who used the services were supposed to show their level of satisfaction by choosing from almost satisfied, almost dissatisfied, and full dissatisfied, and those who had never used the services, were supposed to express their reasons by choosing from poor announcement, unwillingness to receive the service, undesirability of the service, and undesirability of the receiving process of the service. Main parts and the main purpose of asking questions for each part in the second part, the respondents were provided with a list of not delivered wellbeing services to choose which of them is essential, neither essential nor non - essential or non - essential. For making sure if the questionnaire was valid content - wise, first a comprehensive collection of wellbeing services delivered both in tums and in other organizations was identified according to the comprehensive definition of wellbeing services . Next, according to the main purpose of the research, they were put into classified groups to form the research tool . Furthermore, to make sure about the appropriateness and transparency of each question and also the whole tool, in addition to its comprehensiveness, the questionnaire was sent to five professionals and experts in the relevant field . The reliability of the questionnaire was determined after thoroughly calculating the cronbach alpha, which was reported to be around 0.85 . Subsequently, after improving and clarifying the instrument, the participants of sample group were asked to fill in the questionnaire . The data was gathered by means of filled questionnaires, and were analyzed with the excel software and spss, by calculating the frequency rate of the staff that used the welfare services and those who did not use the services . In addition, the t - test was used to calculate the satisfaction score for each service used by the staff . Among all the 98 respondents, almost 46 employees with a mean satisfactory score of 48.46 used the existing services, for which the satisfactory rate was distributed as shown in table 2 . Satisfactory scores of categorized wellbeing services the result shows that financial services, including loans for buying a car, emergency loan, loan for buying commodities, awards for staffs children, and two other kinds of non - profit loans in comparison with other categories is considered as the most satisfactory service (average t = 61.83). Among respondents who did not use the categorized services (52 persons on an average), as shown in the table 3, the most frequents reasons for not receiving services was, staff's unwillingness to receive the service (38.75 in average) and poor announcement (unawareness on the wellbeing services) (37.6 in average). Frequency rate of reasons for not receiving welfare services table 4 presents the frequency of wellbeing services and necessity (delivered by other public / private organizations), which were asked to be judged by respondents (to be added or replaced by the delivered wellbeing services list), as follows: frequency distribution of wellbeing services necessity educational subvention for children of the staff, who were under their patronage is the only non - financial service, with the highest frequency (92.85), identified as the most necessary wellbeing service among all . Among other services that were mentioned by respondents (61 persons of the sample group), the provision of discount cards for using other organizations services (frequency = 12), providing employees with free health care services (frequency = 7), and contracting with adult higher education institutions (frequency = 6) had the most frequency among other items . As mentioned, the objectives of the study were to measure the staff's satisfaction with the wellbeing services received, identify the reasons for not receiving the existing wellbeing services, identify the wellbeing services that were not delivered, but were essential as per the staff, and then ask the staff about other wellbeing services that were possible to be delivered and sort them according to their priority . With regard to the first purpose, the results showed that almost half of the respondents used the wellbeing services provided by the wellbeing office of the university, among which almost half of them were satisfied with the services . The results suggested that not only were most of the staff not interested in using the provided services, but also more than half of those respondents who used them were not really happy . With regard to the second purpose, the results showed that among those who did not use the services, most of them decided not to use it by themselves . This was recommended to be surveyed in a separate study in the future . As was evident, it can be concluded that the functional desirability of the wellbeing service delivery system of tums was not evaluated as satisfactory enough, because of the almost high dissatisfaction level of services that were used . With regard to the third purpose, the results indicated a big gap between the required wellbeing services (by employees) and what was provided by the office, as almost all the respondents agreed with the necessity of services such as health, education, and safety . The existing gap can result from lack of managerial attention to differences in the demographical characteristics and expectations of employees . Consistent with the arguments of boudreau and ramstad, dencker et al . Argued that market segmentation of employees (offering market segments have different benefits) may strengthen the benefits return - on - investment. One of the most significant reasons for the dissatisfaction and unwillingness of employees to use the services refers to the different priorities and type of services they prefer to include, according to their individual differences . Need assessment as a useful technique can help planners to pay more attention to employee's priority differences in wellbeing services and what they really need to include, by designing the appropriate interventions, as davis and gibson emphasized . Considering the employees as the most important organizational stakeholders makes them to participate in the wellbeing decision - making process, such as, asking them to add what services they prefer to be included within the wellbeing package . Although the wellbeing service delivery system of tums has not been effective enough to meet the real needs of the staff by delivering the appropriate services, the most prioritized service type preference of most respondents can be identified . As in the first place the satisfactory score of educational and training services among those who used them was rather high, and second, the most necessary identified services were related to educational services, and of course, among the identified wellbeing service priorities, provision of educational opportunity was one of the preferences with high frequency, therefore, it was clear that educational and training opportunities were important for a majority of the respondents . The findings show that the functional desirability of tums wellbeing services is not satisfactory enough by the staff's view point . The following conclusions can be drawn from the present study; the first major finding is the existence of a big gap between the required wellbeing services (by employees) and what is provided by the wellbeing office of tums . The second and the most obvious finding of this study is the importance of the educational and training opportunities from the point of view of the staff, which has not been paid attention to by the wellbeing service provider in tums . Although we tried to assess the overall quality and functional desirability of the wellbeing service delivery system of tums, some further studies are required in the researcher's view . It is recommended that the factors that cause dissatisfaction and unwillingness to use services in employees be identified, and also the real needs and wellbeing requirements (based on employees preferences, job categories, genders, age, and other considerable individual and social factors) as a strategic basis for redesigning the current wellbeing service delivery system that appears not to be desirable enough in the employees view, be determined.
The clinical manifestation is typical and it is characterized by abduction of the affected arm, flexion of the elbow and pronation of the forearm . Bilateral luxatio erecta represents an extremely rare condition, and just a few cases were reported in medical literature . Because of a high incidence of related complications such as neurovascular damages early recognition and treatment of this type of dislocation is required by physicians . In this article we describe a case of a bilateral luxatio erecta that was successfully treated without long - term complications . A 70-year - old female was accepted in our emergency department because of a bilateral shoulder pain that onsets after a sudden fall happened in her house . The anamnestic research showed that the patient tumbled downstairs; while clinging to both lateral banisters, she slipped down banging her face and chest to the ground . At physical examination both shoulders were abducted and elevated approximately 90100 from the horizontal plane, with flexed elbows and with hands below the head . The patient was unable to adduct and lower the arms, and there was immediate pain when attempting passive movements . No vascular deficit was found, and the patient complained of slight paresthesias on both arms, with superficial and deep sensation deficit on both arms . An x - ray examination was performed showing a bilateral inferior dislocation of humeral heads in relation with the glenoid fossa; no fracture was associated (fig . 1). 1bilateral luxatio erecta bilateral luxatio erecta close reduction under sedation was achieved without difficulty by pushing the humeral head superiorly with an axial traction - countertraction maneuver on both shoulders . X - ray control revealed a correct anatomic reduction of dislocations and after the reduction no vascular damage were found while paresthesias were still present on the left arm (fig . 2). Both arms were immobilized with bilateral slings in total adduction, intrarotation with the supinated arms . 2post - reduction x - ray the progressive mobilization of the shoulders started after 2 weeks with passive movements and pendulum codman s exercises . After 3 weeks the patient gave up complaining paresthesias on the left arm and active movements were allowed . After 8 weeks she recovered the complete function of both shoulders and after a one - year follow - up treatment the patient showed a complete range of motion of both arms . Inferior dislocations are rare (0.5% of all shoulder dislocations) and bilateral inferior shoulder dislocations are exceptional: only 10 cases have been described so far in the literature . According to the literature, luxatio erecta is due to an indirect mechanism such as a violent abduction force on abducted limb resulting in impingement of the proximal shaft of the humerus against acromion . Direct axial loading from superior direction is another different mechanism, though less common . In our case the first mechanism has to be taken into consideration; in fact, while the patient was sliding in a sitting position downstairs, her hands were holding onto both banisters and the arms were abduced . When she reached the ground floor the hands were blocked around the end of the banisters and her head and body were thrown forward by gravity . The clinical presentation of an inferior shoulder dislocation is typical: the extremities are held over the head or abduced in a fixed position with the elbow flexed . Any passive movement is possible, and the humeral head is palpable on the chest wall . X - ray shows the humeral head located below the rim of the glenoid and the humeral shaft is parallel to the scapular spine . In order to reduce inferior glenohumeral dislocation, it is recommended a traction counter - traction maneuver under sedation followed by immobilization in a desault bandage for at least 2 weeks . There is a high incidence of associate tears of the rotator cuff (12% of patients) and a slap lesion has been described by schai and hintermann after a post - reduction arthroscopic assessment view . Fracture of the acromion, clavicle, inferior glenoid fossa and greater tuberosity represent a complication that should need a surgical treatment . Neurovascular damages are also commonly associated with inferior shoulder dislocation [5, 7]; garcia et al . Described a case of bilateral luxatio erecta complicated by an axillary vein thrombosis . In general, neurologic injuries (60% of cases) of brachial plexus, axillar, radial and ulnar nerves are more common than vascular injuries but they tend to resolve after reduction with an excellent prognosis, suggesting the neuropraxia as the main mechanism of the injury [5, 9, 10].

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