This article considers one specific strand of discussion around HIV/AIDS, in order to think about the uses and limitations of human rights discourse in late twentieth century Britain. HIV/AIDS presented particular problems for prisons, which were initially presented as breeding grounds of infection. This shifted with the rise of human rights language and laws in the 1990s, but talk of rights for prisoners was not as comfortable a fit as talk of equivalence of care. This story is situated in the broader context of attitudes towards crime, HIV/AIDS, and rights in late twentieth century Britain. The Lancet in 2007, typically for public health and human rights practitioners, pinpointed the 1980s as a particular turning point: the two previously parallel but separate issues of health and human rights were finally and definitively brought together, they proposed, by the emergence of AIDS. This new and deadly international epidemic, disproportionately affecting marginalised communities, prompting discrimination, and highlighting existing health inequalities, had been "instrumental in clarifying the ways that health and rights connect".Health as a human right is a relatively new concept. Its inscription into some of the foundational statements of the United Nations (UN) and World Health Organisation (WHO) marked the beginnings of a new way of perceiving healthcare, as ideas of human rights started to expand to encompass social and cultural as well as political and civil rights.not proved useful), and how have its meanings changed as the idea has been deployed?This article considers whether and how, in the midst of the AIDS crisis, the idea of health as a human right was successfully applied to the question of healthcare for people in prison in Britain. This reflects upon two questions raised by recent histories of human rights and summarised by Robert Brier: why, in recent times, has the idea of human rights proved useful to historical actors , various Ministers and Chief Inspectors of prisons, individual doctors, addiction workers, and researchers, and the staff of the prison service itself. These were also subjects addressed by the mainstream media, whose coverage of HIV/AIDS often focused on what Matt Cook has described as “headline grabbing outrage from assorted self-appointed moral guardians".Initially, prisoners were perceived first and foremost as a risk to the wider community, presenting prisons – not for the first time – as sites of contagion. Arguments focused upon the need for prisons to fulfil their duty of protecting the community, ensuring that prisoners did not leave more dangerous than when they arrived. As the worst fears of AIDS began to abate and discussions of human rights, including health rights, became more frequent in the early 1990s, the needs and rights of prisoners received more attention. However, this was limited: rights talk was constrained by the absence of enforceable rights, by the nature of the health rights model of the 1990s, and by a failure to perceive suffering within prisons. Although these factors were likely common to many western nations, they were particularly pronounced in Britain. Prisoners’ rights struggled to find purchase, as the last section of this article will discuss. Firstly, though, it is worth setting out the background to both prison and HIV/AIDS policy in the 1980s.The cause of penal reform had enjoyed a relatively low profile in Britain throughout most of the twentieth century. Occasional enquiries and campaigns took place, but prisoner numbers were low and significant upheavals few. In the 1960s, radical prison reform in the United States (US), often driven by prisoners themselves and addressing matters of health, achieved some prominence and was mirrored by the formation in Britain of the groups Radical Alternatives to Prison (RAP) and the National Prisoners’ Movement . These were most active during the 1970s, and PROP in particular explicitly addressed prisoners’ rights. Yet, as one of PROP’s founders has suggested, the organisation was not particularly successful, especially when compared to similar movements in the US.and prisoners’ rights on this side of the Atlantic.There was less mention of both healthcare in prisons everyone, rather than just those engaging in higher risk activities, and was particularly evident from 1986 to the end of the 1980s when fears of a so-called heterosexual epidemic were at their height. The realisation that HIV could affect anyone provided much of the political impetus for action in Britain: the Chief Medical Officer remembered that the news that HIV could be transmitted through heterosexual sex was a "bombshell" that changed everything.As HIV/AIDS policies were developed at the national level, two positions were adopted by those arguing for similar outcomes, sometimes in combination. Firstly, fears that marginalised groups at high risk of infection, such as injecting drug users and gay or bisexual men, would spread the disease to low-risk groups prompted calls for a dramatic re-evaluation of approaches to addiction and public health education.Secondly, arguments emphasising the rights of those at risk to live free from discrimination and to protect their own health began to emerge.These two approaches, stressing the threat posed by those at high risk of HIV to others, and the dangers and problems faced by those at high risk themselves, mirrored patterns within health writing and reporting which have been identified elsewhere. As described by sociologists of health, these forms of writing situated vulnerable and minority groups as both "at risk" personally, and "a risk" to others. However, the extent to which these groups were seen as either "at risk" or "a risk" varied according to the disease in question. Generally, the language of human rights which coalesced around HIV/AIDS in the 1980s and 1990s was sufficiently powerful to include vulnerable and marginalised groups and to position them as "at risk" first and foremost, whereas in the case of tuberculosis, for example, they remained primarily "a risk" to others.There were concerns that the confidentiality of test results was not respected within prisons, where doctors may have to note HIV status within files that were accessible to all staff. In some prisons, those known or suspected to have HIV or AIDS were placed in separate accommodation, leading to isolation and stigmatisation and acting as a disincentive for testing or for those already diagnosed elsewhere to make themselves known. Health promotion and harm reduction measures within prisons were also criticised; education efforts were often seen as insufficient, condoms only became available in limited circumstances from 1995, treatment for drug addiction was found wanting, the introduction of bleach for cleaning injecting equipment faced numerous stumbling blocks, and needle exchanges were never seriously countenanced.Oxford History of the Prison. "In the early days of recognition of the threat of AIDS", criminologist Norval Morris wrote in his chapter on the contemporary history of the prison, "prisons and jails were seen as likely to be fertile fields for expediting the spread of this plague – and so it has proved".Prisons were highlighted as a location where AIDS might very quickly spread. Internationally, one of the earliest reports on prisons came in 1983 from New York. ‘Many features of prison populations make prisons a favorable setting for the development of the acquired immunodeficiency syndrome’, it concluded. Prisoners were predominantly young men, the population in which AIDS was by far most often seen, and importantly, "[h]omosexual activity during confinement and a past history of illicit drug use are common". Research from the Council of Europe echoed this, explaining that injecting drug users were often in prison and may have "occasional homosexual contacts" there. Sex may occur "even between prisoners who have heterosexual orientation outside prison", meaning that this was not restricted to incarcerated drug users and gay men.The prison was itself seen as a site of contagion, but this was not new to AIDS. John Howard’s investigations into prison conditions in the late eighteenth century had exposed the extent of "gaol fever", usually typhus, which could thrive in the close confines of prisons. In the nineteenth century, concerns over contagion shifted to address the spread of moral corruption within prisons, and the separate system was introduced to keep inmates apart from one another. Efforts to avoid contagion attained an extra layer, as prisoners were isolated from each other as well as their wider communities.It was, however, the impact upon the world outside prison walls that was most frequently cited as a reason for concern about infection. In the case of gaol fever, the fact that it affected  prison warders and court staff became an important stimulus for prison reform.British Medical Journal in 1988, "they may after release act as a source of infection to the community".The debate within the medical community about the provision of condoms to prisoners highlighted the same concern. "If prisoners participate in activities likely to put them at risk of infection with HIV", wrote a genitourinary specialist from Edinburgh in the This moment in the history of AIDS from around 1986 to the end of the decade was, as Matt Cook as suggested, one characterised by suspicion and a sense of "threat and doom", and government campaigns were "inflected by the emotions that they expected from an abstractly conceived 'public'".The Lancet about AIDS and condoms for prisoners was also concerned about sexual violence, believing that young or "mentally retarded" inmates were often sexually exploited by other prisoners. His argument in favour of condoms and conjugal visits was based on familiar ground: the fact that the alternative presented a"potentially significant route of infection into the heterosexual community outside".In this context, the well-being of inmates was rarely of concern, even as arguments were made for education or services that would have the effect of reducing HIV transmission and thereby benefiting their health. Labour MP Harry Cohen raised the question of the liability of the prison service in the event that an inmate was raped and infected with HIV, but as with the arguments above, the point he wished to drive home was that "society will pay", this time in terms of compensation to the injured party as well as through the spread of the virus amongst the "community as a whole".This focus on the impact of the health of prisoners upon the health of society as a whole could, of course, be a useful line of argument to attract the attention of those with little interest or sympathy towards prisoners. Even in articles written by lawyers and doctors who were campaigning on behalf of prisoners, and were preparing to sue the Home Office for failing to provide adequate services to injecting drug users, the first line of attack was that "the uncontrolled spread of HIV infection between prisoners threatens the government’s successful HIV control programme and presents a grave threat to the nation’s health".Overt talk of human rights was becoming increasingly frequent in Britain as the 1990s dawned. Conservative Prime Minister John Major’s ‘Citizen’s Charter’ was launched in 1991, as was a proposed police code of ethics which referred explicitly to human rights, and pressure was beginning to mount over the lack of human rights protections in law. Campaigns calling for civil rights for disabled people hit the headlines in 1992, and eventually led to a Disability Discrimination Act in 1995. A new group, Physicians for Human Rights (UK), was founded in 1989 and their 1992 meeting was dedicated to prison medicine, signalling that connections were being made between pre-existing concerns over the quality of prison healthcare and the language and concepts of human rights.The relevance of human rights to HIV/AIDS was also being discussed more widely both nationally and internationally, by lawyers, ethicists, and campaigners. In July 1989, the first international consultation on AIDS and human rights was organised by what was then the United Nations Centre for Human Rights. A key driving force behind this interest in AIDS amongst human rights groups, as Elizabeth Fee and Manon Parry, have shown, was Jonathan Mann as director of the WHO’s Global Programme on AIDS. The WHO began "ying the control of HIV/AIDS to human rights",At the same time, though, public outputs and debates surrounding HIV, AIDS, and prisons were tailing off. A new drug, AZT, offered some prospect of treatment for AIDS, and the numbers of diagnoses in Britain had not been as high as the worst predictions had foretold. HIV/AIDS was no longer feared on quite the same scale. As historians during the 1990s observed, its metamorphosis into a chronic condition dramatically affected the way in which it was conceptualised and discussed.Assessments and critiques of established prison policies were emerging, particularly in relation to England and Wales. Studies of prevalence in Scotland found very low rates of HIV infection amongst prisoners and attributed this to proactive education and intervention inside and outside prisons; studies of ex-prisoners in England found much higher rates and were correspondingly critical.The question of prisoners’ rights in relation to HIV/AIDS was taken up most forcefully in Britain by the Prison Reform Trust and its Deputy Director, Una Padel. She pointed out that discussions about AIDS in prisons tended to emphasise the risk of spread to the community and not the risk to prisoners themselves. What, she asked, of prisoners’ "basic needs" for safety, medical care, confidentiality, and quality of life? Padel emphasised that prison was an opportunity to reach people who might miss other public health messages, who might have less support and information, and whose own health was at risk, and highlighted the fear and hostility surrounding prisoners with HIV/AIDS, leading to isolation and stigmatisation.Such overt references to prisoners’ rights and needs were rare, however. What did become more common was an explicit acknowledgement, alongside anxiety about unhealthy prisoners infecting the community as a whole, that prisoners should, therefore, receive care and attention in relation to HIV/AIDS themselves. Conservative peer Lord Ferrers reflected this modification in language in 1991, saying that "we continue to be committed to doing all that we can to prevent the transmission of infection within prison, to reduce the risk of prisoners infecting others when they go back into the community and to provide care and support for prisoners whom we know to be infected".The Woolf Report’s references to justice were not confined to the treatment of people with HIV or AIDS. It concluded that the riots of April 1990 with which it was most directly concerned "occurred because three elements that ensure stability in the prisoner service were out of balance: security, control and justice. Our report focuses on the third element, justice, which refers to the prison service’s obligation to treat prisoners with humanity and fairness".Healthcare was also addressed, with emphasis upon providing the same standards of care that were available in the wider community. Following the publication of a report by the Chief Inspector of prisons entitled "Prisoner or Patient?" in 1995, discussion began in earnest to integrate prison healthcare with the NHS.The idea that prison medical care should be equivalent to the care available elsewhere became a fixture of debates over prisons and AIDS. It was in the name of achieving equivalence that the prison medical service was integrated finally and fully with the NHS in 2006, that segregation was eliminated, and that efforts continued to try to introduce facilities for sterilising injecting equipment in prisons and even needle exchange schemes.This can be explained by three interconnected factors, common to many  places with prisons but exacerbated in the British context. Firstly, concerns over healthcare, HIV prevention, and how people with HIV/AIDS were treated in prison were difficult to actually address through the mechanisms of rights because, as Una Padel argued and as had been the case for decades, prisoners had almost no rights that could actually be enforced.Researchers in criminology added further weight to this view, pointing out that a lower quality of care for prisoners had been explicitly endorsed by the courts in 1990.Secondly, people in prison in Britain were perceived as lacking individual responsibility, which was fundamental for the model of human rights which had evolved around HIV/AIDS. As psychiatrist and medical ethicist Dr Timothy Harding wrote in 1987, if prisons were to follow the same strategies that were adopted in the wider community to combat HIV/AIDS, this "implies an approach based on individual responsibility, in which each prisoner is treated as being autonomous and personally responsible for his own health and for the consequences of his behaviour".did lack agency. They pursued legal remedies, for example, altered drug-taking habits, and fashioned home-made prophylactics from whatever was available,The problem went deeper for those in prison. As feminist and postcolonial scholarship has argued, human rights depend (at least in their recent iterations) upon a problematic view of what it means to be human that valorises the autonomous and self-determining individual – the active and independent citizen who makes wise choices in relation to HIV risk behaviours, for example. In the words of Shenila S. Khoja-Moolji, the "acquisition of more rights by individuals is assumed to be the only way to secure development and emancipation. This, however, clearly has consequences for those who are excluded from or made non-existent", by the project of human rights.Thirdly, ideas of rights failed to take root in relation to people in prison because any suffering on their part, whether directly or indirectly associated with HIV, AIDS, or any other health matter, was not perceived. As Lynn Hunt has argued, for human rights talk to emerge, some types of human suffering must come to be seen as unacceptable.As Alison Liebling has argued, Woolf’s concept of justice quickly became confused with ideas of leniency and liberalism and was abandoned. Prisoners’ voices, never heard very clearly, were silenced – or redirected towards "long-winded and cumbersome" official channels.What was more effective, in the end, was the call for people in prison to receive healthcare that was equivalent to that available outside. The idea of equivalence had been influenced by calls to recognise prisoners’ rights, but as a concept, it was imbued with much greater flexibility. It was rhetorically softer, with less insistence upon empathy and a less obvious relationship to the world of (American) litigation and (European) courts of human rights. It permitted the regional and local variations that were found outside prisons and the discretion of governors and health-care managers. Importantly, by referring to healthcare outside prisons it also referred back to the wider community, as so much discussion about prisoners had always done. Whereas talk of rights depicted prisoners as a distinct and demanding collective, talk of equivalence connected prisons to the needs and standards of the wider community.The ways in which debates about HIV/AIDS in prisons were articulated reflected changing ideas about the disease. Prisoners were primarily seen as a risk to others, especially in the 1980s as fears of a heterosexual epidemic were at their height. Calls for prisoners to be recognised as a risk to the wider public drew upon the idea of prisons as sites of contagion. As public panic abated and talk of human rights and health rights became more common in the 1990s, the idea of these rights for those in prison was raised but gained little traction. Health rights could not easily be translated into a setting in which individuals were seen as irresponsible and lacking in autonomy, and this alongside the absence of enforceable rights and a changing political mood in relation to crime, punishment, and prisons in Britain restricted the uses of human rights discourse in relation to prisons.The idea of health rights for prisoners had to be adjusted to accommodate this situation, coming to focus instead on the principle of equivalence of care. This was used to argue for reform, but its adoption as a prison service mantra did not mean that prisoners had the same rights as others to obtain, for example, condoms or sterile injecting equipment. This disrupts any teleological narrative of progress for health rights, and for prisoners’ rights as well. "This is not to say that a health and human rights approach is futile", as Anne-Emmanuelle Birn has affirmed, "only that in order to operate effectively, it must be accompanied by large-scale social justice movements aimed at political change".


Escherichia coli isolated from urinary samples.The majority of studies that link antibiotic usage and resistance focus on simple associations between the resistance against a specific antibiotic and the use of that specific antibiotic. However, the relationship between antibiotic use and resistance is more complex. Here we evaluate selection and co-selection by assessing which antibiotics, including those mainly prescribed for respiratory tract infections, are associated with increased resistance to various antibiotics among E. coli records from urine samples in English primary care  between April 2014 and January 2016 were obtained from the Second Generation Surveillance System. Elastic net regularization was used to evaluate associations between prescribing of different antibiotic groups and resistance against amoxicillin, cephalexin, ciprofloxacin, co-amoxiclav and nitrofurantoin at the clinical commissioning group (CCG) level. England is divided into 209 CCGs, with each NHS practice prolonging to one CCG.Monthly primary care prescribing data were obtained from National Health Service (NHS) Digital. Positive Amoxicillin prescribing (measured in DDD/ 1000 inhabitants / day) was positively associated with amoxicillin  and ciprofloxacin  resistance. In contrast, nitrofurantoin prescribing was associated with lower levels of resistance to amoxicillin . CCGs with higher levels of trimethoprim prescribing also had higher levels of ciprofloxacin resistance .E. coli causing urinary tract infections. Our findings suggest that when predicting the potential impact of interventions on antibiotic resistances it is important to account for use of other antibiotics, including those typically used for other indications.Amoxicillin, which is mainly (and often unnecessarily) prescribed for respiratory tract infections is associated with increased resistance against various antibiotics among  In England, approximately three-quarters of antibiotics are dispensed in primary care . A substHowever, the relationship between antibiotic use and antibiotic resistance is more complex. A particular antibiotic may not only select for resistance against that same antibiotic i.e. ‘selection’, but also for resistance against other antibiotics i.e. ‘co-selection’. There may be cross-resistance between antibiotics, such as observed for ampicillin and amoxicillin . ResistaThe vast majority of studies that link antibiotic usage and resistance at the population level focus on simple associations between the resistance against a specific antibiotic and the use of that specific antibiotic or antibiotic group, or alternatively group all antibiotics together . There iEscherichia coli isolated from urinary samples across these same areas in England, thereby taking into account prescribing of other antibiotics groups. Because we only had data on antibiotic prescribing in primary care, we focused on E. coli sampled from the urinary tract by general practitioners. We used elastic net regularization [We therefore evaluated associations between prescribing levels of antibiotic groups in primary care across geographical areas in England and resistance against amoxicillin, cephalexin, ciprofloxacin, co-amoxiclav and nitrofurantoin, among rization ,16, becarization  and ridgrization –works parization ,16. Thisrization .E. coli infections and uropathogenic E. coli strains are often part of the human intestinal microbiota. Given the systemic nature of systemic antibiotics, this research may shed light on the question whether and to what extent antibiotics typically being used to treat  respiratory tract infections, such as amoxicillin [The vast majority urinary tract infections are caused by xicillin , may resEscherichia coli bacteria sampled from the urinary tract by comparing antibiotic prescribing and resistance in different geographical areas in England. We evaluated variation in prescribing by geographic area in lieu of linked data on antibiotic prescribing and antibiotic resistance at an individual patient level. However, unmeasured confounding may actually be larger at the individual patient level. While patient characteristics that are also prognostic factors of having a resistant infection are influencing decisions to prescribe an antibiotic to an individual patient [The work presented in this paper provides evidence about which antibiotics are associated with higher and lower levels of antibiotic resistance against common antibiotics among  patient ,20, vari patient ,22. In aOur models show that amoxicillin, the most commonly used antibiotic in England and mainly used for respiratory tract infections, is associated with increased resistance against several other antibiotics among bacteria causing urinary tract infections. The methods used in this study, that overcome several of the limitations of previous studies, can be used to explore the complex relationships between antibiotic use and antibiotic resistance in other settings.http://digital.nhs.uk/). Antibiotic groups were created based on the first five characters of the Anatomical Therapeutic Chemical (ATC) classification system    prescribed in the month before the monthly measured resistance prevalence (expressed in DDDs per 1000 persons per day), month of the year, calendar year and the test rate. The test rate was defined as the number of esistant .Antibiotesistant . After pAll elastic net analyses were performed using the ‘glmnet’ package in R version 3.4.3 . To reduIn a secondary analysis we varied the lag time between antibiotic prescribing and the resistances of interest. First, instead of using antibiotic prescribing in the month before the resistance measurement as a potential covariate, we evaluated the association between antibiotics used in 1–3 months before the resistance measurements. Further, we assessed the association between antibiotics used in the year (1–12 months) before the resistance measurements.In addition, we performed a sensitivity analysis, restricting to months with at least 20 measurements to assess the potential influence of potential random error and systematic error due to low sampling rates.Flucloxacillin) [The antibiotic groups that were used most intensively with ≥1 daily defined doses (DDD) per 1000 inhabitants per day, were tetracyclines, penicillins with extended spectrum (mainly amoxicillin) [While we evaluated the association between resistances of interest and all antibiotic groups, E. coli urinary samples from general practice patients sent in for laboratory testing were tested for resistance against nitrofurantoin. The percentages of samples tested for resistance against the other included antibiotics varied between 78% for amoxicillin and 90% for co-amoxiclav.Between April 2014 and January 2016, nearly all  E. coli urinary isolates that were resistant to the antibiotics tested   in the mE. coli samples that tested resistant to nitrofurantoin  (A similar direct association was seen in that CCGs that used more nitrofurantoin had a higher percentage of to 2.24) . The datto 2.24) .However, it should be noted that a substantial proportion of these specific antibiotics are used in the hospital settings, for which no data was available . BesidesThere were also some antibiotics that had negative associations with antibiotic resistances. Nitrofurantoin use was associated with decreased amoxicillin resistance (RR 0.92 95%CI 0.84 to 0.97) . PreviouResults were very similar when restricting the analyses to months with at least 20 measurements  and rarely for urinary tract infections (~2%) , is assoE. coli urinary pathogenic clonal group ST131 are often non-susceptible to both fluoroquinolones and trimethoprim-sulfamethoxazole and/or β-lactam antibiotics [We found that use of amoxicillin and trimethoprim were associated with resistance against ciprofloxacin, which suggests that co-selection may be occurring. Isolates from the common ibiotics ,30, whicibiotics . This liibiotics .7 Nitrofurantoin resistance genes are, in contrast to trimethoprim and amoxicillin resistance genes, not frequently found on mobile genetic elements with multiple resistances or correlated with multiple resistances in other ways [E. coli that are susceptible to amoxicillin and trimethoprim. Collateral sensitivity  has previously been observed for resistance against several antibiotics among E. coli isolates in an experimental setting [E. coli strains [Nitrofurantoin had a negative association with amoxicillin resistance. This is in line with our previous finding that areas with relatively high nitrofurantoin use have lower trimethoprim resistance levels.her ways ,11,31. T setting . Selecti strains , suggestGiven the high fitness cost of nitrofurantoin resistance , the posE coli are unlikely to frequently harbor resistance mechanisms against antibiotics like macrolides, which could make such a synergistic combination therapy particularly effective against Gram-negative bacteria resistant to multiple antibiotics including ciprofloxacin [E. coli strain [The negative association between prescribing of tetracyclines or macrolides and ciprofloxacin resistance is harder to explain. Macrolides are typically active against Gram-positive bacteria, although they can be effective against Gram-negative bacteria when used in combination with antibiotics that do have outer-membrane disruptive activity . Given tfloxacin . Howeveri strain . This maAmoxicillin is the most frequently used antibiotic for respiratory conditions which are responsible for the largest share in inappropriate antibiotic prescribing in primary care –3. BasedIn many countries nitrofurantoin has been adopted as the first-line treatment for uncomplicated urinary tract infections ,34. CompRecent recommendations are to prescribe nitrofurantoin as the first choice treatment for uncomplicated urinary tract infections . We founE. coli.Our findings therefore suggest that a shift towards more nitrofurantoin instead of trimethoprim for uncomplicated urinary tract infections could potentially reduce antibiotic resistance among Patterns of co-resistance and co-selection likely differ between various parts of the world as there is substantial variation in selection pressure by antibiotics and infection prevention and control between countries ,36. HoweThe most obvious limitation of this work is that the associations we found are not necessarily causal. As with any observational study we could not take into account confounding by unmeasured factors, such as antibiotic use in hospitals and other potential selective pressures. Although we only included samples taken from patients presenting in primary care, the unavailability of hospital prescribing data may have especially affected the analyses focusing on co-amoxiclav and cephalexin as less than half of co-amoxiclav prescribing and approximately half of cephalosporin prescribing occurs in the general practice setting . In addiBesides the influence of unmeasured confounding, we cannot exclude the possibility that prescribing differs as a consequence of resistance rather than the other way around, i.e. reverse causation. We tried to reduce such reverse causation by looking at antibiotic prescribing happening before the resistance measurement. Nonetheless, reverse causation may reduce the strength of a positive association or even reverse the association. In addition, some of our results may be partly due to other types of model misspecification.If we were only interested in the influence of one particular antibiotic, a regularization approach that penalizes all coefficients except the antibiotic of interest might provide better estimates . HoweverGiven the limitations described above and the relatively small effect sizes observed, one should be cautious in making policy recommendations based on our observations alone. Nevertheless, our results add to the increasing body of evidence that suggest that co-selection may play a relevant role in driving antibiotic resistance rates.Amoxicillin prescribing is associated with increased resistance to amoxicillin, ciprofloxacin and trimethoprim. Amoxicillin is the most frequently used antibiotic for respiratory conditions, which are responsible for the largest share in inappropriate antibiotic prescribing in primary care. These findings suggest that there is a potential to reduce selective pressure via (co-)selection with unnecessary use of amoxicillin for viral and self-limiting respiratory tract infection.E. coli. The methodology used in this study, that can cope with correlated antibiotic use, can be used in other settings to further explore the complex relationships between antibiotic use and levels of antibiotic resistance.Nitrofurantoin prescribing is associated with lower levels of resistance to amoxicillin and trimethoprim, while trimethoprim prescribing is associated with increased levels of amoxicillin, ciprofloxacin and trimethoprim resistance. This suggests that replacing trimethoprim prescribing with nitrofurantoin prescribing where possible for uncomplicated urinary tract infections may also be associated with a reduction in trimethoprim, amoxicillin and ciprofloxacin resistance among S1 FileE. coli resistant to antibiotics.Figs A-G. Variation in antibiotic prescribing and proportion of urinary samples with (DOCX)Click here for additional data file.S2 FileE. coli urinary samples and antibiotic prescribing.Tables A-J. Associations between antibiotic resistances among (DOCX)Click here for additional data file.S3 File(CSV)Click here for additional data file.


Pharmacists’ knowledge about the clinical and legal aspects of antibiotic supply has an impact on appropriate dispensing practice. There are limited studies evaluating community pharmacists’ knowledge of antibiotic dispensing in low and middle-income countries, including Sri Lanka. We aimed (i) to evaluate community pharmacy staff’s self-reported knowledge about antibiotics and dispensing behaviour of antibiotics without a prescription, and (ii) to identify possible factors impacting their antibiotic dispensing behaviour.A cross-sectional survey was conducted among a random sample (n = 369) of community pharmacies across all nine provinces in Sri Lanka using a self-administered questionnaire on their antibiotic knowledge and dispensing practice. Data were analysed using descriptive and inferential statistics including; t-test, one-way ANOVA or chi-square test, and binary and multiple logistic regression.p = 0.017, specific knowledge about antibiotic resistance (ABR) t(262) = 4.98, p = 0.021 and legal aspects of antibiotic dispensing χ2 = 8.55, p = 0.003) were significantly higher among pharmacists than assistants. One in every three pharmacy staff reported that they dispensed antibiotics without a prescription on patient request; however the proportion was close to half when the patient was known to them. About 30% of the staff reported to have supplied antibiotics for minor infections in the week prior to the survey. However, there was no significant difference in the supply between pharmacists and assistants except for acute sore throat . Those pharmacists with higher ABR knowledge were less likely to give out antibiotics without a prescription for viral infections in adults  and children . Awareness of legal aspects of antibiotic supply reduced overall dispensing , and specifically for bacterial infections in adults . Knowledge about antibiotic use and misuse reduced the likelihood of illegal dispensing for common cold  and acute diarrhoea .A total of 265 pharmacy staff (210 (79%) pharmacists and 55 (21%) assistants) responded. Overall mean antibiotic knowledge score was 26.1 . The overall mean knowledge score t(263) = 2.41, Despite the law prohibiting provision, antibiotic dispensing without a prescription continues in community pharmacies in Sri Lanka. Appropriate antibiotic dispensing was associated with high levels of pharmacists’ legal and clinical knowledge about antibiotics. Strategies to change the current practice are urgently needed. Antibiotic resistance (ABR) is growing at an alarming rate and poses a major threat to the clinical efficacy of antibiotics . ABR incABR is an inescapable consequence of antibiotic use , but it Although knowledge and professional competency of community pharmacists in antibiotic dispensing are vital to ensure appropriate antibiotic supply to the community, limited studies (and none in Sri Lanka) have evaluated these characteristics in LMICs where antibiotic supply without a prescription is high. Therefore, this study aimed to investigate the antibiotic dispensing behaviour of Sri Lankan community pharmacies. A national survey was conducted to (i) evaluate community pharmacy staff’s self-reported antibiotics knowledge and dispensing behaviour (without a prescription), and (ii) identify possible factors impacting such antibiotic dispensing behaviour.A cross-sectional study was conducted using a validated, self-administered and structured questionnaire among community pharmacy staffs throughout Sri Lanka between December 2016 and September 2017.A systematic sampling of community pharmacies in Sri Lanka, comprising of all types of community pharmacies in the country, including semi-government  and private pharmacies  covering all nine provinces of the country was conducted. The study sample was achieved using a proportionate sampling technique in each province, as described below and elsewhere .The original sample size (n = 369) was estimated using standard error of proportions equation with a 95% confidence interval using margin of random error of ±5% based on the results of a previous pilot study conducted in Sri Lanka measuring knowledge about antibiotics among community pharmacists. Sample size estimation has already been reported in detail elsewhere . The resPharmacies for this survey were randomly selected from all nine provinces of the country (sampling frame). A simple random sampling technique was used in the sample selection. The proportionate sample of pharmacies for each province was derived from the list of pharmacies from the Pharmacy Directory maintained by the National Medicine Regulatory Authority (NMRA), which includes all pharmacies that had legal permission to function (as at April 2016) .The selected community pharmacies in each province were visited by trained research assistants who were either pharmacy undergraduates or recent graduates.The questionnaire was distributed to either consented community pharmacist or pharmacy assistant (in the absence of a pharmacist at the time of the visit) in their preferred language . The most senior pharmacy assistant was approached in the latter case. Community pharmacy staff either completed the questionnaire on the spot or asked the research assistant to collect it at a mutually agreed time and a day. A reminder was made over the phone a day before the agreed collection day. If the questionnaire was not ready on the agreed day, respondents were given an addressed envelope with the postal charges paid to post it to us after filling.This study was approved by the Ethics Review Committee, Faculty of Medicine, University of Ruhuna, Sri Lanka (Reference number 16.11.2016:3.1). Informed written consent was obtained from all participants for the self-reported survey after explaining about the study and its objectives. The consenting pharmacy staff were asked to complete the structured questionnaire described below. The questionnaire was anonymous with no personal identifiable information collected.The structured questionnaire  used in The face and content validity of the questionnaire was assessed using a panel of six experts from Australia and Sri Lanka, including two researchers, a pharmacologist, a clinical psychologist, a clinician and a pharmacist. The reliability, clarity, comprehension and time of completion of the modified questionnaire were assessed among ten purposely selected community pharmacists and who were excluded from the final survey. The initial questionnaire was developed in English and translated into the two primary languages spoken in Sri Lanka using forward-backwards and reconciliation process , 20.Socio-demographic and professional characteristics; including age, gender, geographical location, level of pharmacy education, years of working experience in the community pharmacy settings, employment type, employment status, type of pharmacy, and number of pharmacists and pharmacy assistants working at a given time in the pharmacy, were included.Knowledge was assessed using four sub-groups (concepts) including knowledge about antibiotics, ABR, use/misuse of antibiotics and legal aspects of antibiotics dispensing. Each sub-group had statements to measure the concept with “Yes”, “No” and “Unsure” response options. Knowledge about antibiotics was measured using three statements. For each statement a score of one was assigned to a correct response, and these scores were summed to get an overall score (0–3). Since the index score of this variable was normally distributed, this variable was treated as a continuous variable to predict antibiotic dispensing behaviours of pharmacists , 21. KnoAntibiotic dispensing behaviour was assessed using the following six statements: (1) I dispense antibiotics without a prescription if a patient requests it; (2) I give antibiotics without a prescription for a child with minor viral infections; (3) I give antibiotics without a prescription for an adult with minor viral infections; (4) I give antibiotics without a prescription for a child with minor bacterial infections; (5) I give antibiotics without a prescription for an adult with minor bacterial infections; (6) If I know the patient, I dispense antibiotic without a prescription on patient’s request. Each of these items had five response options on a Likert scale: (1) never, (2) some time, (3) half of the time, (4) most of the time, and (5) always.Additionally, self-reported rates of antibiotic supply without a prescription in the previous week by pharmacy staff for specific minor infections; including acute sore throat, common cold, wound infections, UTI and diarrhoea were also assessed using a Likert scale with six points .Pharmacist: Those who reported possessing a degree in pharmacy, proficiency or diploma in Pharmacy, efficiency (apprentice pharmacy training) qualification were categorised as pharmacists and the rest were considered as pharmacy assistants .v. Definition of antibiotics: This item was created based on responses of two statements asked from the respondents: (1) Antibiotic is an agent used to kill or inhibit the growth of microorganisms . (2) Antibiotic is an agent to kill or inhibit the growth of bacteria. Both statements had three response options: (1) Yes, (2) No, (3) Unsure. Respondents were considered having knowledge about the definition of antibiotic if they gave the correct responses to both statements.Completed questionnaires were coded, reviewed for accuracy, entered into a database in the SPSS , and analysed using descriptive and inferential statistics. All categorical variables, including respondents’ socio-demographic and professional characteristics and antibiotic dispensing practices, were expressed as frequencies and percentages. The knowledge scores were calculated as the mean (±SD) or frequency (%). The influence of respondents’ professional and demographic factors on knowledge were tested using t-test, one-way ANOVA or chi-square test. Binary logistic regressions and multiple logistic regressions were performed to examine the association of knowledge about antibiotics, and socio-demographic and professional characteristics on reported antibiotic dispensing practice .All the predictors adjusted in the multiple regression models were entered simultaneously to predict the unique contribution of the variables to the outcomes of interest. The predictors adjusted in the models including gender , geographical area , age , years of community pharmacy experience (continuous), employment type , employment status , knowledge about antibiotics , knowledge about ABR , knowledge about antibiotic use/ misuse  and knowledge about legal aspect of antibiotic use  were entered into the models.p-values presented are two tailed and level of significance was set at p < .05.Odds ratios (OR), adjusted odds ratios (Adj. OR) and 95% confidence intervals (CI) were calculated where appropriate. All In total, 267 pharmacy staff (pharmacists and pharmacy assistants) were surveyed  and 265 participants were included in the final analyses as two questionnaires were incomplete. Of the participants included in this study, 21% (n = 55) were categorised as pharmacy assistants and 79% (n = 210) as pharmacists.2  = 26.85, p < 0.001). About two-thirds of pharmacy staff were male , aged between 30–49 years  and with more than five years of community pharmacy work experience . More than two-thirds of community pharmacies were in urban (71.3%) areas. Only about 2% of community pharmacists had a formal university degree in pharmacy and over half of the (53%) pharmacists had an efficiency (apprentice) pharmacy training.p = 0.021. Furthermore, a significantly higher proportion of pharmacists were aware of the legal aspects of antibiotic dispensing compared to the assistants  = 8.55, p = 0.003)  . Howeverp>0.05) in general. However, a significantly higher proportion of the pharmacy assistants reported having dispensed antibiotics without a prescription compared to the pharmacists in the past seven days for sore throat  = 4.20, p = 0.040).As p = .027) and children . Knowledge of the legal aspects of antibiotic dispensing also significantly decreased the likelihood of non-prescription antibiotic dispensing for unspecified patients , the patients known to the pharmacist  requesting an antibiotic and adults with bacterial infections   .p = .015)  . All othp = .011) and acute diarrhoea . However, other knowledge items and demographic characteristics such as geographical location of the pharmacy, years of community pharmacy experience, employment status and employment type, once adjusted in the regression model were not significantly associated with antibiotic dispensing practice without a prescription for the minor infections.As To the best of our knowledge, this is the first nationwide study from Sri Lanka evaluating the self-reported antibiotic dispensing practice of community pharmacy staff (pharmacists and pharmacy assistants) and the impact of pharmacists’ knowledge and professional and demographic characteristics on antibiotic dispensing.This study, demonstrated that whilst pharmacists showed a greater knowledge about ABR and legal aspects of antibiotic dispensing than the pharmacy assistants, they had an overall poor knowledge of antibiotics (definition), appropriate antibiotic use and antibiotic resistance. This poor knowledge was reflected in the self-reported behaviour, with a large proportion of participants providing antibiotics to patients on request, and inappropriately in situations where patients may have had viral infections. The study findings also demonstrated that pharmacists who reported knowing about the legal requirements for supplying antibiotics with a prescription and who were knowledgeable about ABR and the use/ misuse of antibiotics, were less likely to dispense antibiotics without a prescription.et.al, in 2008, found that around 24% of Thai pharmacists had inadequate knowledge about antibiotics [Our findings regarding poor knowledge about antibiotics are similar to findings from other studies conducted in LMICs –14, 22. ibiotics . The knoibiotics . Thus, libiotics , 24, 25.Targeted education of practising pharmacists and other staff may also specifically address other issues highlighted in this study; for example, provision of antibiotics more readily to patients known to the pharmacy staff. Familiarity with the patient appeared to be a reason why antibiotics were more likely to be provided without a prescription, with no significant difference observed between pharmacists and assistants. These findings are consistent with similar studies conducted to evaluate the inappropriate dispensing practice in LMICs , 27. It In addition to inadequate clinical experience and knowledge about antibiotic dispensing contributing to the illegal and inappropriate antibiotic supply reported in this study, there may be other reasons explaining the behaviour of the pharmacists and pharmacy assistants. For example, pressure from the public to supply an antibiotic, the profit motive, disregard for their legal obligations related to antibiotic supply, lax law enforcement, lack of awareness about existing antibiotic dispensing laws and fear of losing customers .However, our present study also revealed that having pharmacists with a better knowledge of ABR and use/ misuse could reduce antibiotic supply without prescription for viral infections (appropriate practice); and better knowledge related to dispensing regulations may reduce antibiotics supply without a prescription  to both known and unknown patients and for bacterial infections. Recent literature from other countries has also found similar relationships , 14, 28.However, the control of ABR cannot be the sole responsibility of health professionals and scientists. The public and other stakeholders have a major role to play as well. Public awareness should be encouraged to minimise patient demand for antibiotics and optimise appropriate use. A survey of parents of school children in Italy found that about 10% and 21% correctly knew the definition of antibiotics and ABR, respectively, and one in every third person surveyed was willing to self-medicate with an antibiotic . InterneThe government should consider ABR as a major public health issue. Policies and regulations should be put in place to enforce appropriate access, minimise the public’s demand from health professionals and reduce inappropriate use of antibiotics. The media professionals should also be adequately trained on how to convey medical and scientific information in lay language to inform the populations on practices that promote the appropriate use of antibiotics. The empirical evidence may facilitate the development of educational and behavioural interventions, dispensing guides, and strengthening the policies which can be undertaken to combat against this public health issue.Like most surveys, our study also has some limitations. There is a possibility of social desirability bias on the antibiotic dispensing practice-related issues, where the respondents may give more favourable responses about their antibiotic dispensing practice . HoweverApproximately 20–30 minutes was taken to complete the questionnaire, which could have impacted the responses through survey fatigue .Another possible limitation of this study is non response bias caused by those who did not respond. However, in our study, the response rate was very high (72%), when compared to similar research  with resDespite Sri Lankan law prohibiting provision of antibiotics to the public without a prescription, this study revealed that pharmacy staff continue to provide antibiotics without a prescription. Pharmacists did not report an overall lower provision rate. The illegal and inappropriate antibiotics supply was associated with lower levels of pharmacists’ legal and clinical knowledge about antibiotics.There is room for improvement in community pharmacy staff’s basic knowledge about antibiotics, antibiotic resistance, antibiotic use and misuse. It is important that the government strictly enforces the law regarding antibiotics supply. Furthermore, standards should be implemented and adopted on the appropriate provision and supply of antibiotics. Educational programmes and interventions should be developed and implemented to ensure that both newly qualifying pharmacists as well as currently practicing pharmacists in the community settings are well-informed about antibiotics, appropriate use and antibiotic resistance, as well as the legal requirements for supply.S1 Appendix(PDF)Click here for additional data file.


In silico formulation optimization of the cubic phases was done, and the corresponding formulations were prepared and characterized. The optimized formulations were screened based on the stable microstructure and proper fluidity. Highly efficient mouse skin permeability of Met was found with the cubic phases compared to Met solutions. High antimelanoma effect of transdermal Met-loaded cubic phases also was shown by the significant decrease of tumor volume and the improvement of melanoma cell apoptosis on the B16 melanoma mice. Met-loaded cubic phases are a promising topically applied medication for local therapies of melanoma.Metformin hydrochloride (Met) is commonly used for antidiabetic therapy though its antimelanoma action is also reported. Conventional oral administration method of Met is not appropriate for therapy of melanoma because of large dose, adverse reactions, and low efficiency. Here, a transdermal Met-loaded cubic phase was developed for local treatment of melanoma.  Malignant melanoma is one of the most aggressive forms of skin cancers, which is characterized by early metastasis, rapid development, poor prognosis, and high mortality  is a drug commonly used for the treatment of type-2 diabetes. Moreover, numerous studies have demonstrated that Met exhibits anti-melanoma activity. The anti-melanoma effect of Met is mediated through p21- and AMPK-independent cell cycle arrest . GMO was supplied by Dansico Co., Ltd. . All other materials were of pharmaceutical or analytical grade.Male Balb/c mice  were supplied by the SPF Co., Ltd. . All animal handling and surgical procedures were conducted in strict accordance with the NIH Guidelines for the Care and use of Laboratory Animals.The cubic phase was transparent but stiff and viscous which limited its potential use as the delivery system  and dissipative particle dynamics (DPD) simulations. All simulations were performed in Materials Studio 7.0 .The basic components of Met-loaded cubic phases include Met, GMO, water, and ethanol. These molecules were put into the simulated system in our previous research . Whereafter, MD simulations were performed in order to obtain the Flory-Huggins parameters (χ) of binary components in all formulations . The structures of the samples were determined in a small-angle X-ray scattering instrument  method referred to other reports  side facing toward the donor cell. A Met solution or a Met-loaded cubic phase (sample F4) was put into the donor cell, which directly contacted the surface of SC. The administered dose was 1.5 g, containing 71.4 mg of Met and 85.5 mg of ethanol. At predetermined time points , 1 ml of saline was withdrawn from the receptor cell, and the same volume of fresh saline was injected into the receptor cell so as to reach the sink condition. The withdrawn samples were measured with the HPLC method illustrated as the reference  was calculated according to the V (ml) is total volume of saline in the receptor cell, Cn (μg/ml) is the concentration of Sample n, Ci (μg/ml) is the concentration of Sample i, V0 (ml) is the volume of withdrawn saline and A (cm2) is the effective diffusion area. The nQ was plotted as a function of time.The dorsal skins of Balb/c mice were obtained after sacrifice. The vertical Franz diffusion cells with an effective diffusion area of 0.785 cm−2·h−1) and lag time , respectively. The activity of cubic phases was expressed as enhancement ratio (ER) that was the ratio of Q5h in the cubic phase group to that in the solution group.The slop and intercept of the linear portion of the profile were presented as . The xenograft volume (V) is measured with a caliper and calculated as 0.5 L × W2. where L is the largest superficial diameter and W is the smallest superficial diameter of the xenograft. The tumor sizes were measured and recorded every day. The formulations were initially applied to the mice at the tumor sites topically once daily after the tumor size reached about 50–100 mm3. The melanoma-bearing mice were divided to four groups of six mice in each group. Group I was composed of tumor-bearing mice without treatment and served as the control. The other groups included the tumor-bearing mice that were treated with the Met solutions (Group II), the blank cubic phase (Group III) and the Met cubic phase (Group IV). The mice were sacrificed after 10 days. The tumors were dissected and weighed to calculate the tumor inhibitory rates, which were defined as (Wblank − Wtest)/Wblank×100%, where Wblank and Wtest were the average tumor weights of the control group and the therapeutic groups, respectively.Every Balb/C mouse was subcutaneously injected into the hip using the B16 cell suspension  followed by Student–Newman–Keuls test. The value of p < .05 was considered significant.The statistical significance of the differences was analyzed by Supplementary Equation (1), because it is impossible for fragment B to form the hydrogen bonding with other molecules due to nonpolar structure. As listed in Supplementary Table 1, the estimated χB/H2O=5.1 indicated fragment B was hydrophobic which made up the liquid lyophobic region of surfactant bilayer in the liquid crystal systems below. The estimated χB/ethanol = 0.5 suggested fragment B/ethanol was weakly miscible. In contrast, all other components can form mutually hydrogen bindings. Therefore, their values of χ were calculated by adopting Supplementary Equations (2) and (3), respectively. The χH2O/A was predicted to be −1.1, suggesting fragment A was insoluble in H2O but there was a certain amount of attraction force between them. With the number of molecules ratio of fragment A/ethanol decreased, the predicted Flory–Huggins parameters were more close to 0 (Supplementary Tables 2–6), indicating fragment A could be soluble in a certain amount of ethanol. The absolutes of χMet/ethanol decreased as the increase in ethanol content (Supplementary Tables 2–6), suggesting Met/ethanol could be easily miscible with the increasing of ethanol content.In the study, MD stimulations were carried out to estimate the Flory–Huggins parameter χ. The Flory–Huggins parameters (χ) between fragment B and other components were predicted by Six samples, labeled as F1, F2, F3, F4, F5, and F6, were selected to build the internal structure of them using DPD stimulations. The calculated ‘dot-and-line’ models (series 1) and isodensity profiles of the water particles (series 2) for liquid crystal systems were shown in The water diffusivity increased linearly with the increase of ethanol concentration , suggestThe surface tension between the liquid channels and the water channels was obtained from the various liquid crystal systems . The surIn conclusion, the former four formulations were cubic phases, and sample F4 exhibited the proper viscosity among them. Thus, the sample F4 were finally selected as the optimized formulation.The images of F1, F2, F3, and F4 just displayed dark background under PLM . All the6：8 , which s6：8 . Further6：8 . The lat6：8 , respect6：8 . The SAX6：8 , whose c6：8  showed tThe gels of F1, F2, F3, and F4 were homogeneous and transparent, where their flowing ability increased in turn. The F5 and F6 gels had better flowing ability than the F4 gel. However, the former two gels were not cubic phases, but lamellar gels. Therefore, the F4 gel was finally selected after considering the stable microstructure and proper fluidity. Furthermore, the computing technologies are extremely reliable for optimizing the formulations of the cubic phases.Supplementary Table 7, there was significant difference on the cumulative amount per unit area within 5 hours between the cubic phase (Qn = 6088 ± 663 μg/cm2) and the solution . In contrast to the Met solution (Jss=936.8 ± 121.5 μg·cm−2·h −1), the steady-state flux of the cubic phase (Jss=1259.6 ± 312.6 μg·cm−2·h −1) was obviously high. These results demonstrated the high Met transdermal improvement of cubic phases. Here, in our previous research, we explored the transdermal delivery mechanisms of cubic phases for Met with in silico and in vitro methods. Firstly, Met mainly diffuses from the water channels of cubic phases to the surface of stratum corneum. Next, GMO highly enhances Met permeation through skin by disturbing the interaction between Met and skin proteins, and disordering the arrangement of skin lipid molecules  and the control Group I, . The resin vivo, we performed tunnel assays on tumor sections from treated mice. A large positive tunnel staining area was observed in Group IV, while few areas were observed in other groups (To determine whether the pro-apoptotic effects of the Met cubic phases on melanoma cells occur r groups . These rr groups . Moreover groups . In addir groups . Therefoin silico modeling to optimize the Met formulation. The experimental methods demonstrated this technique was highly accurate. Most importantly, the Met cubic phases led to the reduction in the size of the melanoma due to the high skin permeation and cytotoxicity. Taken together, our work provides an alternative strategy in optimizing the formulations of drug delivery systems. Cubic phases are an ideal transdermal delivery system of Met as potential therapeutics for the clinical treatment of melanoma.Cubic phases are very popular transdermal delivery systems. Moreover, Met has recently been proved to have high therapeutic efficiency of antimelanoma and good skin permeation. Therefore, the novel contribution of this study was the optimization and the fabrication of a Met cubic phase for local therapy of melanoma. The GMO, the matrix for cubic phases, possess a rich array of temperature and composition sensitive behavior (Reese et al.,


The aim of this study was to investigate two enkephalin-degrading enzymes, aminopeptidase N (APN/CD13) and endopeptidase (NEP/CD10), gene and protein expression levels in sperm samples of fertile and heroin-addicted men, and the correlation between their expressions and semen quality.In this case-controlled study, semen was collected from 24 normozoospermic healthy(as a control group) and 24 heroin-addicted men donors (as case or addiction group). Sperm cells isolated by CookMedical gradient (40-80%) and followed up by swim-up techniques were used for real-time quantitative polymer-ase chain reaction (qPCR) and flow cytometry techniques to assess APN/CD13 and NEP/CD10 genes and proteinssubsequently. Semen parameters were analyzed by computer-assisted sperm analysis.The findings revealed that there were significant differences in sperm total motility , progressive motility  and viability  in the addicted group vs. control ones. APN and NEP gene expression levels in the addictedgroup decreased compared with the control ones . Flow cytometry analysis showed that the average percent of APN/CD13 in heroin consumerssignificantly decreased compared with the healthy ones, while NEP/CD10 rate between two groups was similar. Wealso observed that duration of drug dependence is correlated with sperm viability  and motility, NEP , and APN  gene expression levels.We conclude that semen quality and enkephalin-degrading enzymes were altered in heroin-addicted men.other confirming the internal validity of our estimates. Infertility is one of the most serious social problems thathas an effect on a huge percentage of couples. In general,the basic cause of infertility refers to the male partnerin approximately 40-45% of cases . In someThe activity of the opioid system is mediated byendogenous opioid peptides (EOPs). EOPs or opiatealkaloids carry out their function through three typesof opioid receptors  on membranes , 9. EOPsThe effect of opiates on spermatozoa in some aspectsis still unknown or controversial. Some studies havebeen performed on the effect of certain drug abuse onthe human and mouse sperm parameters -15. In oSperm motility is considered a key functional parameterthat controls reproduction  and is wSince decreased motility (asthenozoospermia) is acommon abnormality among opiate drug addicts ,it is lIn this case-control study, participants were interviewedafter written informed consent. The data on personalinformation ,history of addiction , and medical status  were obtained via a structuredquestionnaire.Based on the medical files and questionnaires, twentyfour 20-50-year-old men with normal body mass index(BMI) who just used heroin for at least 12 months-without using other drugs during that interval- wereselected as a case or heroin-addicted group. They wereintroduced from addiction treatment centers beforeentering treatment programs and should have met theDiagnostic and Statistical Manual of Mental Disorders(DSM-V) criteria for addiction.Also, 24 age-BMI-matched men with a normal semenanalysis according to World Health Organization (WHO)2010 criteria volunteered to participate in this study as acontrol group. They were male partners of married coupleswithout any illicit drug use who had attended the ShahidAkbar-Abadi Obstetrics and Gynecology Hospital of theIran University of Medical Science for female infertilityconsultationSubjects with medical problems known to be associatedwith subfertility in both groups, illicit-drug usersin controlgroup, and individuals that started treatment with otherdrugs in addicted ones were excluded from the study.Semen samples were obtained from donors bymasturbation after 2 to 3 days of abstinence into sterilecontainers and allowed to liquefy at 37˚C for 30 minutesbefore processing. Semen volume, as well as spermconcentration, viability (by eosin B staining), morphology(by Papanicolaou staining method), and motility weremeasured in each sample by CASA .For eosin B staining , 20 microlitersthe sperm suspension was mixed with 7 µl eosin andobserved under a light microscope (×400 magnification).Then, 200 sperm were counted and the percentage of livespermatozoa was recorded. With the staining, red spermheads were considered as dead , 24.Sperm morphology was assayed by the Papanicolaoumethod. First, smears were prepared and stained withthe Papanicolaou method based on the protocol. Then,the morphology of 200 spermatozoa was surveyed under ×1000 oil immersion lens. With the staining, thenuclei turn blue, and the acrosome and tail become pink.Abnormal morphology wasreported in five fields of visionrandomly, and the percentage of abnormal morphologywas recorded . Before RNA extraction or flow cytometry, spermatozoawere isolated from semen on cook medical gradient(40-80%), followed by a swim-up in washing mediumsupplemented by Albumin 10% to recover motile cellswithout any contaminant leukocytes or other debris,and then they were visually examined under a lightmicroscope. All primers  were desThe RNA of spermatozoa was isolated with theRNeasy Mini kit . First strand cDNAsynthesis was carried out using QuantiNova ReverseTranscription Kit . Then real-timequantitative polymerase chain reaction (qPCR) wasperformed using QuantiNova SYBR Green PCR Kit. β-actin) and compared with a calibrator(control group) by the comparative Ct method (2-∆∆ct)  values were averaged. Expression valueswere normalized to the average expression of the housekeeping gene ((2-∆∆ct) .6sperm in 2 ml phosphate bufferedsaline  was centrifuged in 4˚C for 10minutes (×300 g). The pellet was suspended in 100 µlwashing medium and then was added 20 µl antibody [PEMouse Anti-Human CD13 and PE Mouse Anti-HumanCD10 (BD Biosciences USA)]. Samples were incubatedat 4˚C for 1 hour. Next, sperm was centrifuged three timesat 4˚C for 10 minutes (×300 g). Finally, the pellet wassuspended in 300 µl PBS and analyzed using the flowcytometer.Flow cytometry was performed on a BD BiosciencesFACSCalibur in order to rate of presences of surfaceexpression of enkephalinase and aminopeptidase onsperm cells. Sperm was stained directly for APN andNEP. Briefly, 1×10Statistical analysis was done with statistical software. The normal distributionwas evaluated with the Kolmogorov-Smirnov test.The results were analyzed by performing independentsamples t test and Mann-Whitney U test. P≤0.05 wasconsidered as statistically significant and mean ± SE wasalso calculated for each variable. The partial correlationand multiple regression analyses were done between APNand NEP levels and other parameters. This study has been approved by the medical EthicsCommittee of Iran University of Medical Science (code:IR. IUMS.rec.1394.9211313202). All human trails werecarried out in accordance with the Declaration of Helsinkiguidelines.2)was significantly lower than the healthy ones (28.69± 0.91 kg/m2) (P≤0.01). All subjects in the addictedgroup were smoker, so there was a statistical differencebetween groups in this case (P≤0 .01).Twenty-four healthy and twenty-four men addictedto heroin participated in the study. The mean agesin addicted and control groups were 35.44 ± 1.3years and 33.91 ± 1.79 respectively, which there wasno significant difference in average age. Althoughthe range of BMI was normal in both groups, thisparameter in the addicted men decreased compared with the control ones  (P≤0.01)As shown in the Figure 1 shows flow cytometry analysis of cell surfaceprotein enkephalinase and aminopeptidase in sperm ofhealthy and addicted men. The results demonstrated thatthe mean APN/CD13 rate in the control group (63 ± 12)was significantly higher than addicted ones (24 ± 12)(P≤0.05), while NEP/CD10 ratio in the control (4.3 ± 1)and addicted (3.7 ± 2) group did not show a significantdifference.APN  gene expressionlevels were significantly negative correlation withduration of heroin dependence  and sperm viability and motilitywere reduced in men addicted to heroin. We also showedthat there was a significant negative correlation betweenInfertility is one of the most important problems ofhuman societies throughout the world. Considering therole of recreational heroin consumption as an idiopathicetiology of male infertility and increasing consumptionof illicit drugs, especially among young people ofreproductive age, socio-medical studies on this issuehas been done less yet. The living conditions, lack ofcooperation, simultaneous use of various drugs and legaland ethical problems in sampling in addicted peoplemake the research difficult and complicated in this area.Therefore, research in this area can be very valuable.Our findings suggest a remarkable association amongheroin addiction, asthenozoospermia and decreased APNand NEP mRNA levels. In addition, the duration of drugdependence is one of the main factors contributing to thedetrimental effects of heroin on impaired male fertility.Decreased in heroin users BMI may be caused by caloricmalnutrition , inhibitIn this study, reduced sperm motility was observed inaddicted men. Our finding is in line with other studieswho mentioned that opioids such as heroin, kerack, andmorphine can impair sperm parameters in mice and human.They also showed those alterations were dose-dependent, 16, 17.APN gene andsubsequently, its protein in heroin consumers may resultin opioid accumulation in the semen microenvironment.Indeed, the occupation of receptors and degradingenzymes deficiency or inactivity may affect the spermquality and male fertility. Agirregoitia et al. heroin consumers usually take various addiction andnarcotic drugs like opiate, kerack or methamphetamines,(b) the majority of addicted men had no trends to providesemen. Hence, we selected persons who used only heroinfor at least 12 months -without using other drugs, ii. Inaddition; cigarette smoking was a major interferencevariable, which was controlled by partial correlation, iii.Scio-economic and family backgrounds were differentamong the participants. For example, diet may affectsperm parameters, and iv. We could not follow up thefertility status after treatment.NEP and APN gene expression levels with theduration of heroin dependence. This study may increaseour understanding of the effects of drugs and toxins onmale infertility.We conclude that semen quality and enkephalindegrading enzymes were decreased in heroin-addictedmen and there is a significant negative correlationbetween


Purpose Approximately 3% of lung cancer bears mutations leading to MET exon 14 skipping, an oncogenic driver which is further evidenced by case reports of patient response to MET kinase inhibitor treatment. Approximately 15% of tumors harboring MET exon14 skipping have concurrent MET amplification. Experimental Design Merestinib is a type II MET kinase inhibitor. Emibetuzumab, a bivalent anti-MET antibody, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET exon14 skipping and MET amplification. Results Merestinib inhibited Hs746t cell proliferation (IC50=34 nM) and totally eliminated pMET at 100nM. Emibetuzumab showed little anti-proliferative activity against Hs746t cells (IC50>100nM), did not reduce pMET, and slightly reduced cell surface MET. In the Hs746t xenograft model, dose dependent differences in durability of response were seen with merestinib including durable tumor regression (91.8%) at 12 mg/kg qd. Emibetuzumab treatment (10mg/kg qw) provided transient tumor regression (37.7%), but tumors re-grew while on treatment. Concurrent combination of merestinib (6 mg/kg qd) and emibetuzumab resulted in 85% tumor regression, while a sequential combination (initiating merestinib first) resulted in longer duration of treatment response. Conclusions Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). As a type II MET kinase inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or to patients who progress on type I MET inhibitor treatment. Data also support clinical evaluation of the sequential combination of merestinib with emibetuzumab when patients progress on single agent merestinib.The online version of this article (10.1007/s10637-017-0545-x) contains supplementary material, which is available to authorized users. MET amplification or gain-of-function mutation in MET are the various mechanisms for which MET pathway dysregulation is implicated in cancer development and progression. Mutations that lead to the skipping of exon 14 in MET have been detected in several cancers and are most prevalent in lung cancer at an incidence of about 3% in both Western and Asian countries . Crizotinib was formulated as a solution in 10% acacia/0.05% antifoam. Emibetuzumab was diluted in PBS. Animals were removed from the study when tumor burden exceeded 2000 mm3.All animal studies were performed in accordance with the International Association for Assessment and Accreditation of Laboratory Animal Care institutional guidelines. All in vivo experimental protocols were approved by the Eli Lilly and Company Animal Care and Use Committee. Female athymic nude mice were obtained from Envigo . Hs746t cells were expanded in culture, harvested, and washed in HBSS. Approximately 5 × 10Tumor volumes and body weight were measured bi-weekly. Statistical analysis was performed on the last study day when no more than half of the animals in the vehicle group were removed from the study due to tumor burden.Tumor volume was transformed to the log scale to equalize variance across time and treatment groups. The log volume data were analyzed with a two-way repeated measures analysis of variance by time and treatment using the MIXED procedures in SAS software (Version 9.3).The correlation model for the repeated measures was spatial power. Treated groups were compared to the control group at each time point. The MIXED procedure was also used separately for each treatment group to calculate adjusted means and standard errors at each time point. Both analyses account for the autocorrelation within each animal and the loss of data that occurred when animals were removed or lost before the end of the study. The adjusted means and standard errors were plotted for each treatment group versus time.50 = 34 nM [50 of 33.4 nM. The anti-proliferative effect of several MET kinase inhibitors was compared and IC50 values were found to be similar to merestinib   Fig. . ReductiMET-targeting kinase inhibitors, both highly specific PF04217903) and non-specific  totally inhibited pMET in Hs746t cells, leading to a reduction of pAKT, pERK   after 21 days of dosing (Day 32). As expected, merestinib administered at a suboptimal dose of 6 mg/kg once daily (insufficient to cover the MET target), resulted in only transient tumor regression, followed by tumor re-growth while on treatment. After 21 days of dosing, the T/C was 18.3% (p = 0.033) as compared to the vehicle group. Crizotinib treatment, at 25 mg/kg once daily, resulted in 92.9% tumor regression (p < 0.001) after 21 days of dosing. There was no significant difference in the antitumor effect of merestinib (12 mg/kg dose) and crizotinib (p = 0.76). Emibetuzumab (10 mg/kg once weekly dosing) showed delayed tumor regression relative to either single agent kinase inhibitor. Tumor regression was transient and tumors re-grew while on treatment. Tumor regression was 37.7% in the emibetuzumab group (p < 0.001) at dosing completion (Day 32)  . Thus, statistical analysis of the anti-tumor effect of the treatments was performed on study day 27, although the dosing period continued through day 41. After 14 days of dosing, merestinib (6 mg/kg once daily) resulted in significant anti-tumor effect with T/C of 11.8% as compared to vehicle group (p < 0.001). As a single agent, emibetuzumab treatment (10 mg/kg once weekly) resulted in 38.6% tumor regression (p < 0.001). Concurrent combination of merestinib (6 mg/kg once daily) and emibetuzumab (10 mg/kg once weekly) resulted in 83.1% tumor regression (p < 0.001) and the combination was well tolerated. Single agent merestinib (12 mg/kg once daily) was shown previously   was compared to the concurrent combination of merestinib (6 mg/kg once daily) with emibetuzumab (10 mg/kg once weekly) Fig. b. In theMET exon14 skipping and MET amplification. The response was comparable to that of the type I MET inhibitor, crizotinib, dosed at 25 mg/kg once daily in this same model. The observed in vivo anti-tumor effect of merestinib and crizotinib is consistent with the potent in vitro anti-proliferative effect, inhibition of pMET and downstream signaling of the activated MET pathway in Hs746t cells. No evidence of acquired tumor resistance (tumor re-growth) to either single agent merestinib (12 mg/kg) or crizotinib was seen after 64 days of treatment. Treatment response in both groups was also durable, as no tumor growth was observed in mice 5 weeks after treatment termination.This is the first preclinical study reporting the evaluation of a MET-targeting kinase inhibitor in combination with an anti-MET antibody in a tumor model bearing MET exon 14 skipping. In this study, treatment with merestinib, a MET-targeting kinase inhibitor, at the once daily dose of 12 mg/kg resulted in durable tumor regression in mice bearing Hs746t xenograft tumors with MET amplification, but MKN45 cells bear no MET exon 14 skipping. This data would suggest that the degradation of MET internalized by emibetuzumab may be CBL-mediated. The in vitro reduction of total MET in Hs746t cells was enhanced when emibetuzumab was combined with merestinib, with about 50% reduction in cell surface MET receptor. In vivo anti-tumor effect in the Hs746t xenograft model with the MET-targeting antibody and kinase inhibitors reflects the same trend as the in vitro data in this MET exon 14 skipping cancer model. Hs746t xenografts treated with emibetuzumab showed a 7–10 day delay in tumor regression as compared to merestinib and crizotinib. Tumor regression was transient with emibetuzumab treatment and tumor outgrowth continued while on treatment.The anti-MET antibody emibetuzumab showed much less in vitro anti-proliferative activity and pMET inhibition as compared to the MET kinase inhibitors evaluated. The extent of MET internalization by emibetuzumab was considerably less in Hs746t cells than in MKN45 cells. Both cell lines have 3; the dosing period was only 14 days and the animals were sacrificed 4 days later. Tumor regression was observed during the 14 day dosing period. However, tumors were smaller when treatment was initiated, and without longer tumor monitoring, it is not known if the tumor regression observed was durable. The DN30-Fab is a MET antibody that induces the shedding of the extracellular domain of MET without MET agonistic activity [1 antibodies also did not show in vitro anti-proliferative activity in the Hs746t cells [The in vivo and in vitro data in this study consistently demonstrate that MET kinase inhibitors such as merestinib and crizotinib result in better anti-tumor response in the MET exon 14 skipping Hs746t model than the anti-MET antibody emibetuzumab. Similar in vitro and in vivo data in Hs746t were reported for another bivalent anti-MET antibody ABT-700. ABT-700 showed little or no effect in vitro in reducing total MET or pMET , while Aactivity . DN30-Faactivity . In vivoactivity . In spit6t cells , but didIn vitro FACS data showed that a low concentration of merestinib (40 nM) was sufficient when combined with emibetuzumab  to enhance the reduction of total MET in Hs746t cells. Each single agent by itself did not induce the reduction of total MET. Increasing merestinib to 100 nM did not further enhance the reduction of total MET when combined with emibetuzumab. These data are consistent with the in vivo findings of concurrent low dose merestinib  combined with emibetuzumab, showing enhanced anti-tumor effect over each single agent at their respective dose.While single agent merestinib (12 mg/kg once daily) proved to be highly efficacious in the Hs746t xenograft model, with no further benefit observed with the addition of emibetuzumab, clinical evaluation of the combination may still be warranted. Preclinical data may or may not be entirely translatable to patients with tumors bearing MET exon 14 skipping. Data from this study showed that sequential treatment combination of merestinib (6 mg/kg once daily) with emibetuzumab provided more durable antitumor effect than concurrent combination . The data from this first reported preclinical study of the combination of merestinib, a MET kinase inhibitor with emibetuzumab, an anti-MET antibody, also support clinical evaluation of sequential combination treatment of single agent merestinib first followed by the addition of emibetuzumab when patients progress on merestinib.Crizotinib is a type I MET kinase inhibitor. Several case studies of cancer patients with MET exon 14 skipping or MET amplification who progressed on crizotinib treatment had either pre-existing or acquired secondary point mutation(s) in the MET kinase domain –30. ThesESM 1(DOCX 99 kb)


The emergence and dissemination of antibiotic resistance is now understood as an unavoidable aspect of bacterial evolution following the consumption of antibiotics  resulting from mutations possibly selected by disinfectants and other antibiotics in veterinary practices , Tackling the spread of antibiotic resistance will surely require a better usage of antibiotics in order to slow down the emergence of resistant variants associated to antibiotic therapies. But, considering the indispensability of antibiotics in modern medicine, antibiotic resistances will continue to be released in anthropogenically-impacted environments where ARBs can persist, accumulate, transfer their resistant genes (ARGs) to indigenous microbes, and finally re-enter the food chain and contaminate human and animal guts for a new round of selection  , the restriction of their availability/delivery, and the confinement of particular antibiotic usages to human medicine are important measures aiming at preventing the emergence of particular resistances in the animal husbandry sector and their dissemination in the human health sector EUR-Lex, . NeverthCollateral effects of antibiotics at sub-inhibitory concentrations and trace metal elements clearly highlight the fact that the antibiotic resistance risk should not be associated with the sole antibiotic therapy practices, and should rather be considered as a multifactorial problem where co-selection and stimulation of horizontal gene transfer also fully applies. Further in depth epidemiological studies should allow determining the extent of such collateral effects outside the context of a Petri dish, and may explain why some antibiotic resistances escape any reduction of occurrence while reducing the corresponding antibiotic consumption. On the other hand determining exhaustively which antibiotic molecules exhibit collateral effects, and at which concentrations, would be an additional step toward antibiotic risk assessment, whether for therapeutic practices or for the effect of antibiotics once diluted in the downstream environments.CM wrote this opinion paper.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.