To design a maternal handwashing intervention for the newborn period, this qualitative study explored drivers of handwashing among mothers and other caregivers of neonates and infants in two rural areas of Bangladesh., we performed thematic analysis to explain determinants of maternal handwashing behaviors.We conducted 40 semi-structured observation sessions to observe handwashing behaviors of primiparous and multiparous mothers of neonates, and to understand the contextual factors that facilitated or hampered those behaviors. We then conducted 64 interviews with mothers of neonates and mothers of infants and 6 group discussions with mothers of infants, other female caregivers and fathers to explore perceptions, beliefs, and practices related to handwashing in the neonatal period. Based on a conceptual model and the Theory of Reasoned Action/Theory of Planned Behavior, we developed a conceptual model a prioriWe conducted 200 h of observation among mothers of neonates. The age range of participating mothers varied between 17 and 25 years and their maximum education was up to 10th grade of schooling. Mothers, other female caregivers and fathers perceived a need to wash hands with or without soap before eating or before feeding a child by hand to prevent diarrhea. Mothers expressed the importance of washing their hands before holding a baby but were rarely observed doing so. All respondents prioritized using soap for visible dirt or feces; otherwise, water alone was considered sufficient. Lack of family support, social norms of infrequent handwashing, perceptions of frequent contact with water as a health threat and mothers’ restricted movement during first 40 days of neonate’s life, and childcare and household responsibilities adversely impacted handwashing behavior.Addressing emotive drivers of handwashing within existing social norms by engaging family members, ensuring handwashing facilities and clarifying neonatal health threats may improve maternal handwashing behavior in the neonatal period.The online version of this article (10.1186/s12889-018-5365-1) contains supplementary material, which is available to authorized users. Hand hy5% CI: 0.9, 1.48, According to the 2014 Bangladesh Demographic and Health Survey (BDHS), the neonatal mortality rate was 28/1000 live births that comprised 61% of all under-five deaths . PreviouThis qualitative study was conducted in the rural areas of Matlab sub-district and Habigonj district in Bangladesh among mothers of neonates and mothers of infants. In rural Bangladesh, women’s social status is typically predominantly linked to traditional social and cultural norms that sometimes limit women’s autonomy in decision making or accessing specific health services . TherefoMatlab is a rural sub-district of Chandpur district in south-eastern Bangladesh where icddr,b  maintains a Health and Demographic Surveillance System (HDSS), with added maternal and child health (MCH) interventions in some villages. We conducted this study in 18 villages where the MCH interventions did not occur and the rate of institutional delivery was similar to other rural areas in Bangladesh [Table sh Table , 14. Frosh Table . In addiHabigonj district under the Sylhet division in the north-eastern part of Bangladesh  Semi-structured observationsii) In-depth interviewsWe conducted single 5-h semi-structured observation sessions (from 8 am to 1 pm) in households of mothers of neonates to observe their typical activities. Mothers were informed that the major objective was to observe their daily practices and routines of feeding, cleaning and overall caring behaviors for their neonates. In a semi-structured observation form, we recorded events of potential pathogen transmission and whether or not mothers washed their hands with or without soap at those opportunities. Events of potential pathogen transmission included feeding or touching the baby, cleaning a child’s bottom, mothers eating and going to the toilet. We also took detailed handwritten notes of the physical environment and setting of each event to understand the possible contextual factors that could facilitate or hamper handwashing behavior, such as mothers involved in multiple tasks, no help from attendants etc. . In subsWe conducted in-depth interviews with both mothers of neonates and mothers of infants to elucidate their perceptions and opinions about the health of their children, healthy practices, prevention of illness, and taking care of children. We interviewed the same mothers of neonates whom we also observed. After each observation session, we asked mother of neonate for appointment for an interview within the following two days. All the interviews were conducted in native Bengali in a private setting chosen by the informants and each lasted 60–90 min.iii) Group discussionsAfter completing all the observation sessions and in-depth interviews, we organized group discussions to further explore the themes related to childcare and hand hygiene that emerged from the observations and in-depth interviews. In both sites, we conducted group discussions of 90–120 min duration with three different groups: mothers of infants, female caregivers other than the mother, and fathers of neonates or young infants. We did not conduct any group discussion with mothers of neonates since we assumed that they might wish to confine themselves to their household compounds, given the social norm of restricted movement outside the household during the postnatal period in rural areas of Bangladesh , 19 [ForBased on the Health Belief Model and the Theory of Reasoned Action/Theory of Planned Behavior, we developed a conceptual model a priori to explain determinants of maternal handwashing behavior in the neonatal period Fig. . As elucTo analyze the data from the semi-structured observations, the same research team expanded field notes taken during observations. We also counted the total handwashing opportunities at critical times and calculated the proportion of handwashing events occurred. We transcribed all audio recorded interviews and discussions verbatim in Bengali. Then we developed a code list based on the conceptual model and data collected, and coded all the Bengali transcriptions using the qualitative text organizing software Altas.ti 5.2.We performed thematic analysis of data from mothers, secondary caregivers, and fathers according to the themes identified in the conceptual model to describe perceptions related to hand hygiene behaviors, physical environment to perform required handwashing, and social dimensions that promote execution of mothers’ intended actions. We identified common trends and patterns from the responses and prepared summaries of each theme. We also compared the common or specific practices and driving factors to perform handwash that recoded in the observations with mothers of neonates and also reported by the same mothers. The comparisons allowed us to triangulate the data and confronted the evidences with the reporting.Among 64 mothers we interviewed in both study sites, all were married; the age of primiparous mothers ranged from 17 to 22 years and the median age for multiparous mothers was 25 years Table . In totaIn both sites, typically the cooking area and toilet was separated from the bedrooms or living rooms of the main household, such as in one corner of yard. The handwashing station was generally situated in the outside of toilet. Usually, soap was not observed to keep in the common handwashing area shared by multiple families, but for single household, sometimes soap was observed available by the handwashing station.The hand hygiene behavior is a complex issue, combination of multiple overlapping factors. The maternal hand hygiene behaviors often influenced by her individual’s perceptions, physical environmental, or multiple social determinants to execute required hand hygiene:In in-depth interviews and group discussions, respondents described that their typical handwashing practice involves the use of water alone, generally without mention of soap. Respondents reported typically washing their hands, face and feet at least twice a day to clean themselves, after waking up in the morning and after finishing daily household chores or after returning home from daily work (for fathers) in the evening. Most mothers and other caregivers stated that they usually wash their hands if they are involved in cooking, sweeping, cutting vegetables or fish, or when hands are ‘visibly dirty’ . Respondents perceived that after cutting when they washed vegetables, fish, or meat with water alone, or washing utensils or clothes, their hands were also being washed and thus did not need to be washed again before cooking. In addition, fathers reported that they washed their hands with soap after handling cow dung as such contact is considered profane; they also reported washing hands with soap after agricultural work in the field, such as harvesting or spraying pesticide. According to all types of respondents, hands need to be washed with soap after eating to remove bad smells, chilies, or spices; otherwise, the mothers/other caregivers’ hands would irritate a child’s skin. If a mother cared for her child during her leisure time, she usually did not wash her hands at any of the event identified above, as she perceived her hands as clean. Only two mothers mentioned handwashing as part of a ritual ablution before performing prayers, 5 times each day, customary among many Bangladeshi Muslims.n = 2) after visiting the toilet.We observed that washing hands with soap and water was rarely performed Figs.  and 3. IIn in-depth interviews, few mothers reported perceiving that a child was at higher risk of having illnesses during the first month(s) of life. Their common belief was that the risk is greater during later infancy as the child begins crawling which could increase exposure to illnesses or accidents. As mothers reported that those exposures mainly leading to diarrhea but they did not directly link with other infections. Mothers commonly perceived that neonates are very vulnerable to catching colds (respiratory infection) through ways unrelated to hand hygiene: if the baby is kept in a wet bed soiled with urine or if the mother gets a cold through frequent contact with water or by eating cold food and passing it to her breastfeeding baby through breastmilk. Mothers are also commonly advised to eat warm food and warm water, particularly up to 40 days after delivery to prevent the neonate from getting a cold. Related to these humoral concerns, mothers reported that they avoided frequent contact with water, including decreasing handwashing; mothers avoid handwashing after cleaning a neonate’s anus by wiping with a wet piece of cloth instead of washing the anus by hand. Fathers also reported the similar concern that a mother’s cold can be transmitted to her child. Otherwise, fathers perceived that children get affected by viruses during any seasonal change during the year. However, as we talked, both mothers and fathers did not link the neonate’s ‘cold with ‘respiratory infection’ or other health risks that could lead to neonatal mortality compared to mortality of older infants.Respondents perceived that cleanliness could protect children from getting ill. Soap was perceived as necessary for cleanliness; it removes visible dirt, germ and bad smells. Mothers reported that they typically wash their hands with or without soap before feeding a child with bare hands, after using the toilet, cleaning a child’s bottom, contact with an older child’s feces, and before preparing food for an infant or family that needs contact with bare hands and is not cooked further (e.g. mashed potato).To mothers, handwashing is important during these events to prevent children or other family members from getting diarrhea from dirty hands. Mothers perceived that, if one held the child with ‘dirty hands’, the child could get diarrhea, skin irritations, cold and fever. Mothers said that ‘visible dirt’ in hands always persuaded them to wash their hands. When hands were ‘visibly dirty’, they reported washing their hands with soap prior to holding a young child to prevent those illnesses. Fathers and others caregivers endorsed the drivers that described above for their or a mother’s handwashing practice. Our observational data also supported this reporting that “visible dirt” (mothers were handling biomass fuel or cooking spices) facilitated required handwashing.Only one disadvantage of handwashing was cited: one mother in Habigonj stated that if she washes her hands with soap before mashing foods by hand, a common food preparation process in Bangladesh, the mashed item would have a soapy smell.We defined subjective norm as the perception that a given behavior is practiced widely in one’s reference network (e.g. neighbors or other mothers of young children). The predominant existing social norms related to childcare contributed to mothers’ normative beliefs. As reflection, mothers performed the expected hygiene behavior for caring her child from the perceived expectation of her close surroundings. According to mothers, feces of an infant who already started eating semi-solid foods including fish and meat, contains germs and smells foul like adult feces; so after defecation or cleaning a child’s anus, they were motivated to wash their hands, mostly with soap, to remove the foul smell from hands and alleviate perceived disgust. In contrast, feces of a young child who breastfeeds, does not contain any germs nor bad smells, and is not ‘disgusting’ to mothers; so handwashing after cleaning the anus of a young breastfeeding child was often perceived as unnecessary.alga batash) or “evil eyes” (evil spirit) during the first 40 to 45 days after birth. All types of respondents typically perceived that an evil spirit could enter the room of an unattended baby and could harm or kill the baby; harm might be reflected in the baby subsequently developing a lack of interest in breastfeeding or having stomach pain. To mitigate this supernatural risk, mothers reported being advised not to go outside, or to leave neonates unattended, especially at mid-day and after dusk. This mobility restriction prevented new mothers’ movement away from the newborn, such that, if the newborn defecated at night, mothers were unable to wash hands because handwashing facilities were typically located outside the sleeping space. This existing normative believe compounded by physical environment that negatively influenced mother’s required handwashing behavior. A mother of a neonate said,A normative belief unique to the neonatal period was the possible harmful effect of “bad air” (It is not good to keep the baby unattended in his bed (room) during his first 40 days. Satan may come to the child during this time. It would be better to stay in the post-natal room with the baby....so if required, I wipe up my hands with cloth diaper after cleaning him.In Habigonj, we observed two events, when two mothers were about to go outside for handwashing after cleaning a child’s anus but family elders warned mothers about postnatal risk of leaving neonate unattended; as a result, mothers did not go. In both cases, elders were busy with household chores and no one was available to attend the newborn.However, some social norms also encourage handwashing. Most mothers reported that elder female family members advised and reminded new mothers to wash hands if eating after breastfeeding; otherwise it is believed a mother ingesting her own breastmilk from her hands could cause the death of the child.Mothers reported that except when elders’ hands were visibly dirty or soiled with dirt, they could not request them to wash hands before holding a child. Mothers generally believed that elders usually do not hold a child with dirty hands.Many mothers and secondary caregivers perceived that handwashing is not important before holding a child; though it is important before feeding a child. They further explained that a young child is usually wrapped with clothing and does not come into direct contact with one’s hands. Fathers in contrast mentioned that it is important to wash hand first after coming from outside and then hold a young child.Even though respondents identified the importance of handwashing, they reported not washing hands out of “carelessness” (lack of motivation). One mother of an infant said,…from the beginning, Ma advised me to wash hands before breastfeeding…but it (the newborn) breastfeeds frequently… it defecates frequently… (I) wash (only) if hands are visibly dirty, otherwise I do not… I know I should wash hands before every feed…after cleaning (the newborn)…I do not have any problem to wash hand; but I feel idle (lack of motivation/careless) to wash hands every time…I just do not go (for handwash) indolently.A number of mothers indicated that, even if they wished to adopt improved handwashing practices during the neonatal period, they would be teased by others. A primiparous mother of neonate said,Washing hands before and after every task is not the system (practice) in our place….If I wash hands frequently before or after doing tasks and handling baby, elders (mother in-law/aunts) tease and say, ‘she only has a newborn baby, (as if) we never had’!Fathers also added that after coming from outside if anyone wanted to hold a child, it would not possible to ask for a handwash beforehand, otherwise s/he could mind.While some mothers intended to practice a good handwashing behavior in their daily activities, mothers’ existing habit of handwashing within a certain culture drove them to define when handwashing was required. One multiparous mother of a neonate said,I do not wash hands all the time; if my hands are (visibly) dirty, I wash them. If they are less dirty, we wipe them on the door (smiling). We are living in rural area, we cannot do like you people (referring interviewer as urban people); even if you give me so much advice!Some mothers, other female caregivers and fathers indicated this habit of infrequent handwashing as a social norm in their rural area, unlike urban culture. One mother of an infant said,….(I do not wash hands) because it is not in my habit. If everybody practiced it, I would also follow.All respondents, regardless of educational and economical status, reported that washing hands with or without soap was habitual only at certain times, such as before and after a meal; they reported developing these habits during early childhood. Despite reported practices, field researchers observed that mothers rarely used soap to wash their hands Figs.  and 3.Mothers perceived that a “good mother” always keeps herself and her child clean and soap is a necessary item to maintain cleanliness. One primiparous mother of a newborn who passed sixth grade of schooling said,I have to take care of my baby in a good manner... keep him clean (with soap) so that he will get less illness…need to wash him with water after defecation; so he (child) would get less germs. A good mother keeps her child’s clothes, and feeding dishes clean….With these characteristics fathers also added that when a child becomes sick, a good mother gives it medicine. Fathers perceived that mother is mainly responsible for nurturing a young child and other family members support her in this process.Mothers used bar soap (e.g. available brands of antiseptic soap) when bathing or cleaning babies and detergent for washing baby clothes. Mothers underscored that sometimes they might have financial crisis, they always tried to ensure buying soap for a young infant. Only a few mothers said that they could not buy required quantity of soap due to their financial ability. The fathers of young infants and secondary caregivers also mentioned that they know soap is important to keep the baby clean but could not always afford required soaps for their young children due to their financial crisis.The majority of mothers reported that their handwashing behaviors had increased because of the additional responsibilities of new motherhood, particularly by washing children’s clothes, bathing or sponging the child, washing hands after putting on lotion or an oil massage, after breastfeeding and feeding the infants.We observed some mothers were simultaneously occupied in multiple chores while caring for their newborns, confirming their reports during interviews that the responsibilities of nurturing a newborn added to regular household chores sometimes prevented mothers from washing their hands. This condition multiplied for mothers who have multiple children. For instance, in the Habigonj site, mothers of multiple children sometimes had to feed the neonate while serving food or cleaning the bottom of an older child after defecation; they often neglected to wash hands after each task. The social construction of woman role being a wife and a mother, often impacted her child nurturing behaviors that restricted her intent to improve handwashing.Mothers in Matlab reported that they customarily delivered the baby at their parents’ house and that the mother of a newborn was not usually asked to do any household chores when she was at her parents’ house. However, she was expected to perform certain chores at her marital home. During the neonatal period, these mothers often got support from their family for handwashing, e.g. bringing a handwashing station (jug or bucket of water) or soap, or attending to neonates so that the mother could wash hands. Mothers in Habigonj more commonly delivered at their marital home Table ; during Mothers reported and we also observed in both study sites that when a secondary caregiver was absent, mothers were struggling to carry out varieties household chores and at a time caring a neonate. In such situations, mothers were observed not washing their hands at critical times, for instance after cleaning child’s anus, because the baby became fussy and no one there to attend the baby, mother soothed her child first by breastfeeding. Multiparous mothers in Habigonj who belong to a nuclear family were observed to face this barrier more frequently with the lack of secondary caregivers while they were tending to numerous daily household responsibilities, including caring for neonates and older children.Many of our mother respondents from both sites reported that if they wished to adopt improved handwashing practices or keep any handwashing materials inside their bedroom during this period for an enabling physical environment, they would be scolded by family elders and neighbors. In this regard, one mother of an infant stated,They (in-laws) wouldn’t like it… they wouldn’t allow me to keep it (bucket near bedside)… …They would say ‘they are doing all the tasks and daughter-in-law is not doing anything; she does not even go that little distance (i.e. to the handwashing station)’… she (mother-in-law) will be angry with me.Moreover, mothers said that although they often identified items deemed necessary for the child, they didn’t have the power of purchasing materials themselves within existing family roles in social structure. Many of the mothers in Habigonj specifically said that their husbands or sometimes in-laws made the final decisions and purchased related to items needed for childcare. Two mothers of neonates also reported that, when they use too much soap, their husbands scold them as they cannot afford it. Fathers, from the other end confirmed this decision making process and told that they were mainly responsible for outside tasks and also led the treatment related decision (e.g. where to take) when a child got sick.Mothers in both sites discussed that presence of handwashing materials sometimes facilitated mothers as cues to action for handwashing at times of potential pathogen transmission. When family members supported the mother by bringing required water or soap to her location, she would have been more able to do required handwashing. Sometimes, family members reportedly reminded mothers to wash hands before nurturing. We also observed that when family elders were reminding mothers, mothers washed their hands after household chores and before breastfeeding and holding the child.Mothers reported and field researchers also observed that, in some cases, mothers avoided some handwashing with their stored water or used only a little water because of the distance to the water source. Some mothers identified lack of soap availability in the places where soap was needed as a hindrance to practicing handwashing. Lack of affordability of soap was also cited as a barrier for some mothers and fathers respondents.There are sizable opportunities to improve maternal handwashing behavior in the neonatal and infant periods in the Matlab and Habigonj study sites. The lack of handwashing practice during the neonatal period confirms findings from other low-income countries , 21. ConA multi-country analysis of drivers of handwashing behavior among mothers of children less than 5 years old has shown that perceptions of risk usually did not motivate maternal handwashing, except in outbreaks such as epidemic cholera . Yet, ouOur conceptual model attempted to capture many of the motivators and barriers related to handwashing influenced by physical environment and a number of social dimensions throughout this qualitative research. Some common perceptions in the community, such as elders’ reminders to mothers to wash their hands after breastfeeding to avoid any risk to the child from the mother’s excessive exposure to cold food or cold water , are unsWe found that handwashing appears to be an aspirational behavior rather than a health concern, with some respondents indicating that urban people (connoting a higher level of sophistication) have the habit of washing hands, unlike rural people. The perception of the habit of handwashing is critical to behavior change in other settings and can be deeply embedded in culture . We founThese factors suggest that interventions that promote handwashing to mothers of neonates or young infants need to address the broader context by engaging her family and community to make her physical and social environment conducive to handwashing. Focused handwashing instructions on a few specific key times that represent the greatest risk of pathogen transmission may be more practical for mothers to follow in their busy daily schedule. The focused handwashing instructions also need to be streamlined with caregivers’ cultural context and biomedical context with credible interventions, such as waterless hand sanitizer option to minimize time constrain to mothers of neonates.We observed that the availability of handwashing materials where the mother-baby dyad are located (particularly during mothers’ restricted mobility in first 40 days of baby’s life) and reminders from elders to wash hands before nurturing a baby facilitated mothers’ handwashing. However, mothers noted that a simplistic approach of just providing a handwashing station will not overcome all barriers. There must be buy-in from elders who otherwise may scoff at the notion of handwashing before holding the baby, or who may ridicule the mother for prioritizing materials to facilitate her child-rearing. Although this model does not directly mention poverty or lack of agency of mothers to secure soap and a handwashing station, affordability and supply of materials were cited as important barriers in many households of similar settings [One of the limitations of our study is the possibility of courtesy bias during observation. While direct observation is commonly used to measure hand hygiene, the presence of an observer has been shown to increase handwashing behavior in another study in Bangladesh . ParticiThis qualitative study identified a number of important barriers and facilitators to handwashing behavior among mothers and other caregivers of neonates and young infants in the complex social fabric of Matlab and Habigonj, Bangladesh. Designing context-specific interventions based on the dominant social norms and contextual barriers and facilitators identified in our study, rather than blanket approaches based on health messaging that principally targets mothers to the exclusion of other family actors, may yield interventions that are more effective at motivating handwashing behavior change.Additional file 1:Guideline for semi-structured observation, The semi-structured observation data collection guideline has been included to better understand how the data was collected and therefore results were presented in Figs.


Reduced cerebrospinal fluid (CSF) production and increased resistance to CSF outflow are considered to be associated with aging, and are also characteristics of Alzheimer’s disease (AD). These changes probably result in a decrease in the efficiency of the mechanism by which CSF removes toxic molecules such as amyloid-β (Aβ) and tau from the interstitial fluid space. Soluble Aβ is potently neurotoxic and dysfunctional in CSF circulation and can accelerate the progression of AD. Current therapies for AD exhibit poor efficiency; therefore, a surgical method to improve the homeostasis of CSF is worthy of investigation. To achieve this, we conceived a novel device, which consists of a ventriculo-peritoneal shunt, an injection port and a portable infusion pump. Artificial CSF (ACSF) is pumped into the ventricles and the ACSF composition, infusion modes and pressure threshold of shunting can be adjusted according to the intracranial pressure and CSF contents. We hypothesize that this active treatment for CSF circulation dysfunction will significantly retard the progression of AD. Alzheimer’s disease (AD) is an age-related dementia that represents a serious social problem in the aging population worldwide , which is the internal environment of the brain, include amortization, acid-base buffering and transport of electrolytes, molecules and micronutrients  and accurately control the infusion process , the difference can be adjusted to 20–40 cm H2O.The main operation mode of the device allows injection of the ACSF into the lateral ventricles to supplement the insufficient CSF, as well as drainage of the supernumerary CSF through the shunt tube. An infusion bottle containing 150 ml ACSF, which is approximately equivalent to the total amount of CSF in normal adults, is placed in the infusion pump. The ACSF is pumped continuously and slowly into the ventricles over a period of 8–12 h during the daytime. The amount of the ACSF is adjusted by measuring biological markers in the CSF such as Aβ and tau, with the aim of restoring the composition of the CSF. The difference between the intracranial pressure and the valve pressure is set at 0–20 cm HThe hypothesis that dysfunctional CSF circulation is involved in the pathogenesis of AD presents a novel therapeutic target for this disorder. Here, we describe a device that was designed for the treatment of AD according to this hypothesis. The therapy promotes CSF circulation by direct intervention in all stages of the circulation: production, turnover and clearance. CSF production in patients with AD insignificantly decreased to approximately 300 ml per day; about half the amount of normal people  and National Natural Science Foundation of China (Grant No. 81271395).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glucocorticoids (GCs) are potent anti-inflammatory drugs whose mode of action is complex and still debatable. One likely cellular target of GCs are monocytes/macrophages. The role of GCs in monocyte survival is also debated. Although both granulocyte macrophage-colony stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) are important regulators of macrophage lineage functions including their survival, the former is often associated with proinflammatory functions while the latter is important in lineage homeostasis. We report here that the GC, dexamethasone, induces apoptosis in GM-CSF-treated human monocytes while having no impact on M-CSF-induced monocyte survival. To understand how GCs, GM-CSF, and M-CSF are regulating monocyte survival and other functions during inflammation, we firstly examined the transcriptomic changes elicited by these three agents in human monocytes, either acting alone or in combination. Transcriptomic and Ingenuity pathway analyses found that dexamethasone differentially modulated dendritic cell maturation and TREM1 signaling pathways in GM-CSF-treated and M-CSF-treated monocytes, two pathways known to be regulated by ERK1/2 activity. These analyses led us to provide evidence that the GC inhibits ERK1/2 activity selectively in GM-CSF-treated monocytes to induce apoptosis. It is proposed that this inhibition of ERK1/2 activity leads to inactivation of p90 ribosomal-S6 kinase and Bad dephosphorylation leading in turn to enhanced caspase-3 activity and subsequent apoptosis. Furthermore, pharmacological inhibition of GC receptor activity restored the ERK1/2 signaling and prevented the GC-induced apoptosis in GM-CSF-treated monocytes. Increased tissue macrophage numbers, possibly from enhanced survival due to mediators such as GM-CSF, can correlate with inflammatory disease severity; also reduction in these numbers can correlate with the therapeutic benefit of a number of agents, including GCs. We propose that the ERK1/2 signaling pathway promotes survival of GM-CSF-treated proinflammatory monocytes, which can be selectively targeted by GCs as a novel mechanism to reduce local monocyte/macrophage numbers and hence inflammation. Exogenous GCs are very potent anti-inflammatory and immunosuppressive agents broadly used in therapy, albeit with adverse side effects associated with long-term usage4. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action on immune cells.Glucocorticoids (GCs) are stress hormones that modulate a wide range of physiological processes, including metabolism and inflammation5. M-CSF, which circulates at high levels, is expressed in many tissues with evidence to suggest it can govern steady state macrophage numbers and be involved in resolution of an inflammation9. In contrast, GM-CSF circulates normally at low levels but has often been implicated in inflammatory/immune responses during which its levels are elevated9. Currently, GM-CSF and its receptor are being targeted widely in clinical trials to treat autoimmunity/inflammatory diseases10.Monocytes and macrophages are key components of the immune system. It has become apparent that these populations can be quite heterogeneous and plastic depending on the milieu to which they are exposed during their activation and/or differentiation. Two cytokines that can regulate the development and function of monocyte/macrophage populations are macrophage-colony stimulating factor  and granulocyte macrophage-CSF (GM-CSF)11 or act indirectly by binding to other transcription factors 12. There is a broad consensus that GCs exert their anti-inflammatory actions on monocytes/macrophages by primarily inhibiting the transcription of proinflammatory genes  possibly by suppressing the NF-κB activity13. Accumulating evidence suggest that, in addition to their ability to suppress the production of proinflammatory mediators by monocytes/macrophages, GCs can induce specific changes in cell survival, proliferation, and phagocytosis to resolve inflammation18. We have proposed that GM-CSF and M-CSF may contribute to the progression of an inflammatory reaction in part by enhancing tissue macrophage numbers by promoting their survival and/or local proliferation, for example, in the rheumatoid synovium19; in contrast, one of the dramatic effects of GC during rheumatoid arthritis therapy is a striking reduction of synovial macrophage numbers20. However, the effects of GCs on the survival of human monocytes are controversial21, warranting a detailed investigation of their regulation of survival/death signaling pathways.The potent anti-inflammatory and immunosuppressive effects of GCs are mediated mainly by its binding to a cytosolic glucocorticoid receptor (GCR). Translocated GCR can bind directly to DNA to a canonical GC response elementIn the present study, we identified a novel mechanism of GC-induced apoptosis of proinflammatory monocytes. We provide evidence that GC treatment selectively promotes the apoptosis of GM-CSF-treated monocytes by inhibiting ERK/12 phosphorylation, which in turn leads to dephosphorylation of p90RSK resulting in the pro-apoptotic activities of Bad and caspase-3. We propose that inhibition of ERK activity, leading to apoptosis of proinflammatory monocytes, is a novel mechanism by which GCs are capable of selectively downregulating inflammatory processes.Reagents included human GM-CSF , human M-CSF , dexamethasone (Dex), and mifepristone . Antibodies for Western blotting were against phospho-p38 (D3F9), p38 (D13E1), phospho-JNK1/2 (81E11), JNK1/2 (9252), phospho-ERK1/2 (D13.14.4E), ERK1/2 (137F5), caspase-3 (9662), caspase-8 (D35G2), Bcl2 (50E3), Bcl-xL (54H6), Bid (2002), Bak (D4E4), Bax (D2E11), Bim (C34C5), phospho-Bad S112 (40A9), Bad (D24A9), phospho-RSK S380 (D3H11) and RSK (32D7) , and β-actin (A5316) (Sigma-Aldrich).22. Monocytes were treated simultaneously with M-CSF (5000 U/ml), GM-CSF (10 ng/ml) or Dex (100 nM), either alone or in combination for indicated time periods.Buffy coats were sourced ethically and their research use was in accord with the terms of the informed consents obtained by Australian Red Cross Blood Service. Human monocytes were isolated and cultured as described beforeTotal RNA was extracted using ISOLATE II RNA Mini Kit  and reverse transcribed using SuperScript III reverse transcriptase (Invitrogen). Quantitative PCR (qPCR) was performed using the ABI PRISM 7900HT sequence detection system  and predeveloped TaqMan probe/primer combinations for human HLA-DOA, HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, HLA-DRA, HLA-DRB, CIITA, TREM1, and HPRT (Applied Biosystems). Threshold cycle numbers were transformed to cycle threshold values, and the results were plotted using GraphPad Prism version 5.04.23. Microarray analyses were preformed following recommended protocols supplied by Agilent Technologies  as before22. Microarray intensity data were read using the read.maimages function from the Limma Bioconductor R package with background correction using the normexp method (https://www.bioconductor.org/packages/release/bioc/html/limma.html). Fold changes and p values were obtained using the RUVinv function from ruv R package25 . The list of control genes was from Eisenberg et al.26 The data set and technical information compliant with minimum information about a microarray experiment (MIAME)27 can be found at the ArrayExpress Archive Web site .RNA was isolated from three independent cell preparations, each derived from a single donor as before6) were cultured in 6-well BD Falcon™ adherent tissue-culture plates following various treatments at 37 °C in a humidified, 5% CO2 atmosphere. Cells were collected and viable cells were determined by Trypan blue exclusion method. Apoptotic cells, displaying externalized phosphatidylsterine, were measured by flow cytometry staining with a FITC-conjugated annexin V detection kit . The percentage of apoptotic cells is determined as a ratio of annexin V positive cells, but propidium iodide (PI) negative cells, to the total number.Human monocytes (1 × 106) were lysed and cytosolic extract (200 μg) was incubated at 37 °C for 2 h with 200 μM substrate, DEVD-pNA, is composed of the chromophore, p-nitroanilide (pNA), and a synthetic tetrapeptide, DEVD , which is the upstream amino acid sequence of the caspase-3 cleavage site in poly (ADP-ribose) polymerase. Upon cleavage of the substrate by caspase-3-free pNA light absorbance was quantified using a microplate reader at 405 nm.Proteolytic activity of caspase-3 in lysates of monocytes was determined using the ApoTarget™ Caspase-3 Colorimetric Protease Assay kit (Invitrogen). Human monocytes (5 × 10Human monocytes were treated with treated M-CSF (5000 U/ml), GM-CSF (10 ng/ml) or Dex (100 nM), either alone or in combination for indicated periods of time. Cells were stained with fluorochrome-conjugated mAbs specific for human CD14 PerCP-Cy5.5 (M5E2), CD80 APC-H7 (L307.4), HLA-DR PE (G46-6), CD95 PE-Cy7 (DX2), CD178 PE (NOK-1), and their isotype controls  and then analyzed using a CyAn ADP analyzer .22. Protein concentrations of the samples were determined with a Bio-Rad protein assay kit. Equal amounts of protein were loaded on 10% NuPAGE gels (Invitrogen). The separated proteins were transferred onto a polyvinylidene fluoride membrane and then Western blotted with appropriate Abs. Western blots were quantified by densitometry using Quantity One version 4.6.9 (Bio-Rad) and resulting data were plotted as bar graphs as mean ± SEM.Whole-cell extracts were lysed and Western blotted as described previouslyt-test or one-way ANOVA Tukey post-test as indicated (GraphPad Prism 5.04). The p values < 0.05 indicate significance. Data were plotted as mean ± SEM from at least three independent experiments using GraphPad Prism version 5.04.Statistical analyses between groups were performed using either Student’s paired Human monocytes were treated with 100 nM dexamethasone (Dex) or PBS over 3 days. Viable and apoptotic cells were assessed by the Trypan blue exclusion method and annexin V staining, respectively. A representative dot plot illustrating the gating strategy is provided in Supplementary Figure To assess whether the presence of a pro-survival factor would still maintain the survival of monocytes in the presence of Dex, monocytes were treated with GM-CSF (10 ng/ml) or M-CSF , either alone or together with Dex for 1 and 3 days. After day 1, GM-CSF and Dex co-treated monocytes showed some increased cell death compared to monocytes treated with GM-CSF alone, an effect not observed with M-CSF and Dex co-treatment Fig. . These dTo demonstrate that the Dex-induced apoptotic effect in GM-CSF-treated monocytes is mediated via the glucocorticoid receptor (GCR), the steroid receptor antagonist, mifepristone was utilized. Pre-treatment of monocytes with 1 µM mifepristone for 30 min before stimulation with either GM-CSF alone or together with GM-CSF and Dex resulted in abrogation of GC-induced apoptosis in GM-CSF-treated monocytes Fig. , an effeGiven the striking difference noted above in the effects of Dex on the pro-survival action of the two CSFs and given the transcriptional role of the GCR, a whole-genome-wide transcriptomic analysis was carried out for monocytes undergoing the various treatments for 16 h. Microarrays of human monocytes treated with Dex found 685 differentially expressed genes, with 183 upregulated genes and 502 downregulated genes Table . GM-CSF To understand further the effects of Dex on GM-CSF-treated and M-CSF-treated monocytes, microarray analyses of human monocytes co-treated with either GM-CSF and Dex or M-CSF and Dex were performed. GM-CSF and Dex co-treatment resulted in 190  differentially expressed genes Table . On the Interestingly, among the number of commonly regulated genes between Dex and M-CSF a number of MHCII molecules were found to be downregulated Table . Further28. Although GM-CSF and M-CSF were both found to upregulate TREM1 expression, Dex significantly downregulated it only when monocytes were co-treated with GM-CSF and Dex  family are known to modulate monocyte survival and deathnts Fig. ; also thnts Fig. . No channts Fig. . On the nts Fig. . Total Ents Fig. . The incnts Fig. . NotablyIn order to confirm that GC-regulated ERK1/2 deactivation in GM-CSF-treated monocytes was mediated via the GCR, mifepristone was again utilized. Pre-treatment of monocytes with mifepristone restored ERK1/2 phosphorylation in GM-CSF and Dex co-treated monocytes Fig. . SignifiC31. Levels of both anti-apoptotic and pro-apoptotic proteins were measured in order to determine if Dex can regulate their expression to promote apoptosis selectively in GM-CSF-treated monocytes. Expression levels of the anti-apoptotic members, Bcl2 and Bcl-xL, did not change under the different treatments , a downstream effector in the MAPK signaling cascade, has been shown to induce Bad phosphorylation34. As Dex selectively inhibited ERK1/2 activation in GM-CSF-treated monocytes, we investigated whether such inhibition could lead to Bad dephosphorylation and the subsequent increase in apoptosis observed above. Human monocytes were treated with GM-CSF alone or together with Dex for 16 h, in the absence or presence of a prior treatment (30 min) with mifepristone. As noted earlier, monocytes had a basal ERK2 activation , a downstream effector in the MAPK signaling cascade, inactivates Bad by phosphorylation at serine 112. Phosphorylated Bad is sequestered in the cytosol by binding to 14-3-346. When not phosphorylated, Bad induces apoptosis by interacting with the anti-apoptotic Bcl2-family members, Bcl-xL and Bcl2, thereby allowing two other pro-apoptotic proteins, Bak and Bax, to aggregate on the mitochondrial membrane and induce release of cytochrome C, followed by caspase activation and apoptosis31. It is generally believed that survival factors induce activation of specific anti-apoptotic kinases, which modulate the activity of Bad47. Indeed, we show here that both M-CSF and GM-CSF by themselves can activate the Raf/MEK/ERK signaling cascade that results in Bad phosphorylation and monocyte survival. Co-treatment of monocytes with M-CSF and Dex also sustained ERK1/2 and Bad activities, as well as monocyte survival. However, co-treatment of monocytes with GM-CSF and Dex resulted in ERK1/2 deactivation leading to Bad dephosphorylation, the subsequent increase in caspase-3 activity and eventually apoptosis  or agonists 50. IPA analysis and subsequent validation of MHCII molecule expression confirmed also the ability of GCs to suppress antigen-presenting pathways as previously documented52, possibly forming part of their immunosuppressive and anti-inflammatory role. GCs may thus be acting on GM-CSF-treated monocytes and suppressing their maturation into a potent antigen-presenting cell. The expression of MHCII molecules is controlled by the master transcription regulator, CIITA. Of potential mechanistic relevance, GM-CSF treatment of human monocytes increased the amount of CIITA associated with HLA-DR promoter49. In addition, our microarray and subsequent qPCR analyses revealed that both M-CSF and Dex downregulated CIITA expression, which may contribute to the decreased MHCII gene expression in the monocytes treated with either of these two factors with implications for dendritic cell maturation.In addition to promoting survival, the role of GM-CSF in stimulating human blood monocytes for increased antigen-presenting functions is widely reported19; increased macrophage survival is considered to be one possibility for the proinflammatory action of GM-CSF19. We speculate that the selective GC-mediated apoptosis in GM-CSF-treated monocytes could be a mechanism for preventing excessive inflammation by eliminating monocytes/macrophages that are responding locally to a proinflammatory stimulus. Interestingly, in this context, in rheumatoid arthritis patients the benefit of a number of therapeutic agents, including GCs, correlates with a reduction in synovial tissue macrophage numbers20. The selective GC-induced apoptosis of proinflammatory monocytes/macrophages may thus have clinical significanceIn summary, what is notable from our data is that GM-CSF-treated monocytes and not the M-CSF-treated cells gradually die in the presence of a GC. We have delineated above a molecular mechanism which is proposed to account for this GC-mediated apoptosis. We provide evidence that, by selectively inhibiting the Raf/MEK/ERK signaling pathway, GCs induce apoptosis of GM-CSF-treated monocytes, while failing to do so in M-CSF-treated monocytes. Macrophage numbers in inflamed tissues can correlate with disease severity, for example, in the rheumatoid synovium, and one mechanism could be enhanced survivalSupplemental Figure S1Supplemental Table S1Supplemental Table S2Supplemental Table S3Supplemental Table S4Supplemental Table S5Supplemental Table S6


Surface electromyography (sEMG) signals are closely related to the activation of human muscles and the motion of the human body, which can be used to estimate the dynamics of human limbs in the rehabilitation field. They also have the potential to be used in the application of bilateral rehabilitation, where hemiplegic patients can train their affected limbs following the motion of unaffected limbs via some rehabilitation devices. Traditional methods to process the sEMG focused on motion pattern recognition, namely, discrete patterns, which are not satisfactory for use in bilateral rehabilitation. In order to overcome this problem, in this paper, we built a relationship between sEMG signals and human motion in elbow flexion and extension on the sagittal plane. During the conducted experiments, four participants were required to perform elbow flexion and extension on the sagittal plane smoothly with only an inertia sensor in their hands, where forearm dynamics were not considered. In these circumstances, sEMG signals were weak compared to those with heavy loads or high acceleration. The contrastive experimental results show that continuous motion can also be obtained within an acceptable precision range. Stroke is one of the most harmful diseases which results in adults losing their ability to read, speak, understand and move. Patients may even lose their lives. Based on reports from seven countries , stroke occurs in an average of 214 out of 100,000 people per year, with the incidence rate growing annually by 1.9% due to an aging population . One of Generally speaking, there are two main parts to process sEMG signals according to previous literature: feature extraction and feature classification. Moreover, from the perspective of processing, the method of feature extraction can be grouped into three types: time domain, frequency domain and time–frequency domain . The metIn the past several decades, many researchers have studied motion pattern recognition based on sEMG signals, such as in prosthesis. However, much of the research has focused on the recognition of motion patterns, namely, discrete patterns. Continuous motion recognition was merely mentioned. On the other hand, most motion pattern recognitions focused on some special motions that can induce high activation of muscle and strong sEMG signals, such as hand grasping, so that they can obtain high recognition accuracy. In this research, we focused on the recognition of human elbow flexion and extension on the sagittal plane for bilateral rehabilitation with only one electrode attached to the bicep muscle. The subjects were required to perform this motion in the sagittal plane without any load on their upper limbs, except for an inertia sensor (MTx) (30 g). It is more difficult to build the relationship between the sEMG signals and human motion. For bilateral rehabilitation, continuous motion recognition is necessary in real-time. In our previous research, we had developed the upper limb rehabilitation robotics and conducted some experiments for processing the sEMG signals with healthy subjects ,16,17,18To build the mapping between the sEMG signals and elbow flexion and extension on the sagittal plane when the motion is performed very smoothly, so that the dynamic efficacy can be omitted, the statics are analyzed, which are easier and simpler than dynamics. It is not difficult to notice that there are two main forces involved in this motion: gravity and muscle extraction force of the biceps. Thus, it is possible to acquire and process the sEMG signals from the biceps to find the relationship between elbow flexion and extension. The most important phase is to extract the features. In the case of discrete recognition, acquired features are classified into several groups, so the recognition results would be correct as long as the features are classified into the right groups. However, in the case of continuous recognition, the features are not classified into certain groups, but mapped into the quantitative values, such as position, torque, and so on. Therefore, more discriminative features are required for continuous recognition than those for discrete recognition. To evaluate our proposed sEMG processing method, four discrete patterns were classified during elbow flexion/extension, with the upper arm remaining static and perpendicular to the ground, with the forearm hanging down (A), flexion towards horizontal plane (B), lifting in the horizontal plane (C), and extension back to the vertical plane (D) . To ensuThe bipolar surface electrodes were used and the sampling rate is 1000 Hz . The eleSixteen subjects  participated in the experiments. They were asked to perform elbow flexion and extension ten times as shown in Wavelet transform was proposed because its advantages not only involve time and frequency domain features of EMG signals, but also exacting certain frequency domain features such as the frequency section of most EMG signals. Wavelet Packet Transform (WPT) generates a full wavelet basis decomposition tree. In each scale, not only the approximation signals, but also the detail signals are filtered to obtain two additional low and high-frequency signals.s(t), whose scaling spaces are assumed as nth subspace on the jth level, can be reconstructed by Equation (3):For given sEMG signals tion (2) :(2){u2nIn this paper, the Daubechies wavelet was used as the mother wavelet. In addition, we chose Daubechies 2 and decomposed raw sEMG signals to the fourth level. The initial features can be obtained via the reconstructed wavelet via Equation (4). WPT generates a high-dimension feature vector. Some research proposed many methods to reduce the dimensions to save on calculation costs, such as principle component analysis and a self-organizing feature map . In thisAfter processing via WPT, sEMG signals were still not used to obtain the features of motion directly. The target of feature extraction is to obtain some typical data, which can reflect the motion patterns of human motion. Currently, most research in motion pattern recognition using sEMG signals focuses on discrete motion patterns such as hand opening, grasping, and so on, and traditional feature extraction methods can obtain the typical data to reflect the motion characteristics, such as MAV.● Mean Absolute ValueIn general, the entropy of the reconstructed sEMG was commonly discussed. Meanwhile, Mahdi et al.  proved tIn this research, motion patterns to be recognized are continuous in the time domain, which required the processed sEMG to have highly distinguishing characteristics related to elbow flexion and extension. To achieve this goal, a new feature extraction method was proposed in this paper with weighted peaks, which involve the following steps:● Zero CrossingAll the reconstructed sEMG signals of zero crossing are saved to obtain peaks and valleys among them.● Trend Acquisition with Weighted PeaksHuman motion information is immersed in the peaks of sEMG signals, not in one peak, but in many series of peaks. Thus, groups of peaks are chosen and processed to find the implied information:If P(i) are the peaks or valleys between the data of zero crossing, and valleys are transformed into positive numbers (see Else if bers see .According to the comparison between the peaks of the reconstructed sEMG signals and human elbow flexion and extension, it was found that the higher peaks reflected the trend of motion more than the lower peaks. Therefore, the method of weighted peaks was proposed to increase the component of higher peaks and decrease the component of lower peaks to obtain features close to the motion of the subject’s forearm. ● Adapted Weighted Peaks MethodFrom If If Though traditional feature extraction methods perform well in discrete pattern recognition, they would not deal with the continuous pattern recognition using weak sEMG signals. In order to compare the proposed method of feature extraction via weighted peaks to obtain the sEMG trend with the traditional MAV method, LIBSVM library  developeBecause each subject was required to perform elbow flexion and extension with his upper arm relaxed in the sagittal plane at a very low and constant speed, the dynamics of the forearm can be omitted. The processed sEMG signals derived via weighted peaks and adapted weight peaks show a trend of signal variation consistent with the human motion at a certain degree; further details are given in the following section. Therefore, it is possible to use simple linear fitting to build the relationship between the sEMG signals and elbow flexion and extension. Namely, the activation of the bicep muscle is considered to be proportional to the motion of the subject’s forearm. On the other hand, to implement the real-time continuous recognition, we adapted a simple proportional fitting line to map the weighted peaks of sEMG signals to the motion:a and b. For example, according to the ten-time experimental results from subject 1, the average of a is 710, and the value of b is −43.The least squares method was used to obtain the parameters Four of the subjects were required to perform elbow flexion and extension in the sagittal plane with their upper arms relaxed. The rational angle of the forearm is about 90 deg in the sagittal plane from a posture with the forearm vertical to the ground to the posture of the forearm being parallel to the horizontal plane. One example of the first level of experiments was shown in B was not set low enough. However, if the certain value B was set too low, the variation of sEMG peaks would be reduced, and the posture of the subjects’ forearms could not be predicted accurately. From With the increasing demands of rehabilitation for patients who have lost motor function, preliminary research for bilateral rehabilitation based sEMG signals was conducted in this paper, where human elbow flexion and extension were recognized continuously with only sEMG signals from biceps used during the experiments conducted. Wavelet transform packet is used to filter the raw sEMG signals. To obtain the variation trends of sEMG signals which are relative to human motion, a new method of processing of weighted peaks of sEMG signals is proposed as well as adapted weighted peaks. Moreover, the linear fitting method was used to map the features calculated by the proposed methods to the motion of elbow flexion and extension, so that the continuous motion of the forearm in the sagittal plane can be obtained within an acceptable range. According to the contrastive experiments, the proposed methods obtained the trend of sEMG signals with a higher recognition rate for both subjects.SEMG signals have the potential to be used to estimate the dynamics of human limbs, which is of benefit to rehabilitation, especially to bilateral rehabilitation, where hemiplegic patients can train their affected limbs following the motion of unaffected limbs via some rehabilitation devices. Traditional methods to process the sEMG focused on motion discrete pattern recognition, which is not satisfactory for use in bilateral rehabilitation. To overcome this problem, in this paper, the mapping between sEMG signals and human motion in elbow flexion and extension on the sagittal plane was proposed. Participants were required to perform elbow flexion and extension on the sagittal plane smoothly, with only an inertia sensor in their hands, without considering forearm dynamics, where sEMG signals were weak compared to those with heavy loads or high acceleration. The contrastive experimental results show that continuous motion can also be obtained within an acceptable precision range. In the future, these results will be improved and used to develop the exoskeleton device in order to aid bilateral rehabilitation for upper limbs and lower limbs.


Streptococcus pneumoniae and Neisseria meningitidis. This study therefore shows that the bacteria capable of causing meningitis are already able to do this upon entering the blood, and no further sequence change is necessary to cross the blood–brain barrier. Our findings place the focus back on bacterial evolution between nasopharyngeal carriage and invasion, or diversity of the host, as likely mechanisms for determining invasiveness.Recent studies have provided evidence for rapid pathogen genome diversification, some of which could potentially affect the course of disease. We have previously described such variation seen between isolates infecting the blood and cerebrospinal fluid (CSF) of a single patient during a case of bacterial meningitis. Here, we performed whole-genome sequencing of paired isolates from the blood and CSF of 869 meningitis patients to determine whether such variation frequently occurs between these two niches in cases of bacterial meningitis. Using a combination of reference-free variant calling approaches, we show that no genetic adaptation occurs in either invaded niche during bacterial meningitis for two major pathogen species,  CNV, copy-number variation; CSF, cerebrospinal fluid; HPD, highest posterior density; MLST, multilocus sequence type; NRLBM, Netherlands Reference Laboratory for Bacterial Meningitis; SNP, single-nucleotide polymorphism.www.ebi.ac.uk/ena/data/view/ERP004245).Read data, assembled and annotated contigs are deposited in the European Nucleotide Archive (ENA): study accession number ERP004245 .Sample metadata used has been deposited in Figshare: DOI: 10.6084/m9.figshare.4329809  . These rPreviously, it was thought that mutation rates in bacterial genomes were low, and as such there would be no change within a single host . ThroughSuch within-host variation has been shown to occur through a variety of mechanisms, such as recombination , gene loS. pneumoniae  and 0.98 (95 % HPD 0.35–1.55) in the blood. Looking at each sample pair individually, the difference between diversity in each niche appears normally distributed with a mean of zero . Together, these observations suggest a similar rate of diversity generation in each niche. This is in line with our assumption that the two populations have similar mutation rates, and a similar number of generations between being founded and being sampled.As the speed of inversion is rapid, the subsequent polymorphism of this locus was also used to evaluate our assumptions about diversity of the bacterial population within each niche. We calculated the Shannon index of each sample’s vectors πWe made assumptions about the evolution of bacteria within the host, under which we discuss the power of pairwise comparisons between single colonies taken from each niche to capture repeated evolution occurring post-invasion. (i) There is a bottleneck of a single bacterium upon invasion into the first sterile niche , which then founds the post-invasion population . (ii) A 5 c.f.u.) [Initially the population size is small, so selection is inefficient and the population-wide mutation rate is low. However, the eventual carrying capacity (the maximum number of cells) of the blood and CSF are large enough (>1.5×10 c.f.u.)  for bene c.f.u.) , this ca c.f.u.) .Crucially, population sizes where selection acts efficiently  are reacivr locus (see Methods).Similarity of the bacterial growth rate within blood and CSF is an important assumption because in 45 % of the pneumococcal cases there was evidence that CSF invasion happened before blood invasion  . This alS. pneumoniae, we found that two additional passages of the previous sample pair resulted in one additional insertion. In N. meningitidis, a low rate of variation, no selection on phase-variable regions and no variation of other regions have been observed during culture steps [Finally, we considered whether the culturing process would bias our results. In re steps . We therWe performed comparisons between the pan-genome of each pair of blood and CSF isolates, using reference-free variant calling techniques (see Methods). The method was evaluated using simulated data, giving us confidence that it could detect the small amounts of variation expected between each isolate pair .S. pneumoniae, 452 of 674 paired samples (67 %) were identical. The distribution of number of variants between isolate pairs was roughly Poisson (mean=0.547), excluding outliers. Variation between N. meningitidis pairs also followed a roughly Poisson distribution (mean=2.34), which when compared to S. pneumoniae showed a higher number of variants between blood and CSF isolates  such that most pairs had at least one variant between the blood and CSF samples.For each species, we then counted the number of variants of any type between each blood/CSF isolate pair taken from a patient . In S. pIn both species, the mutations that do exist, if they cause the same functional change, could represent a signal of adaptation. To determine whether this was the case, we counted the number of times each gene contained variation between the blood and CSF isolate over all the pairs collected, and used a Poisson test to determine whether this was more than expected for each gene (see Methods).S. pneumoniae, the results are shown in dlt operon, responsible for d-alanylation in teichoic acids in the cell wall [P<10−10). This occurred in only 5 % of samples, so adaptation to a niche due to variation in genes is not common.For ell wall , was theS. pneumoniae strain, which has a functional dlt operon and was therefore assumed to be the ancestral state. There was no directionality to the mutations: 19 occurred in the blood, and 11 in the CSF (P=0.2). Only seven of the patients infected by these strains showed signs of blood invasion before CSF invasion (sinusitis or otitis); this also did not show directionality. The nature of the mutations is shown in To investigate whether this represented adaptation to either blood or CSF, we annotated the effect of these variants, and determined whether they were specific to a niche. We mapped them to the R6 In all the other genes in N. meningitidis are shown in pilE (19), pilC (6) and pilQ (4). Pilus genes are associated with immune interaction [P=0.024) is consistent with this. The other notable gene with more mutations than expected in N. meningitidis was porA, encoding a variable protein that is a major determinant of immune reaction [The most frequently mutated genes between pairs in eraction , and arereaction , in whicpilE, hpuA, wbpC, porA and lgtB within-host has been observed previously in a single patient with a hypermutating N. meningitidis infection [The mutations in nfection . These cpatAB genes, encoding an efflux pump [Our previous result suggesting adaptation from blood to CSF was an intergenic change affecting the transcription the lux pump . In genelux pump , antimiclux pump  and chanlux pump . Changeslux pump .We therefore separately investigated the mutations in non-coding regions. Analysing the positions of these mutations required a consistent co-ordinate system across all sample pairs. We therefore remapped the co-ordinates each variant discovered in an intergenic region to the co-ordinates of the ATCC 700669 reference genome. We used population-matched carriage isolates as the ancestral state to determine whether these mutations occurred in the blood or CSF isolate.S. pneumoniae. The peaks correspond to mutations in genes described in Fig. S10 shows all mutations plotted genome-wide in N. meningitidis are plotted in Fig. S11, 110 of which were in non-coding regions. We observed enrichment, but no niche specificity, in the upstream region of six genes. These mutations are listed in porA and opc were in phase-variable homopolymeric tracts, which are discussed more fully in the section below. The other mutations were upstream of the adhesins hsf/NMB0992 and NMB1994, which are involved in colonization [frpB/NMB1988, which is a surface antigen involved in iron uptake [The mutations in nization  and immunization , and frpn uptake . DifferePhase-variable regions, which may also be intergenic, can mutate rapidly and are known to be a significant source of variation in pathogenic bacteria . This muN. meningitidis, we observed six samples with single base changes in length of the phase-variable homopolymeric tract in the porA gene’s promoter sequence, and five samples with the single base length changes in the analogous promoter sequence of opc. While changes in the length of these tracts will affect expression of the corresponding genes, both of which are major determinants of immune response [porA expression has previously been found to be independent of whether isolates were taken from CSF, blood or throat [In response , the trar throat .S. pneumoniae, recent publications have highlighted a potential role in virulence for the ivr locus, a type I restriction-modification system with a phase-variable specificity gene allele of hsdS in the host specificity domain  to determine whether any of these alleles were associated with either the blood or CSF niche specifically, which could be a sign of adaptation.In y domain  [18, 19,ivr allele . This simultaneously estimated the proportion of each colony pick with alleles A–F for both each individual isolate (π), and summed over all the samples in each niche (μ). We applied this over i samples and c niches (in this case c can be blood or CSF).As the locus inversion is rapid and occurs within-host, we first ensured that cultured samples were representative of the original clinical samples using PCR quantification of each allele. However, as even a single colony contains heterogeneity at this locus, simply taking the allele with the most reads mapping to it in each sample gives a poor estimate of the overall presence of each allele in the blood and CSF niches. To take into account the mix of alleles present in each sample, and to calculate confidence intervals, we developed a hierarchical Bayesian model for the CSF − πblood was calculated (Table S4). All S. pneumoniae samples had a difference in mean of at least one allele , highlighting the speed at which this locus inverts. While this means that between a single CSF and blood pair the allele at this locus usually changes, it is the mean of μc  that tells us whether selection of an allele occurs in either the blood or CSF more generally. This is plotted in S. pneumoniae isolates.For each pair of blood and CSF samples listed inTable S2, the difference in allele prevalence πivr allele between these niches would therefore not be detected, but we can rule out strong selection for a particular allele (odds ratio >2). Application to read data from a large carriage dataset may help resolve whether the same effect does occur in humans, as it would provide a greater temporal range over the course of pathogenesis.Previous work on a murine invasion model  showed aPrevious studies have shown that substantial levels of genomic DNA sequence variation occur in bacteria colonizing or infecting human hosts  and suggSuch adaptations during invasive bacterial disease could lead to new insights into the processes of pathogenesis with the potential to inform therapies . Therefoivr locus of S. pneumoniae. This is important as it captures some of the genome-wide epigenetic variation that affects virulence, via level of capsule expression [N. meningitidis, or provide a selective advantage outside of the nasopharynx.We found overall that blood and CSF isolates have very similar genetic sequences. The small number of mutations observed are not randomly distributed throughout the genome, but are randomly distributed between blood and CSF isolates. These mutations are therefore an observation of a higher mutation rate in these regions during invasion, but not repeated adaptation to either the blood or CSF niche specifically. This result from a much larger sample size indicates that our previous observation of variation between blood and CSF isolates from a single case of meningitis was a rare event most likely driven by antibiotic selection pressure during treatment . Supportpression . Though dlt locus in S. pneumoniae. This locus controls levels on d-alanylation of cell wall teichoic acids. These are recognized by Toll-like receptors and have been shown to increase the sensitivity of the cytokine response of the host [Staphylococcus aureus, mutation of the dlt locus has been shown to affect host tropism, though levels of d-alanylated teichoic acids did not correlate with infectivity [The most notable of these observations for a locus not already known to be under diversifying selection is the the host , while athe host . In Stapectivity .dlt operon accumulates significantly more damaging mutations than expected, implying a selection pressure against the gene function. This suggests that interaction with host AMPs changes upon shift from the nasopharynx to the invasive niche, a notion that is supported by in vitro observations that clinical isolates of S. pneumoniae are more susceptible to AMPs than carriage isolates [dlt compared to other loci. That d-alanylation can be deleterious in invasive isolates has not been previously observed, though virulent isolates are known to be viable without this modification [We observed that after bloodstream invasion, the isolates . We obsefication .It is still possible that the genetics of the invading bacterium may already determine, prior to invasion, whether CSF invasion is possible, though previous studies at the gene level suggest this is not the case . Future 1. The Wellcome Trust Sanger Institute. European Nucleotide Archive ERP004245 (2016).https://github.com/johnlees/paired-samples (2016).2. Lees JA. GitHub http://dx.doi.org/10.6084/m9.figshare.4329809 (2016).3. Lees JA. FigShare et al. GenBank NC_011900.1 (2009).4. Croucher NJ, Walker D, Romero P, Lennard N, Paterson GK et al. GenBank AE007317.1 (2001).5. Hoskins JA, Alborn W Jr, Arnold J, Blaszczak L, Burgett S et al. GenBank NC_013853.1 (2010).6. Denapaite D, Bruckner R, Nuhn M, Reichmann P, Henrich B


Samples of donor pancreatic tissue (n = 78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m2 (P < 0.001), cold ischemia time more than 8 hours (P < 0.01), hospitalization longer than 96 hours (P < 0.05), higher catecholamine doses (P < 0.05), and edematous pancreases (P < 0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P < 0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P < 0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection.Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful ( Type 1 diabetic patients require intensive insulin therapy to avoid serious complications caused by high blood glucose levels. A small number of diabetic patients experience a very labile form of diabetes with unpredictable and repeated hypoglycemia. The transplantation of pancreatic islets to these patients positively influences metabolic control and helps to prevent hypoglycemic episodes. Insulin independence in diabetic patients can be achieved by whole organ transplantation or by the transplantation of isolated islets from 2 or 3 donors [Despite continuing progress in the practice of pancreatic islet isolation, isolation outcome is highly variable and remains hard to predict. Currently, appropriate donor selection is seen as one of the keys to successful pancreatic islet isolation. The Edmonton group developed a scoring system  to prediBecause of a shortage of organ donors in the last few years, which probably reflects a greater number of people on the waiting list, there are renewed efforts to improve islet isolation from valuable single-donor pancreases. According to previously published studies, the variability of pancreatic donors may significantly influence the success with which we can subsequently isolate islets. The following donor characteristics have all been identified as being critical for isolation outcome: age , 5–8, boCurrently, in most centers, pancreases that are unsuitable for solid organ transplantation  are allocated for islet isolation. The decision as to whether to accept or decline an organ for islet isolation is based on donor characteristics together with a later macroscopic evaluation of the donor pancreas. Unfortunately, islet yield, which is the commonly used indicator of a successful isolation, is largely dependent on the islet mass in the pancreas prior to islet isolation and cannot be accurately determined ahead of pancreas processing.Precise and effective donor selection is needed to avoid discarding potentially useful donations or conversely accepting those with inadequate islet mass. The purpose of this study was to evaluate the donor and pancreas characteristics that influence the outcome of islet isolation and to investigate the value of immunohistological examination of donor pancreases for organ selection.n = 14). Pancreatic islets were isolated from 159 donors according to a modified semiautomatic Ricordi's method [n = 8) for autologous transplantation was also an exclusion criterion.Human pancreases were recovered from 173 deceased donors after brain-dead from January 2010 until December 2014. Accepted but not processed pancreases were excluded , or CIzyme™ collagenase HA  were used. The distended organ was then placed into a digestion chamber at a controlled temperature, with enzyme solution recirculation. During the digestion phase, small samples of the digested tissue from the chamber were collected, stained with diphenyl-thiocarbazone , and microscopically evaluated in real time. Digestion was stopped by cooling and dilution, with the digested pancreatic tissue collected in the washing solution. After pooling, the tissue was placed on ice in University of Wisconsin (UW) solution for approximately 1 hour. A discontinuous gradient was used to determine the pancreatic tissue density and the appropriate density range for separation. Pancreatic islets were separated from exocrine tissue by centrifugation in a continuous Biocoll gradient , using a COBE® 2991 Cell Processor . Islet containing fractions were collected, washed, and counted. Isolated islets were then cultured in supplemented CMRL-1066 medium , in a humidified incubator at 37°C and a 5% CO125I RIA Kit . Results were expressed as stimulation indices.The minimum islet mass considered for islet transplantation is 4000 islet equivalents (IE) per kg body weight of the recipient. Therefore pancreatic islet isolations with islet yields of ≥250 000 IE, a purity of at least 30%, and viability greater than 80%, were considered successful. The quality of isolated islets was assessed by live/dead cells differential staining and by glucose stimulated insulin secretion, as previously described . BrieflyBased on the Edmonton scoring system the selected donor characteristics of age, BMI, cold ischemia time (CIT), the cause of death, intensive care hospitalization, the levels of serum amylase, use of vasopressors, blood glucose levels, organ procurement, and social and medical history were evaluated. The maximum score is 100 donor points (DP), while the selected organ characteristics of size, consistency (edema or fibrosis), damage, quality of flush, procurement, and packaging only serve to negatively influence the donor score. Based on the final score, donors were divided into 6 categories: poor donors (0–49.5 DP), marginal donors (50–59.5), intermediate donors (60–69.5 and 70–79.5 DP), and optimal donors (80–89.5 and 90–100 DP) . The freμm thick sections were deparaffinized in xylene and rehydrated in a graded ethanol series. After rinsing in 0.2% Triton X 100 and PBS, endogenous biotin was blocked using the biotin blocking system , with endogenous peroxidase blocked with 0.3% H2O2 in 70% ethanol. To prevent nonspecific binding, samples were preincubated with 10% horse serum. For beta cell detection, specimens were incubated with a primary anti-insulin antibody  and detected using a Histofine Simple Stain Rat MAX PO . After incubation with Dako Liquid DAB+ Substrate-Chromogen System , specimens were counterstained with hematoxylin. Pancreatic tissue slices (n = 78) were scanned using the EVOS FL Auto imaging system . Images of entire sections were analysed using the ImageJ software . Pancreatic tissue was manually outlined and insulin-positive cells were selected using the color threshold tool. The percentage of insulin-positive area to total pancreatic area was then calculated.From August 2011, pancreatic tissue samples were recovered for histological examination. Pancreatic tissue was taken from the head or neck of the donor pancreas depending on whether one or two cannulas for collagenase perfusion had been applied. The samples were fixed in 10% formaldehyde with chalk at 4°C overnight. Routine hematoxylin & eosin staining was performed for basic histological assessment. For insulin detection, immunohistochemical staining was used. Four U test or Student's t test; differences between proportions were compared using the Chi-square test. To predict isolation outcome based on immunohistochemical evaluation and donor score, receiver operating characteristic (ROC) analyses were performed and area under the curve (AUC) values were calculated. The associations between selected donor characteristics including the area of insulin staining and donor score were analysed using the Pearson correlation coefficient. Multivariate stepwise logistic regression analysis was then used to determine isolation success based on these two criteria. A P value <0.05 was considered to indicate statistical significance.Results are expressed as mean ± SD. To test for a normal distribution of our data, the Lilliefors test for normality was performed. Differences between groups were compared using the Mann–Whitney P > 0.05).From January 2010 until December 2013 our isolation center received 147 donor pancreases for islet isolation. Because of damage during procurement, 8 pancreases were rejected. 139 donor pancreases were isolated and retrospectively evaluated to assess donor points see . The freIn 2014, 26 donors were accepted for pancreatic islet isolation. According to the results from our retrospective study, exclusion of poor donors at our center was prohibited given that just over a fifth (21.4%) of islet isolations from these donors were successful. Donor selection was therefore based primarily on previous experience, which led to the rejection of 5 edematous pancreases. Moreover, one pancreas was rejected because of a procurement-related issue. We found that islet isolation from poor or marginal donor pancreases  was unsuccessful. The frequency of donors with a DP rating of 60 to 69.5 was 15%; from these, 66.7% of islet isolations were successful. The most frequent donor group (45%) was the 70 to 79.5 DP group, for which successful isolations were achieved in 44.4% of cases. Islet isolations from optimal donors (15%) were all successful. In 2014, our overall success rate increased to 45.0%. Over the last 5 years, any donor with a DP of more than 90 was available for pancreatic islet isolation in our center.P > 0.05). However, the quality of the islets, as assessed by glucose stimulated insulin release, revealed significant differences. The stimulation indexes of the transplanted versus nontransplanted islets were 7.6 ± 5.5 versus 5.8 ± 6.0, respectively (P < 0.01).The number of islet isolations per year has continuously decreased in our isolation center (from 46 in 2010 to 20 in 2014); in average we had 32 isolations annually. The average islet yield from all 159 isolations was 223 289 ± 131 276 IE, while 61 successful isolations produced 356 328 ± 87 356 IE. The mean purity of the transplanted islets was 49.7 ± 8.0%. Islet transplantation was performed for 50 recipients; 19 patients received a single infusion, 11 patients received two infusions, and 3 patients received three islet infusions. In vitro analyses of the isolated islets showed comparable viability of the transplanted and nontransplanted islets at 94.9 ± 4.2% versus 91.0 ± 7.1%, respectively (n = 61) and failed (n = 98) islet isolations (see 2 was strongly associated with islet isolation failure (P < 0.001), while islet isolations from donors with a BMI less than 20 kg/m2 (n = 8) were completely unsuccessful (P < 0.05). Higher doses of vasoactive drugs (P < 0.05), hospitalization in excess of 96 hours (P < 0.05), and cold ischemia for more than 8 hours (P < 0.01) all had adverse effects on islet isolation. Selected pancreatic characteristics such as weight, fatty infiltration, and capsule injury were similar for both groups (P > 0.05), although significant differences were observed in pancreas consistency; edematous pancreases negatively influenced isolation outcome (P < 0.01). Digestion time was also strongly associated with the isolation outcome (P < 0.01).To identify the critical factors associated with isolation outcome, selected donor characteristics were retrospectively analysed in successful (ions see . While nn = 3). Histological examination of donor pancreases was performed on 78 samples obtained prior to islet isolation. Basic histological examination of donor pancreases showed no direct impact on isolation outcome  was 1.43 ± 0.6, significantly higher than that for the failed group (n = 41), which was 1.02 ± 0.7 (P < 0.001). An increased insulin-positive area was therefore strongly associated with a successful isolation outcome. To demonstrate the ability to predict isolation outcome using this criterion, a ROC analysis was performed using the insulin-positive areas determined for donor pancreases .Analyses of immunohistochemical data showed that successful isolations were achieved from organs with an increased insulin-positive area. Representative micrographs of pancreatic tissue are shown in ases see . This an0.05 see . The opt r correlation coefficient , BMI , BSA , and donor score  and negatively with digestion time . Multivariate stepwise logistic regression was then performed to show the predictability of isolation outcome with the multivariable use of the immunohistochemistry data with the donor scoring system  provides positive evaluations for BMI values up to 52 kg/m2. We can expect that donors with higher BMI have larger pancreases that contain more islets. Although pancreatic weight cannot be assessed before pancreas acceptance, the study of Kin et al. [Positive correlations between BMI and isolation success have been consistently reported , 8, 16. n et al.  showed tDonor organ preservation has been the subject of multiple studies although the results are controversial. Qin et al. , in multAfter organ acceptance, donor pancreases are macroscopically evaluated in an islet isolation laboratory. Based on previously published data, a large pancreas probably associated with a higher BMI, as well as fatty infiltration, increases the success of islet isolation . A higheIn conclusion, the size of insulin-positive areas in donor pancreases is strongly associated with the islet isolation outcome. Therefore immunohistological examination of donor pancreases might be helpful in guiding organ selection. We found that the prediction of isolation outcome could be improved by combining immunohistological examination with the donor scoring system. A caveat to this is that while this method cannot eliminate all unsuccessful donors, it can enhance isolation success and at the same time decrease costs resulting from failed isolations.