By observing handwashing behavior in 347 households from 50 villages across rural Bangladesh in 2007, Stephen Luby and colleagues found that hand washing with soap or hand rinsing without soap before food preparation can both reduce the burden of childhood diarrhea. Standard public health interventions to improve hand hygiene in communities with high levels of child mortality encourage community residents to wash their hands with soap at five separate key times, a recommendation that would require mothers living in impoverished households to typically wash hands with soap more than ten times per day. We analyzed data from households that received no intervention in a large prospective project evaluation to assess the relationship between observed handwashing behavior and subsequent diarrhea.Fieldworkers conducted a 5-hour structured observation and a cross-sectional survey in 347 households from 50 villages across rural Bangladesh in 2007. For the subsequent 2 years, a trained community resident visited each of the enrolled households every month and collected information on the occurrence of diarrhea in the preceding 48 hours among household residents under the age of 5 years. Compared with children living in households where persons prepared food without washing their hands, children living in households where the food preparer washed at least one hand with water only , washed both hands with water only , or washed at least one hand with soap  had less diarrhea. In households where residents washed at least one hand with soap after defecation, children had less diarrhea . There was no significant association between handwashing with or without soap before feeding a child, before eating, or after cleaning a child's anus who defecated and subsequent child diarrhea.These observations suggest that handwashing before preparing food is a particularly important opportunity to prevent childhood diarrhea, and that handwashing with water alone can significantly reduce childhood diarrhea.Please see later in the article for the Editors' Summary The resurgence of donor interest in regarding water and sanitation as fundamental public health issues has been a welcome step forward and will do much to improve the health of the 1.1 billion people world-wide without access to clean water and the 2.4 billion without access to improved sanitation. However, improving hygiene practices is also very important—studies have consistently shown that handwashing with soap reduces childhood diarrheal disease—but in reality is particularly difficult to do as this activity involves complex behavioral changes. Therefore although public health programs in communities with high child mortality commonly promote handwashing with soap, this practice is still uncommon and washing hands with water only is still common practice—partly because of the high cost of soap relative to income, the risk that conveniently placed soap would be stolen or wasted, and the inconvenience of fetching soap.Handwashing promotion programs often focus on five “key times” for handwashing with soap—after defecation, after handling child feces or cleaning a child's anus, before preparing food, before feeding a child, and before eating—which would require requesting busy impoverished mothers to wash their hands with soap more than ten times a day.In addition to encouraging handwashing only at the most critical times, clarifying whether handwashing with water alone, a behavior that is seemingly much easier for people to practice, but for which there is little evidence, may be a way forward. In order to guide more focused and evidence-based recommendations, the researchers evaluated the control group of a large handwashing, hygiene/sanitation, and water quality improvement program—Sanitation, Hygiene Education and Water supply-Bangladesh (SHEWA-B), organized and supported by the Bangladesh Government, UNICEF, and the UK's Department for International Development. The researchers analyzed the relationship between handwashing behavior as observed at baseline and the subsequent experience of child diarrhea in participating households to identify which specific handwashing behaviors were associated with less diarrhea in young children.The SHEWA-B intervention targeted 19.6 million people in rural Bangladesh in 68 subdistricts. In this study and with community and household consent, the researchers organized trained field workers, using a pretested instrument, to note handwashing behavior at key times and recorded handwashing behavior of all observed household at baseline in 50 randomly selected villages that served as nonintervention control households to compare with outcomes to communities receiving the SHEWA-B program. The fieldworkers recruited community monitors, female village residents who completed 3 days training on how to administer the monthly diarrhea survey, to record the frequency of diarrhea in children aged less than 3 years in control households for the subsequent two years. The researchers used statistical models to evaluate the association between the exposure variables (household characteristics and observed handwashing) and diarrhea.Using these methods, the researchers found that compared to no handwashing at all before food preparation, children living in households where the food preparer washed at least one hand with water only, washed both hands with water only, or washed at least one hand with soap, had less diarrhea with odds ratios (ORs) of 0.78, 0.67, and 0.19, respectively. In households where residents washed at least one hand with soap after defecation, children had less diarrhea (OR = 0.45), but there was no significant association between handwashing with or without soap before feeding a child, before eating, or after cleaning a child's anus, and subsequent child diarrhea.These findings from 50 villages across rural Bangladesh where fecal environmental contamination, undernutrition, and diarrhea are common, suggest that handwashing before preparing food is a particularly important opportunity to prevent childhood diarrhea, and also that handwashing with water alone can significantly reduce childhood diarrhea. In contrast to current standard recommendations, these results suggest that promoting handwashing exclusively with soap may be unwarranted. Handwashing with water alone might be seen as a step on the handwashing ladder: handwashing with water is good; handwashing with soap is better. Therefore, handwashing promotion programs in rural Bangladesh should not attempt to modify handwashing behavior at all five key times, but rather, should focus primarily on handwashing after defecation and before food preparation. Furthermore, research to develop and evaluate handwashing messages that account for the limited time and soap supplies available for low-income families, and are focused on those behaviors where there is the strongest evidence for a health benefit could help identify more effective strategies.http://dx.doi.org/10.1371/journal.pmed.1001052.Please access these Web sites via the online version of this summary at PLoS Medicine, called “Water and Sanitation,” provides information on water, sanitation, and hygieneA four-part collection of Policy Forum articles published in November 2010 in improving hygiene practicesHygiene Central provides information on  Intervention trials promoting handwashing with soap in communities with high child mortality consistently report a reduction in childhood diarrheal disease A second common characteristic of handwashing promotion programs is a focus on five “key times” for handwashing. These include handwashing after defecation, after handling child feces or cleaning a child's anus who had defecated, before preparing food, before feeding a child, and before eating Two steps that might improve the effectiveness of handwashing promotion interventions would be to encourage handwashing only at the most critical times for interrupting pathogen transmission and clarifying whether handwashing with water alone, a behavior that is apparently much easier for people to practice, should be encouraged. There are however, few data available to guide more focused recommendations.In 2007, the Government of Bangladesh Department of Public Health Engineering in collaboration with UNICEF and with support from the Department for International Development (DFID) of the British Government launched a program, Sanitation, Hygiene Education and Water supply-Bangladesh (SHEWA-B) that is among the largest intensive handwashing, hygiene/sanitation, and water quality improvement programs ever attempted in a low-income country. The intervention targeted 20 million people in rural Bangladesh. As part of the assessment of the program's impact, fieldworkers conducted household structured observations at baseline in 50 randomly selected villages that served as nonintervention control households to compare with outcomes to communities receiving the SHEWA-B program. Community monitors assessed the frequency of diarrhea in control households each month for the subsequent 2 y. We analyzed the relationship between handwashing behavior as observed at baseline and the subsequent experience of child diarrhea in these households. The objective of this analysis was to identify which specific handwashing behaviors were associated with less diarrhea.UNICEF publicly requested bids for the evaluation of the SHEWA-B program. The International Centre for Diarrhoeal Disease Research, Bangladesh  was selected through a competitive process and signed a contract with UNICEF for the evaluation. From UNICEF's perspective, and the perspective of the Government of Bangladesh, this was not a research contract. It was a contract to evaluate a US$90 million program targeting 20 million people across rural Bangladesh. The Government of Bangladesh separately contracted with 58 organizations to implement the intervention across 68 rural subdistricts on an aggressive launch schedule. The program evaluation required a preintervention baseline survey. If the evaluation team postponed field work for the 12- or more wk process that is characteristic for local human study participant protocol review and approval, ICDDR,B would have been unable to provide a preintervention measurement. This would have reduced the ability of ICDDR,B to assess the program, and would represent a failure to meet contractual obligations. We received ICCDR,B administrative approval to classify this activity as a nonresearch program evaluation that did not require independent human study participant review because the primary goal of this activity, particularly from the sponsor's perspective, was program evaluation and not generation of new generalizable knowledge.The plan for the evaluation was reviewed by UNICEF and the Government of Bangladesh Department of Public Health Engineering, but was not reviewed by an independent human study participant committee. Each field worker received formal training in taking informed consent. As part of the consent process the field worker clarified how much time we were requesting from prospective participants. Field workers explained that there was no individual benefit or compensation for participation, that there would be questions about use of water, toilet facilities, and handwashing, and noted that these topics may be uncomfortable or that it may be uncomfortable to have a stranger in their home observing household activities. Twice during the consent process the field workers specified that participation was voluntary. They explained that even if the participant originally provided consent, he or she could withdraw consent at any time. Field workers secured written informed consent from each participant. Fields workers provided participants with contact information for the study coordinator and the research administration office of ICDDR,B if they had any questions. All collected information was kept in locked rooms. Only staff whose responsibilities included working with the data had access to the data. Study supervisors made unannounced visits to field teams to ensure that field workers properly implemented the enrollment and consent process.upazilas) in 19 districts. The government and UNICEF selected the specific intervention upazilas because of the perceived need and the absence of other active programs addressing water, sanitation, and hygiene in these communities. Upazilas are further subdivided into unions. We listed all of the unions and their populations in the 68 targeted subdistricts and randomly selected 50 unions with the probability of selection proportional to the size of the union. For each SHEWA-B intervention upazila where a union was chosen for evaluation we selected a control upazila that had similar geography, hydrogeology, infrastructure, agricultural productivity, and household construction, and where the government confirmed that no other major water-sanitation-hygiene programs were ongoing. We selected unions for evaluation in the control upazilas using the same probability of selection proportional to size used to select unions for evaluation in the intervention upazilas.The SHEWA-B program targeted 68 subdistricts  from each evaluation community who was at least 18 y of age and who had completed at least 8 y of formal education to visit each of the enrolled households each month and administer a brief questionnaire to collect information on each child <5 y of age. These community monitors participated in a formal training program to learn how to administer the questionnaire. The initial 3-d training included 2 d of classroom instruction with role playing followed by 1 d of field testing. After 12 mo the community monitors participated in a 1-d refresher training session. One of the monthly surveillance questions asked whether the child had diarrhea during the preceding 2 d. Another question asked if the child consumed only breast milk in the preceding 24 h. This surveillance continued for 24 mo following the cross-sectional survey. The community monitors were paid a modest stipend.Drawing water from a hand pump to wash hands or pouring water from a pitcher requires one hand. Rural Bangladeshi residents commonly run water over the “dirty hand” to clean it. If they use soap they sometimes roll the soap within the one hand. For each of the five key handwashing opportunities we classified the handwashing behavior into one of five categories: (1) no observed handwashing, (2) washing one hand with water alone, (3) washing both hands with water alone, (4) washing at least one hand with ash or mud, and (5) washing at least one hand with soap. We selected these categories because across the five key handwashing opportunities these categories displayed a range of handwashing behaviors that generally included sufficient observations to support analysis of associations.Within each household, during the 5 h of observation fieldworkers often observed multiple occasions of the same opportunity for handwashing. For example, because there may be multiple children in the household and multiple snacks in addition to the main meal, the fieldworkers might observe four opportunities to wash hands before feeding a child. The handwashing behavior might be different in each of these four episodes, and fieldworkers recorded each episode separately. We classified the thoroughness of handwashing along a scale: the least thorough handwashing was no handwashing, washing one hand with only water, washing both hands with only water, washing with mud/ash, and washing with soap were progressively classified as more thorough handwashing. The household's handwashing behavior for each key time was classified on the basis of the most thorough handwashing behavior observed.We considered drying hands with a clean towel or allowing hands to air dry before touching another surface as optimal hand drying. We classified drying hands on a visibly dirty towel, drying hands on clothing, or not drying hands before touching another surface, as progressively less optimal hand drying. We classified the household's hand drying behavior for each handwashing opportunity on the basis of the most optimal hand drying behavior observed at that key time.We used principal component analysis of 21 household characteristics  to evaluWe calculated odds ratios to evaluate the association between the exposure variables—household characteristics and observed handwashing—and diarrhea. To account for the repeated observations for diarrhea in single households and the clustering of observations in villages we used general estimated equations to calculate these adjusted odds ratios and 95% confidence intervals p<0.05) with diarrhea in the bivariate analysis. We began with a bivariate model that included handwashing behavior, and sequentially added each of the household characteristics that were associated with diarrhea in bivariate analyses. The final multivariate model retained all those variables that both significantly improved fit of the model (p<0.05) and were independently associated with diarrhea (p<0.05).We constructed a multivariate model for each of the key times when handwashing behavior was significantly associated  Version 9.1 (SAS Institute) for the general estimated equations modeling.The evaluation team completed structured observations, baseline cross-sectional interviews, and initiated monthly surveillance in 347 households that did not receive the SHEWA-B intervention. Community monitors collected data on 465 children who lived in these 347 households for at least 1 mo. In the first month of diarrheal surveillance, there were 379 children <5 y living in these households. Their mean age was 19.2 mo. During 24 mo of follow-up, 66 children were born into these households, 20 children moved into the surveillance households, 24 children moved or dropped out, one child aged out, and 12 children died. The mean age of participating children after 24 mo was 37.7 mo. Among the 10,234 potential monthly child assessments from the time a child was first identified in a surveillance household, community monitors completed data collection for 9,897 (97%).A third of the fathers and more than a quarter of the mothers had no formal education . Over 90Caregivers reported that the child had diarrhea in the 48 h preceding the monthly interview in 947 (9.6%) of the 9,897 monthly assessments. In the bivariate analysis, household characteristics that were significantly associated with less child diarrhea included the mother or father having 7 or more years of education, a wealth index in the fourth quintile and ownership of a television, radio or mobile phone . ChildreMothers reported at least some breast-feeding of their <1-y-old children in the preceding 24 h in 93% of monthly visits and reported exclusive breastfeeding of their <6-mo-old children in the preceding 24 h in 55% of monthly visits. Young children were both more likely to be breastfed and more likely to have diarrhea. After adjustment for age, neither any reported breastfeeding nor exclusive breast-feeding was associated with significantly less diarrhea.Fieldworkers observed at least one opportunity to wash hands before preparing food in 281 (81%) of the households during structured observation. Handwashing before preparing food was associated with less diarrhea in the subsequent 2 y of follow-up . In housFieldworkers observed at least one opportunity to wash hands after defecation in 102 (29%) of the households during structured observation. Fieldworkers observed only a single household where residents never washed their hands after defecation. Handwashing with soap was much more common after defecation than before food preparation. In 25% of households, at least one household resident washed at least one hand with soap after defecation. Among the 27 observed episodes of handwashing with soap after defecation, in eight (30%) both hands were washed with soap. Children who lived in households where fieldworkers observed at least one hand washed with soap after defecation experienced substantially less diarrhea in the subsequent 2 y of follow-up compared with children who lived in households where only one hand was washed with water after defecation .The fieldworkers' observations of handwashing before feeding a child, before eating, and after cleaning a child's anus who had defecated were not associated with subsequent diarrhea .Among household residents observed washing hands, fieldworkers observed optimal hand drying, either allowing hands to air dry or drying hands on a clean towel, uncommonly, ranging from 3% before eating to 17% after cleaning a child's anus who had defecated. Children who lived in households where optimal hand drying was observed had somewhat less diarrhea than other children, but none of the observed differences were statistically significant.In the multivariate analysis of structured observations before preparing food, washing both hands with water only and washing at least one hand with soap were both independently associated with significantly less diarrhea morbidity during 7,999 subsequent monthly assessments for diarrhea . The numIn the multivariate analysis of structured observations after defecation, washing at least one hand with soap was independently associated with significantly less diarrhea in the 2,952 subsequent monthly assessments . With a In 50 villages across rural Bangladesh where fecal environmental contamination, undernutrition, and diarrhea are common, in those households where fieldworkers observed food preparers washing their hands before handling food, children under the age of 5 y experienced less diarrhea over the next 2 y compared with children living in households where food preparers did not wash their hands before preparing food. This observation suggests that before preparing food may be a particularly important time to promote handwashing Shigella and SalmonellaTomatoes, cucumbers, carrots, and various seasonal vegetables and greens are common components of meals in rural Bangladesh. Some of these vegetables are served raw but most are boiled and made into a curry that is commonly served with rice, the primary staple of the Bangladeshi diet. Many foods that are not further cooked, for example boiled root vegetables, fruits including bananas, and dried fish are often mashed and mixed by hand with spices and other ingredients during food preparation. Raw vegetables are commonly contaminated with pathogens and are a common vehicle for gastrointestinal pathogen transmission. Numerous outbreaks of gastroenteritis from a variety of pathogens have been traced to raw vegetables If persons preparing food did not wash their pathogen-contaminated hands before touching raw vegetables and rice, these foods may have become contaminated with gastrointestinal pathogens, which could subsequently multiply in a conducive growth environment before consumption. However, if the vegetables were cooked at a high enough temperature for a long enough time the pathogens would not survive and would not be transmitted. In future research, it would be useful to have fieldworkers specifically code the context of the handwashing opportunity around food preparation, so that the association between handwashing before handling raw vegetables and other foods that were subsequently cooked, handwashing before handling foods that were eaten raw, and handwashing before cross contaminating food that was not further cooked could be separately assessed.In contrast to standard recommendations for handwashing that stress the central importance of using soap and specify detailed techniques for washing underneath fingernails, continuing lathering for over 20 s, and using either a clean towel or air drying to ensure effective handwashing Although the benefits of handwashing with water alone observed in this evaluation conflict with standard recommendations, they are consistent with an older randomized controlled intervention study from urban Bangladesh. Stanton and Clemens used structured observation to observe handwashing behavior and noted an association between washing hands with or without soap and reported childhood diarrhea in a case control study in low-income urban communities in Dhaka Bangladesh Microbiological studies demonstrate that washing hands with water alone reduces the concentration of various bacteria on hands The low proportion of households that followed recommended hand drying procedures suggests that substantial efforts would be required to change community habits to conform with hand drying recommendations. Since children living in households that practiced recommended hand drying behavior did not have significantly less diarrhea than other households, these data suggest that efforts to promote improved hand hygiene would be better focused on behaviors more strongly associated with child health, for example on handwashing before preparing food and after defecation, than on prescribing specific hand drying behavior.People wash their hands more frequently when they know they are being observed But there are two difficulties with this alternative interpretation. First, the strong association of handwashing with water alone before preparing food with diarrhea is less likely to result from social desirability bias, because there is no strong cultural norm for handwashing before preparing food. Indeed, only 1% of households washed hands with soap before preparing food. If household residents washed hands before preparing food because of social desirability bias that was then linked to other behaviors associated with less diarrhea, then we would also expect to find a significant association of diarrhea with handwashing before eating, before feeding a child, and after cleaning a child who defecated, associations that were not significant in this analysis. A second difficulty with attributing the observed associations to a theoretical unknown, unnamed, and unmeasured confounder is that the analysis implies that such a causal pathway for reduced diarrhea was independent of education, wealth, exclusive breastfeeding, and other evaluated household characteristics. An unmeasured personal or household characteristic that is so powerful that it dominates the relationship between handwashing and diarrhea, but is so elusive that we cannot even name it, seems a less likely explanation than pathogen contaminated hands and food, a biologically plausible explanation that invokes a pathway of gastrointestinal pathogen transmission repeatedly demonstrated in other contexts.The observation in this evaluation that children living in households where residents washed their hands with soap after defecation had less diarrhea compared with children living in households where handwashing after defecation was less thorough is consistent with findings of previous intervention studies An important limitation of this study is that measuring handwashing on a single day risks misclassifying exposure. Among mothers in Burkina Faso, observed handwashing behavior after cleaning a child who had defecated was concordant with observations on a different day between 57% and 73% of the time A second limitation is that fieldworkers observed an opportunity to wash hands after defecation in only 29% of households. The resulting limited statistical power precluded a thorough assessment of the utility of washing hands after defecation with water only or with ash/mud, the contribution of other determinants of diarrhea, or a combined model that included both handwashing before preparing food and handwashing after defecation. However, even with limited power there was a strong association between handwashing with soap and less subsequent diarrhea, and the point estimates of the odds ratios are suggestive of less diarrhea for handwashing with water alone.A third limitation is that different gastrointestinal pathogens have different routes of transmission within different contexts, which might limit the generalizability of this study. It is possible that transmission of gastrointestinal pathogens from hands to food during preparation is a less important route of transmission in other settings. Additionally, in settings where water to wash hands is more heavily contaminated with feces than available water in rural Bangladesh, washing hands with water alone may be less protective. However this evaluation was conducted in 50 rural villages in 26 districts across Bangladesh and Bangladesh is the eighth most populous country in the world, so the analysis is not identifying a highly isolated phenomenon. In an assessment of hygiene indicators in rural Nicaragua, washing hands before preparing food was the single hygiene indicator most strongly associated with child diarrhea p-values were <0.005.A fourth limitation is that the program evaluation was not designed to evaluate the hypothesis that observed handwashing behavior was associated with a change in the prevalence of subsequent diarrhea. Because this is a secondary analysis of the data, there is some risk of data mining to identify an interesting but ultimately not robust finding. However, we planned these data analyses at the time we designed the program evaluation. There was a dose effect between thoroughness of handwashing before preparing food and subsequent observed diarrhea and the associated An important flaw in this evaluation was that we did not have the protocol reviewed by an independent human study participant committee. The amount of time we asked from participants, the intensity of the interaction with the field team, and the use of these data to draw generalizable insights to improve global scientific understanding mean that the activity had substantial research components and should have been reviewed by an independent human study participant committee. The study team did implement standard procedures to minimize risks and harms to evaluation participants, but similar future evaluations should be reviewed by human study participant committees. Rigorous evaluations of large public health programs provide insights that can translate into improved programs that save lives and improve community health. However large public health programs in low-income countries often have extremely tight implementation schedules. Human study participant committees in low-resource settings may need to develop additional capacities to provide appropriate independent review more promptly for these type of evaluations.Most people living in low-income settings have apparently concluded that following recommendations that require them to wash hands with soap ten, 20, or more times per day is not feasible The lower prevalence of childhood diarrhea seen in this evaluation among children living in households where residents washed hands with soap are consistent with the many intervention trials that demonstrate less childhood diarrhea in households where residents are encouraged to wash hands with soap


Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a sensorimotor disorder that causes patients to experience overwhelming and distressing sensations in the legs compelling the patient to move their legs to provide relief. The purpose of this study was to determine if biomarkers in the cerebrospinal fluid can distinguish RLS/WED patients from neurological controls.p ≤ 0.05, Av. Ratio ≥ 2.0) between RLS/WED and control CSF samples were identified using MALDI-TOF-MS. Statistical analyses of the validation immunoblot assays were performed using Student’s t-test.We obtained CSF samples by lumbar puncture from 5 early-onset RLS/WED patients and 5 controls. We performed 2-dimensional difference in-gel electrophoresis (2D-DIGE). Proteins that were significantly altered were identified by Student’s t-test. Protein spots that were differentially expressed  were increased and 2 proteins (Apolipoprotein A1 and α-1-acid Glycoprotein) were decreased in RLS/WED patients.Our results reveal a protein profile in the RLS/WED CSF that is consistent with clinical findings of disruptive sleep, cardiovascular dysfunction and painful symptoms. Moreover, protein profiles are consistent with neuropathological findings of activation of hypoxia inducible factor (HIF) pathways and alterations in dopaminergic systems. These data indicate the CSF of RLS/WED patients may provide information relevant to biological basis for RLS/WED, treatment strategies and potential new treatment targets. Restless Legs Syndrome/Willis-Ekbom Disease (RLS/WED) is a sensorimotor disorder that affects between 5-10% of the population . Those wIron dysregulation in the pathogenesis of RLS/WED is supported by a substantial volume of research including several studies reporting decreased ferritin in cerebrospinal fluid (CSF) and decreased brain iron particularly in the substantia nigra using MRI, ultrasound imaging and brain autopsy analysis . A numbeCSF was utilized for this biomarker study because it is most likely to reflect changes in CNS metabolic status due to its proximity to the brain. An advantage of CSF as the biological fluid for proteomic analyses over blood is that the CSF is sequestered behind both the blood–brain and brain-CSF barriers. This isolation permits the identification of biomarkers that are specific to CNS disease processes. Hence, our goal for this study was to identify CSF biomarkers for RLS/WED using two-dimensional difference-in-gel electrophoresis (2D-DIGE) together with tandem Mass Spectrometric (MS) analysis. To further validate the identified biomarkers, we used immunoblot analyses to quantify differences in protein levels between early-onset RLS/WED and age- and gender-matched control subjects. In this study, we identify six proteins whose levels are altered in early-onset RLS/WED subjects.The study was approved by DongSan Medical Center Institutional Review Board in Taegu, South Korea where subjects were recruited and CSF was collected. The two-dimensional Difference-In-Gel Electrophoresis analyses were performed at Penn State Hershey College of Medicine. The diagnosis of RLS/WED was made by a board certified Korean neurologist with expertise in RLS/WED diagnosis (YWC). The diagnosis was based on diagnostic standards set by the NIH workshop on RLS/WED  and was After written informed consent was obtained from the research subjects, approximately 10 mL of CSF was obtained via lumbar puncture with a 25-gauge needle and placed immediately on ice. CSF specimens were spun at low speed in a refrigerated centrifuge to remove any cellular contamination, aliquoted, and stored at −80°C within 1 h. All samples were collected between 9:30 AM and 11:00 AM. The CSF samples were precipitated with trichloroacetic acid/acetone following storage at− 80°C and the protein pellets were resuspended in 10 μL of DIGE labeling buffer -dimethylammonio]-1-propanesulfonate (CHAPS) and 30 mM Tris/HCl, pH 8.5). Protein levels were quantified using the 2D Quant-Kit .Isoelectric pH gradient (IPG) strips, urea, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), dithiothreitol (DTT), Pharmalyte pH 3–11, acrylamide, ammonium persulfate, TEMED, glycine, methylene bisacrylamide, TRIS, thiourea, and 2-D Quant Kit (PlusOne or Ettan™ sample preparation kits and reagents) were obtained from GE Healthcare . ReadyPrep 2D-Clean-up Kit was obtained from Bio-Rad . Iodoacetamide and mineral oil were obtained from Sigma . SDS was obtained from USB . Methanol and glacial acetic acid were purchased from Fisher Scientific .Each of the dry CyDyes  was reconstituted and 50 μg of total protein was labeled with each diluted CyDye solution as described in the manufacturer’s protocol.CSF containing 150 μg of labeled protein was added to an IPG strip. Strips were rehydrated for 20 h followed by isoelectric focusing for 27 h. The following gradient program was used – step 1: 0.01 h, 150 V, 2 mA, 5 W; step 2: 2.00 h, 150 V, 2 mA, 5 W; step 3: 6.00 h, 500 V, 2 mA, 5 W; step 4: 5.00 h, 3500 V, 2 mA, 5 W; step 5: 9.20 h, 3500 V, 2 mA, 5 W; step 6: 5.00 h, 50 V, 2 mA, 5 W. SDS-PAGE was carried out in 12.5% of acrylamide. Following the focusing step, the proteins on the strip were reduced and alkylated with gentle shaking in two subsequent steps for 30 min each in 10 ml equilibration buffer  containing 100 mg DTT and 250 mg iodoacetamide in the second step. Strips were removed from equilibration solution, blotted, and placed on a 1-mm thick 25.5 × 20.5 cm 12.5% acrylamide non-gradient SDS gel. The strips were sealed with 1% agarose, 125 mM Tris–HCl at pH=6.8, 1% SDS. Proteins were separated on the gels using 600 V, 400 mA, 100 W for 4 h at 10°C. Analytical gels were imaged immediately after separation, while the preparative gels were fixed with 7.5% acetic acid/10% methanol overnight and stained with Coomassie Blue R-350. The images from analytical gels were collected as described previously . Intra-gEach of the proteins identified by 2-D DIGE analyses was confirmed by immunoblot analyses. Protein concentration was determined and immunoblot analyses were performed as described previously . An aliqThe immunoblots were quantified using densitometric analysis. Chemiluminescent detection was performed using either anti-mouse or anti-rabbit IgG ECL HRP-conjugated secondary antibodies . FujiFilm imaging system was used for detection, and densitometric analysis was performed with Multiguage software version 3.0.p ≤ 0.05, Av. Ratio ≥ 2.0) between RLS/WED and control CSF samples were identified using MALDI-TOF-MS.The Extended Data Analysis tool (EDA) in the DeCyder 2D software package was used for the statistical analysis of the 2D-DIGE experiments. Proteins that were significantly altered were identified by Student’s t-test. Protein spots that were differentially expressed . These protein spots were chosen for identification by MALDI-TOF. Of the six proteins selected  and 5 female age-matched controls (mean age ± SD 58.8±7.66 years old) . In addition, because it had been reported that the expression of proteins involved in maintaining iron homeostasis in CSF were altered in CSF in patients with RLS/WED, H-ferritin levels were determined in the CSF using immunoblot analyses but the differences did not reach statistical significance.Immunoblot validation was performed on all of the candidate proteins. Cystatin C was able to be validated with immunoblot analyses Figure  and was Prior to this study, only specific iron-related proteins and dopaminergic metabolites have been examined in the CSF of RLS/WED patients ,16. AlthIn this discovery study, we have utilized a 2D-DIGE coupled with tandem MS to identify 6 candidate CSF protein markers for early-onset Restless Legs Syndrome (RLS/WED); four proteins  were increased and 2 proteins (Apolipoprotein A1 and α-1-acid Glycoprotein) were decreased in RLS/WED patients compared to controls.in vivo. It is known to inhibit both endogenous and exogenous cysteine peptidases, regulate the immune system, have antibacterial and/or antiviral effects; and neuroprotective effects during brain injury. Cystatin C has been described previously as a biomarker of pain in lumbar disk herniation, sciatica, and labor [et al. [Cystatin C levels were increased in the CSF of early-onset RLS/WED patients. Cystatin C is a cysteine proteinase inhibitor that has multiple functions nd labor ,18. The nd labor ,20. Alsond labor  and headnd labor . The fin [et al.  suggestsIn this study, we found increased levels of lipocalin-type prostaglandin D2 synthase (L-PGDS) in RLS/WED CSF. L-PGDS is the most abundant protein in the CSF and serves two roles: 1) synthesis of prostaglandin D2 (PGD2) from prostaglandin H2 (PGH2); and 2) binding and transporting small lipophilic molecules. L-PGDS alterations have been reported in a number of sleep disorders including narcolepsy and obstructive sleep apnea with and without daytime sleepiness -26 and tet al. have reported decreased serum 25-hydroxyvitamin D levels in female RLS/WED subject and have postulated that the CNS iron deficiency that occurs in RLS/WED may induce alterations in the dopaminergic pathway via a vitamin D-mediated pathway [In this study, we report elevated vitamin D binding protein in the CSF of RLS/WED subjects. Vitamin D binding protein (VDP) is noted for specifically binding and transporting a number of sterols including vitamin D metabolites to target cells, however, it has been also been shown to be a scavenger of toxins that are released due to cellular injury or death  and a mo pathway . BecauseBeta-Hemoglobin was found to be increased in RLS/WED CSF. β-Hemoglobin is a major component of adult hemoglobin and normally is not present in the CSF. As such, the presence of β-Hemoglobin in the CSF is often considered to be indicative of blood contamination. While, obtaining a CSF tap with no blood contamination is extremely difficult, we have examined the CSF for frank signs of blood to rule out contamination. β-Hemoglobin is known to transport oxygen in the blood, however, it has also been reported to be important in dopamine regulation in the brain . BiagiolApolipoprotein A1 was found to be decreased in the CSF of patients with RLS/WED. Apolipoprotein A1 is the primary protein constituent of high density lipoproteins (HDL). Although the role of Apolipoprotein A1 in the CNS is not completely understood, it is thought to be important for transport of lipids into the brain . To dateAlpha-1-acid glycoprotein levels were decreased in the CSF from RLS/WED patients. Alpha-1-acid glycoprotein (AGP) is synthesized primarily in the liver, but also is reportedly synthesized in the endothelial cells of the blood–brain-barrier. Alpha-1-acid glycoprotein (AGP) is an acute phase reactant that is involved in the inflammation cascade and is thought to provide immediate and effective response to inflammatory damage prior to successive protective mechanisms . Their pOur results reveal a protein profile in the RLS/WED CSF that is consistent with iron deficiency, dopamine dysregulation and inflammation. The APO-A1 finding may be of relevance outside of CNS given the recently reported increased risk of cardiovascular disease in patients with RLS and cardiovascular dysfunction and reports of painful symptoms. The profiles in the CSF are also consistent with neuropathological findings of activation of HIF pathways and alterations in dopaminergic systems. The data indicate that the CSF protein profile, if confirmed in larger sample sizes, may provide support for existing hypotheses about a biological basis for RLS/WED which could prove clinically meaningful in evaluating therapeutic strategies and identifying new targets.Dr. Allen has in the last 2 years served as a consultant for Boehringer Ingelheim, GlaxoSmithKline, Luitpold Pharmaceuticals, Pfizer, EMD Serono, Pharmacosmos, Neurogen, Jazz Pharmaceuticals and UCB Pharma. He also received research support from GlaxoSmithKline, Pharmacosmos and the USA National Institutes of Health. Dr. Earley was a member of the Data Safety Committee for Phase III clinical trial by Merck. Dr. Connor discloses that he is a paid consultant for GlaxoSmithKline and the International Copper Association and has consulted for Neurogen. Drs. Patton and Cho, and Mr. Clardy report no disclosures.SMP carried out the 2D-DIGE analyses, identification of biomarker proteins, and drafted the manuscript, YWC recruited, diagnosed and obtained cerebrospinal fluid samples from RLS/WED and control subjects, TC performed the DeCyder-DIA proteein analyses of 2D-DIGE images, RPA participated in study design and coordination, and assisted in the draft of the manuscript, CE participated in study design and assisted in the draft of the manuscript, and JRC participated in the study design and assisted in the draft of the manuscript. All authors have read and approved the final version of the manuscript.


To functionally characterize pro-inflammatory and vasoconstrictive properties of cerebrospinal fluid after aneurysmal subarachnoid hemorrhage (SAH) in vivo and in vitro.The cerebrospinal fluid (CSF) of 10 patients suffering from SAH was applied to the transparent skinfold chamber model in male NMRI mice which allows for in vivo analysis of the microcirculatory response to a superfusat. Microvascular diameter changes were quantified and the numbers of rolling and sticking leukocytes were documented using intravital multifluorescence imaging techniques. Furthermore, the pro-inflammatory properties of CSF were assessed in vitro using a monocyte transendothelial migration assay.CSF superfusion started to induce significant vasoconstriction on days 4 and 6 after SAH. In parallel, CSF superfusion induced a microvascular leukocyte recruitment, with a significant number of leukocytes rolling (day 6) and sticking (days 2-4) to the endothelium. CSF of patients presenting with cerebral edema induced breakdown of blood vessel integrity in our assay as evidenced by fluorescent marker extravasation. In accordance with leukocyte activation in vivo, significantly higher in vitro monocyte migration rates were found after SAH.We functionally characterized inflammatory and vasoactive properties of patients' CSF after SAH in vivo and in vitro. This pro-inflammatory milieu in the subarachnoid space might play a pivotal role in the pathophysiology of early and delayed brain injury as well as vasospasm development following SAH. Cerebral vasospasm following acute aneurysmal subarachnoid hemorrhage (SAH) is still one of the most feared complications of this disastrous disease. Although recent advances in vasospasm treatment have been reported ,2, the pRecently, an activation of inflammatory pathways has been suggested to be involved in the pathogenesis of secondary brain injury after SAH . For exaTherefore, this study was conducted to evaluate potential vasoconstrictive as well as pro-inflammatory properties of cerebrospinal fluid (CSF) after SAH. For this purpose we established an in vivo model in which vascular reactivity as well as leukocyte endothelial interaction could be observed after exposition to post-SAH CSF. We furthermore applied a cellular in vitro assay to confirm inflammatory cell attraction by post-SAH CSF.Animal experiments were approved by the responsible ethics committee and conducted according to the regulatory guidelines (Regierungspräsidium Karlsruhe). The withdrawal of post-SAH CSF from the critically ill patients was approved by the local ethics committee and conducted throughout the routine CSF sampling . All diagnostic assessments were done on the basis of our institutional guidelines.For the present study, 10 consecutive patients with severe SAH  were included. The screening was done at the initial presentation in our institution. The patient characteristics are displayed in Table radiographical vasospasm was made on the basis of DSA, documenting angiographic arterial narrowing. The diagnosis of clinical vasospasm was defined by clinical signs of ischemia and/or vasospasm-associated infarctions in the CT, as well as a relevant territorial hypoperfusion in Xe-CT. TCD served as screening method only. In repeated native CT-scans cerebral infarctions and edema development were documented ). When blood flow velocity increased over 120 cm/min or by more than 50 cm/min within 24 h, Xenon-cCT was scheduled to evaluate cerebral blood flow with good spatial resolution and to assess the hemodynamic relevance of the suspected vasopasm. Furthermore, digital subtraction angiography (DSA) was performed according to our standard protocol on days 7-9 after SAH (or earlier in case of suspected vasospasm). The diagnosis of As a control group six consecutive patients were assessed, who were referred to our department for myelography diagnostics of lumbar spine degeneration. These were considered healthy individuals in terms of CSF alterations . Although almost all of these patients had medical side conditions due to their age , inflammatory issues were ruled out by standard laboratory blood sampling.The transparent dorsal skinfold chamber in the mouse is a model for studying microvascular function under physiological and pathophysiological conditions -18. ReceBriefly, mature immune competent NMRI mice were anaesthetized using an intraperitoneally applied mixture of ketamine and xylazine (7.5 mg ketamine and 2.5 mg xylazine/100 mg body weight). The hair on the back of the mice was carefully shaved and chemically epilated. The dorsal skin was elevated and implanted into two symmetrically applied titanium frames. In this way the extended double layer of skin was trapped. The titanium frames spared an area of 15 mm in diameter in which the upper skin layer was completely removed and the remaining layer  was covered with a glass slide.In this way direct, non-invasive access to the vasculature was gained for the superfusion experiments and repeated multifluorescence videomicroscopy. For the experimental protocol only chambers without signs for bleeding or inflammatory processes  were included. In parallel, a catheter was placed into the jugular vein and was subcutaneously guided to the dorsal end of the skinfold chamber. This catheter was used for application of the fluorescent dyes.For multifluorescence intravital videomicroscopy, the animals were immobilized in a special mouse restrainer and fixed upon a table with the titanium frame of the skinfold chamber. The microscopic examination was conducted after intravenous application of 1 ml of 5% Fluorescein Isothiocyanate (FITC)-Dextrane . The leucocytes were labelled by 0.1 ml of 0.2% rhodamine 6 G . The chamber was observed using a Zeiss fluorescence stereo-microscope equipped with a high pressure mercury lamp for epi-illumination . By using two dyes with different emission spectrum the process of multi-step leukocyte recuitment, i.e. rolling, sticking, and migration along the endothelium (Rhodamine 6 G) as well as microhaemodynamics (FITC-Dextrane) could be evaluated sequentially in the according observation fields by switching between the filters. A CCD videocamera mounted on the microscope allowed for recording of the data for later off-line analysis.The experimental observation began with a mapping of the microvascular bed of the preparation for later re-assessment of the regions of interest. Five arterioles and five venules (diameter 20-60 μm) were randomly selected for the documentation. The quantitative analyses of the microcirculation were performed using a 10× long distance-objective as well as a 20× immersion-objective with magnifications of 200× and 400× respectively.At the time of initial patient recruitment, the skinfold chamber was prepared. For each patient and for each respective time point one mouse was prepared . To exclude influence of the anaesthesia or the operation trauma the skinfold chamber experiments did not start before day 2 after preparation. On days 2, 4, 6 and 8 after occurrence of the SAH, 3-5 ml CSF were harvested from the EVD of each respective patient. The sample was centrifuged at 3000/min for 10 min and the supernatant was withdrawn. All samples were used freshly for the superfusion experiments, without storage or freezing. One patient who showed signs of bacterial meningitis was excluded from the protocol after diagnosis on days 4.First, FITC-Dextran and rhodamine6G were injected i.v. and a baseline analysis of the microvasculature was performed. 5 distinct pre-capillary arterioles and post-capillary venules (20-60 μm) were identified for further analysis of microhemodynamics and leukocyte/endothelial-interaction. Approximately 30 min later, after removal of the cover glass, the skinfold chamber was superfused with 0.5-1 ml of post-SAH- or control CSF for 10 min. Following superfusion, the chamber was closed again with a fresh glass cover and the microvasculature was visualized using the multifluorescence videomicroscopic setup. The following microhemodynamic parameters were documented quantitatively in the same microvascular segments before and 10, 30, 60, 120, 180 and 240 min after superfusion with CSF: microvascular diameter, erythrocyte velocity and microvascular permeability, the later of which was defined via the extent of extravasation of the low-molecular weight fluorescent marker Rhodamine 6 G. For the evaluation of the leukocyte flow properties, the leukocyte/endothelial interaction was divided into temporarily ('rolling') and permanently adhesive ('sticking') leucocytes. Rolling was defined as a passage time of more than 20 s within the vascular segment . Permanent adhesion was documented for sticking leukocytes that remained in one place for longer than 20 s. The latter were counted as cells per square micrometer. The area was calculated from the diameter and the length of the vessel segment (200 μm).The membrane surfaces of trans-well chambers (pore size 5 μm) were coated with 50 μl 0.1% gelatine, were covered with a layer of human umbilical cord endothelial cells  and incubated for two days at 37°C. Staining with crystal violet (1%) permitted observation of the integrity and the confluence of the cell layer twice during the incubation time course. In a second step, 24 h before starting the assay, the human acute monocytic leukemia cell line THP-1 was incubated with TGF beta-1  and vitamin D  for 24 h. After stimulation these cells yielded a strong expression of CD14+ on their surface, which was verified by FACS analysis, supposing that they had differentiated into mature monocytes.After washing of the HUVEC cell layer, a total number of 400000/200 μl BSA (0.5%) was applied to each trans-well chamber and 600 μl of post SAH CSF was applied to the respective wells. Again, the CSF samples of patients undergoing myelography for spinal diagnostics served as controls. After incubation at 37°C for 90 min the assay was stopped and the total number of CD14+ cells was counted that had transmigrated through the HUVEC cell layer.P-values smaller than 0.05 were considered statistically significant.The statistical analysis of arteriolar diameter, rolling and sticking leukocytes and transmigrated monocytes was done using univariate ANOVA with posthoc Bonferoni correction. Edema/Rhodamine Correlation was evaluated using Wilcoxon ChiSquare test. Eight patients out of 10 developed hemodynamically, angiographically or clinically accessible cerebral vasospasm. The diagnosis of cerebral vasospasm was established 6.6 ± 2.1 days after the onset of the bleeding.The quantitative analysis of the CSF samples revealed a significant vasoconstrictive potential of the post-SAH CSF when compared to control-CSF on days 4 and 6 Figure . The maxLong before clinical manifestation of cerebral vasospasm, analysis of the CSF-samples revealed significant pro-inflammatory properties of post-SAH CSF. Starting on day 2 after SAH, an activation of the leukocyte/endothelial interaction with an increased number of recruited leukocytes, both rolling and sticking fractions, could be shown. A significantly increased number of rolling leukocytes could be shown on day 6, the number of sticking leukocytes was significantly elevated from day 2 through day 4 Figures  and 4.To further assess the pro-inflammatory properties of post-SAH CSF in vitro we established a THP-1 monocyte transendothelial migration assay. The total number of CD14+ cells that migrated through the HUVEC cell layer was significantly elevated after stimulation with post-SAH CSF during the entire 12 days observation period after SAH Figure . Again, On days 6 and day 8 after SAH an increase of the microvascular permeability was documented in terms of extravasation of the low-molecular fluorescent marker Rhodamine G6 from the microvasculature. This extravasation could be predominantly observed in the postcapillary, venular segments of the microcirculation. A correlation with the clinical course of the individual patients showed that CSF-induced leakiness of blood vessels was associated with the development of brain edema and cerebral infarctions Table .In this study we functionally characterized the pro-inflammatory and vasoactive milieu in post-SAH CSF in vivo and in vitro. We described two assays in which the vasoconstrictive and inflammatory properties of human CSF could be evaluated in i) a well-established in vivo animal model for microcirculatory studies and ii) an in vitro monocyte transendothelial migration assay. This experimental setup provided evidence for significant early vasoconstrictive and inflammatory characteristics of human post-SAH CSF. This was displayed by a significant arteriolar vasoconstriction and intravascular leukocyte recruitment in the in vivo assay, that preceded the diagnosis of cerebral vasospasm in our patients. The increased chemotaxis of THP-1 monocytes in the in vitro assay documented upregulated trans-endothelial migration properties of inflammatory cells upon stimulation with post-SAH CSF.The involvement of inflammatory reactions in early brain injury, development of cerebral vasospasm and delayed brain injury after SAH has been more and more discussed within the past years ,6-15,22.Supposing that post-SAH CSF has the capability to attract a significant number of inflammatory cells from the circulation and pool them in the subarachnoid space, an inflammatory milieu might be elicited along the blood vessels and in the basal cisterns. This inflammatory environment might initiate or contribute to cerebral vasospasm. As a possible mediator for vasoconstrictive and inflammatory activity interleukin-6 has already been discussed. An upregulation of this inflammatory cytokine has been observed extraparenchymally in the CSF, but also in the brain parenchyma itself  in patients after SAH . In addiOur study has several limitations. Due to the small number of patients, a correlation analysis of the detected CSF changes with the development of cerebral vasospasm or secondary brain injury could not be performed. Nevertheless, our data were suggestive for a time relationship between the occurrence of vasoconstriction and leukocyte activation in the assays and the clinical onset of vasospasm. Cerebral vasospasm was diagnosed in our patients between days 3 and 9 by TCD, Xe-CT and/or DSA (mean 6.6 ± 2.1 days), which is well in accordance with the known time course of vasospasm in man. Yet, despite the tight diagnostic regime, we cannot exclude, that we missed an earlier development of cerebral vasospasm in our patients and the observed CSF alterations were an epiphenomenon. Furthermore, the evaluation of the dorsal skinfold chamber elicited high standard deviations, due to a high interindividual variability, especially in the leukocyte/endothelial interaction. We therefore faced the following statistical problems. 1. Irregularities in the data curves gave an impression of an unsteady response to the superfusion. 2. Alpha as well as beta errors might be underestimated.Of 10 patients eight developed cerebral vasospasm, which seems much. Yet, all of the patients, included in our study suffered from severe SAH . Thus, the observed high incidence of morphological vasospasm is within the reported range. Furthermore, we used a tight regimen of multiple testing methods, not to miss out on the occurrence of cerebral vasospasm.The present study supports the existing theory that inflammatory mechanisms can contribute to secondary brain injury after SAH. We could functionally demonstrate an inflammatory millieu in post-SAH CSF in vivo and in vitro. The detected inflammatory changes were accompanied by microvascular diameter changes, and preceded the clinical diagnosis of cerebral vasospasm in our patient population.Source of Funding of the project: Centre for Stroke Research, Berlin, Germany. Otherwise, none of the authors has any financial or non-financial competing interests concerning the current study.UCS: conduction of the experiments, analysis and interpretation of the data, composition of the manuscript (full responsibility for the content). JS: conduction of experiments, interpretation of the data, involved in composition of the manuscript . NH: establishment of the technical procedures, conduction of experiments, review of the manuscript . PH: substantial contributions to concept and design, conduction of experiments, interpretation of data . PV: conception and design, interpretation of the data, revision and approval of the manuscript (full responsibility for the content). All authors read and approved the final manuscript.


Large registries of potential unrelated stem cell donors have been established in order to enable stem cell transplantation for patients without HLA-identical related donors. Donor search is complicated by the fact that the stored HLA information of many registered donors is incomplete. We carried out a project that was aimed to improve chances of patients with ongoing donor searches to find an HLA-matched unrelated donor. For that purpose, we carried out additional donor center-initiated HLA-DRB1 typing of donors who were only typed for the HLA loci A and B so far and were potential matches for patients in need of a stem cell transplant. In total, 8,861 donors were contacted for donor center-initiated HLA-DRB1 typing within 1,089 donor searches. 12 of these donors have donated stem cells so far, 8 thereof for their respective target patients. We conclude that chances of patients with ongoing donor searches to find an HLA-matched unrelated donor can indeed be improved by donor-center initiated typing that is carried out in addition to the standard donor search process. Our results also raise questions regarding the appropriate use of incompletely typed donors within unrelated donor searches. Allogeneic hematopoietic stem cell transplantation is a well-established and increasingly used therapy for hematological malignancies and other severe diseases of the blood Full HLA typing, i.e., high-resolution typing of at least the HLA loci A, B, C and DRB1, of newly registered potential stem cell donors is advantageous as it reflects the current standard of donor-recipient matching Available HLA phenotype data of registered donors are routinely extended either by donor center-initiated HLA typing (prospective typing), generally based on selection criteria as donor age or gender or the HLA information available so far, or by patient-related typing requests within actual donor searches. A patient-related typing request is initiated by the responsible transplant physician, forwarded by the national registries involved, and executed by the donor center that has recruited the respective donor. We analyzed 9,478 donor searches between November 2009 and September 2010 that we became aware of due to patient-related HLA typing requests for donors of DKMS German Bone Marrow Donor Center. We then carried out additional donor center-initiated HLA-DRB1 typing of selected donors who were so far only typed for the HLA genes A, B and optionally C but not DRB1 . Donors We did not take into account potentially matching donors outside the DKMS donor file in the worldwide registry as we had no information if these donors were available, were already requested for patient-related HLA typing, or were intentionally not considered by the responsible transplant physicians.HLA typing results of project donors were submitted to registries without references to the specific patients for whom the donors had been included in the project. Furthermore, we did not inform the responsible transplant physicians about the donor center-initiated typing efforts carried out in order to support the donor searches of their patients. This approach was chosen to avoid interferences with the standard search process. Transplant physicians became aware of the extended HLA information of project donors by routinely rechecking registry data during donor searches.Donors who were selected for donor center-initiated typing within the project were followed-up with respect to subsequent requests for confirmatory typing , donor work-up , and stem cell donation.At stem cell donor registration, each donor signed an informed consent form covering sample collection and storage, HLA typing of the sample, data storage and transmission of anonymous data to domestic and foreign registries. All donors who were included in the analyzed donor center-initiated typing project were informed by mail about the continuative HLA typing.In order to avoid delays of the donor search, we initiated HLA typing and informed donors at the same time. Donors who withdrew their willingness to donate stem cells, were temporarily unavailable, or unable to donate for health-related reasons could mark respective statements on a response sheet that was attached to the mail. According to their feedback, they were temporarily or permanently excluded from the donor file. HLA typing results of these donors were included in the analysis regarding 3-locus low-resolution matched donors who were identified within the project.No ethics committee approval was obtained as donor center-initiated typing projects are standard procedures of donor centers that are covered by the consent form signed at recruitment.8,861 donors were contacted for additional donor center-initiated HLA-DRB1 typing within 1,089 of the 9,478 analyzed donor searches. Within 28 days after contact, 382 donors (4.3%) declared their unwillingness or lack of ability to donate stem cells or were excluded from the donor file for other reasons.The maximum number of donors who were contacted for a specific patient as they were potential matches based on their HLA-A, -B low-resolution phenotypes was 70. For 179 patients, we identified in total 236 donors who matched on the 3-locus low-resolution level with their respective target patients.76 confirmatory typing and 17 work-up requests were submitted for donors with donor center-initiated HLA typing, and 12 of these donors donated stem cells so far. 4 donor work-ups are currently processed, one donor was unable to donate for medical reasons. Of these requests and donations, 35 confirmatory typing requests and 10 work-up requests were made for the original target patients. 8 donors donated for their respective target patients, one donor was unable to donate, and one work-up for the target patient is currently processed.Donations for target patients took place between 70 and 357 days  after the respective donor center-initiated typing requests. Interval lengths of the various steps of the donor search process are shown in Details regarding the 8 donors who donated for their target patients are displayed in Our practical study was intended to provide a proof of principle if donor center-initiated HLA typing can improve the success chances of ongoing donor searches. This proof was successfully made.The median and average time from donor center-initiated typing to stem cell donation  suggest that donor-center initiated typing might often be too time-consuming to support donor searches as many stem cell transplantations are urgent. The detailed analysis of sub-intervals in Eight donations resulted from donor center-initiated HLA typing of more than 8,000 donors within the study. These figures raise questions regarding the cost-benefit ratio of the analyzed HLA typing efforts. We did not carry out a formal cost-benefit analysis. Generally, cost-benefit analyses of ongoing donor recruitment or continuative HLA typing of already registered donors are complicated by several practical and ethical problems The 8 donating study donors who were obviously – after additional donor center-initiated HLA typing, confirmatory typing and medical clearing – assessed as best available donors for their target patients by the responsible transplant physicians had originally not been requested for additional typing based on their available HLA information that did not include HLA-DRB1. It is, therefore, doubtful if they had advanced to donation without donor center-initiated HLA typing.This finding raises the question if there are deficiencies of the existing donor search process regarding the use of incompletely typed donors, especially of donors who are typed for HLA-A and -B only. An infrequent use of donors with this typing profile has been reported before The restriction to 3-locus low-resolution matching in the donor selection process and the non-availability of data on patient-related requests for non-DKMS donors within the considered donor searches are the major limitations of the study. It is, therefore, not possible to draw definite conclusions regarding the efficiency of the donor search process from our results. Due to the high relevance of this issue for optimal donor selection and thus for the best possible patient care, further analyses of this question are urgently required.It is obvious that full HLA typing at donor recruitment reduces the probability of non-finding the optimal stem cell donor. As new donors are increasingly typed more completely due to advances in typing technology and related cost reductions, the probability of non-finding the optimal donor should decrease in the future. However, there will be millions of partially typed donors in the worldwide donor file for many years to come. There is a need for strategies that make sure that these donors are utilized properly in actual donor searches. Haplotype frequency-based search algorithms Many donor centers regularly run prospective typing projects in order to increase the completeness of HLA information of their registered donors. Our results suggest it might be indicated to use the funds available for such projects at least partly for donor center-initiated HLA typing as support for currently ongoing donor searches as described in this work. We plan, therefore, to continue with this project with a refined donor selection process that is based on probabilities for 4-locus high-resolution instead of 3-locus low-resolution matching.


This feature may be exacerbated in the case of compact genomes, like the one of bacteriophage λ, used in our study. Transcription that starts at the rightward pR promoter and proceeds through the λ origin of replication and downstream of it was proven to stimulate the initiation of λ DNA replication. Here, we demonstrate that the activity of a convergently oriented pO promoter decreases the efficiency of transcription starting from pR. Our results show, however, that a lack of the functional pO promoter negatively influences λ phage and λ-derived plasmid replication. We present data, suggesting that this effect is evoked by the enhanced level of the pR-driven transcription, occurring in the presence of the defective pO, which may result in the impeded formation of the replication initiation complex. Our data suggest that the cross talk between the two promoters regulates λ DNA replication and coordinates transcription and replication processes.Transcription and DNA replication are tightly regulated to ensure coordination of gene expression with growth conditions and faithful transmission of genetic material to progeny. A large body of evidence has accumulated, indicating that encounters between protein machineries carrying out DNA and RNA synthesis occur  Escherichia coli and metazoans) that transcriptional activity of the neighboring region may have a large impact, both adverse and advantageous, on the function of origins of replication , which serves as a starting-point for synthesis of a short leftward transcript (oop), was previously suggested to influence the replication initiated at oriλ . Plasmids and bacteriophages are listed in Supplementary Table S2 and oligonucleotides in Supplementary Table S3. All genetic manipulations were described in the Supplementary Material.Bacterial strains used in this study are described in the Plasmid maintenance was investigated according to the previously described method .Escherichia coli C600 or C600polA1 strains, bearing a hybrid ColE1-λ helper plasmid, pLamberA, were transformed by a series of plasmids bearing various insertions between pO and oriλ. Efficiency of transformation was estimated by determining a number of transformants obtained per 1 μg of DNA used in the experiment.E. coli cells was measured as described earlier . The purification procedures have been described previously  according to Viguera et al. , with mo , 2% Triton X-100 and 50 ng/μl poly dI-dC, in 20 μl of total volume. The samples were incubated for 10 min at 37°C, and subsequently resolved electrophoretically in 5% polyacrylamide gel  running in 0.5 × TBE (45 mM Tris-borate/1 mM EDTA) at 9 V/cm at 4°C. DNA was visualized using GE Healthcare Typhoon 9200 scanner.Fifty nanograms of a Cy5-labeled DNA fragment (52 bp long) encompassing the Supplementary Material.Density shift experiments were carried on as described earlier . Full depO promoter  increased levels of the pO-derived oop RNA  did not influence considerably copy number of both wild-type λ plasmid and its pO− derivative  and pKB2pO− (bearing the point mutation in the −10 region of pO) that harbor a region essential for λ DNA replication , corroborating the conclusion that the pO−  mutation results in a decreased efficiency of λ plasmid DNA synthesis.Previously reported analysis of the pKB2 replication intermediates revealed the presence of unidirectionally and bidirectionally replicating molecules in similar amounts . ResultsO− . The latter type of replication is suggested by the presence of the bubble arc in the experiment with HindIII- and EcoRV-digested pKB2pO− . The presence of this arc interferes with the comparative analysis of the theoretical schemes with images obtained as results of the experiment. In the case of the EcoRV digest, the double Y signal that is present on experimental pictures is not marked on the theoretical scheme. This signal comes from recombination intermediates, and it is not the same signal as that which appears in the scheme ‘Bi’. Generally, such signals emerging near the single Y arc signal, which comes from DNA fragments that do not contain the origin of replication, are more difficult to interpret owing to their overlapping. In this case, the HindIII digest is the least informative, as the origin of replication is situated almost in the middle of this fragment. Therefore, the choice of other restriction digests, BglII and EcoRV, in which the origin is situated at the left and at the right end of the fragment, respectively, allowed for more precise identification of the directionality of replication. Appearance or disappearance of the long bubble arc, in the case of such digests, enables detection of replication forks proceeding leftward or rightward, respectively. Consequently, the lack of this signal in the case of BglII digestion indicates the absence of plasmid molecules replicating according to the θ unidirectional rightward mode. Conversely, the bubble arc present in the case of the EcoRV digest indicates that plasmid replicates according to the unidirectional leftward mode.One should note that the experimental data presented in O−, in contrast to the wild-type plasmid, replicates mainly bidirectionally and that unidirectional rightward replication is impaired. The observed weak replication signal of pKB2po− plasmid is also in agreement with the decreased efficiency of DNA replication of the λ phage bearing the pO− mutation, and strongly supports the hypothesis that pO promoter’s activity plays an important role in the λ DNA replication.To sum up, these experiments indicated that pKB2ppO promoter mutation affects the efficiency and directionality of replication. The point mutation in the −10 region of the pO promoter dramatically decreases its activity  replicates predominantly in a bidirectional manner . 2D-AGE analysis of replication intermediates resulting from replication initiated at oriλ revealed that both plasmids replicate similarly, with the advantage of bidirectional replication over unidirectional replication in both directions. Interestingly, the signal of unidirectional rightward replication, visible after digestion with BglII as a bubble arc, which was absent in the case of the pKBpo− plasmid, is restored in the absence of the functional Cro protein  plasmid in comparison with the wild-type λ plasmid , controlled by the TetR repressor, and λ replication genes O (together with oriλ located in the middle of O) and P that are positioned downstream of this promoter. Efficiency of replication of this plasmid depends on derepression of the ptet promoter activity by tetracycline or its analogs, for instance, autoclaved chlortetracycline (aCTC) . We used this plasmid as a convenient tool to test whether eliminating transcription from pO has an impact on transcriptional activation of oriλ, mediated by the ptet activity.The results presented in the preceding paragraph imply that a functional interdependence exists between the two promoters in the regulation of replication starting from  inducer . PlasmidpO− mutation was determined after treatment with various aCTC concentrations. The experiment was performed in E. coli recA cells, defective in DNA recombination to prevent formation of plasmid multimers. Importantly, characteristic regulation of the copy number in response to the inducer concentration is preserved in this strain. In the case of pTCλ5, the copy number rose with increasing aCTC concentration, as it was described previously . This observation implies that, under conditions allowing for high activity of the promoter responsible for transcriptional activation of oriλ, a lack of the intact pO promoter may result in an increased plasmid multimerization. It is in agreement with results from the analysis of the pKBlin plasmid replication . This result supports earlier conclusions that decreased activity of pO considerably impairs λ plasmid replication and the very low stability observed for the mutant plasmid may in part result from enhanced multimerization, as it was previously observed with respect to ColE1-like and other plasmids . This result suggests that in the absence of pO activity, λO might bind less efficiently to iterons present in the ori region. Such effect could be a consequence of enhanced transcription from pR, which might remove the O-some more effectively. Alternatively, pO activity could directly influence the efficiency of binding or stability of the initial λO-oriλ complex. We conclude that, most likely either transcription from pO or formation of the transcription complex at this promoter interferes with pR activity, causing impairment of RNA production downstream of the latter promoter. We cannot exclude a possibility that the absence of pO activity results in a stimulation of transcription from the pR promoter at the initiation stage; however, this scenario seems less likely.To test the influence of λO protein overproduction on the transcription starting from the pO promoter has adverse effects on the λO binding to the iterons. Therefore, we asked if the decreased pO promoter activity could affect the formation of λ replication complex, in consequence decreasing the efficiency of replication. In particular, we wondered whether dysfunction of the pO promoter could influence interactions between λO and its binding sites in vivo. To test this, we have constructed two series of ColE1-like plasmids bearing various numbers of iteron sequences, one based on a medium copy number plasmid (pBR322) and the other on a high copy number plasmid (pUC19). We assumed that when λ plasmid DNA is introduced into cells bearing plasmids with iterons, these λO-binding sequences should outcompete iterons located on the λ plasmid in binding the replication initiator protein. This would result in impaired replication and hence reduced number of transformants obtained after transformation of E. coli cells by λ plasmids. Therefore, cells harboring competitor plasmids were transformed with either wild-type λ plasmid (pKB2) or its variant containing the pO− mutation (pKB2pO−). Assessment of the efficiency of transformation revealed that the presence of the iterons on a high copy number plasmid (the pUC19 derivative) resulted in a decreased efficiency of transformation by λ plasmid DNA in all tested experimental systems (Supplementary Table S4). Moreover, the level of transformation impairment was proportional to the number of iteron sequences present on the pUC19-derived plasmid, confirming that the effect was caused by the presence of the λO binding sites. Interestingly, effects on transformation efficiency were significantly stronger for λ plasmids devoid of the functional pO promoter (Supplementary Table S4). Analogous experiments with pBR322-derived plasmids bearing iteron sequences revealed no significant influence on the efficiency of transformation by λ plasmids, most likely reflecting the difference in the plasmid copy number between pBR322 and pUC19. These results strongly suggest that the presence of the intact pO promoter is important for efficient binding of the λO protein to oriλ present in cis.The results presented in the preceding section might suggest that decreased transcription from the pO− plasmid could be corrected in cells containing increased amounts of the λO protein. We addressed this question by measuring relative levels of DNA of λ plasmids, either wild-type (pKB2) or with the pO− mutation (pKB2pO−), in the wild-type host bearing the O gene under the control of an IPTG-inducible promoter on the chromosome . We found that pKB2pO− copy number was significantly decreased relative to that of the wild-type λ plasmid in the absence of the O gene expression in trans. However, on O expression induction, this parameter was significantly increased for both plasmids . In both hosts, the λO protein was provided in trans from a helper plasmid (pLamberA). Results of the experiments presented in pO promoter and oriλ resulted in a drastic reduction in the efficiency of polA mutant transformation by the plasmids carrying such modifications. This suggests that not only the process of transcription per se but also RNA polymerase binding at the specific position relative to oriλ is important for efficient replication of λ plasmids. Alternatively, the formation of a specific DNA structure or nucleoprotein complex is required for the replication. One may expect that such minor changes in the promoter-origin relative location should not be so significant if pO-mediated regulation of oriλ replication relied only on possible effect of the ongoing transcription on the DNA topology at the origin . Therefore, these results imply that the role of pO in the formation of the λ replication complex may not be restricted to the regulation of pR-derived transcription.We compared the efficiency of transformation of in vitro replication assays performed using the series of mutated plasmids as templates confirmed that the pO promoter mutation or increasing its relative distance to the origin have a negative effect on λ plasmid replication. The pO dysfunction resulted in ∼40% decrease in the efficiency of replication . Replication of plasmids containing larger insertions (100 and 500 bp) was also less effective than that observed for the wild-type plasmid, although the effects of those modifications were less pronounced than those observed in the in vivo studies .Results of pR promoter was identified as a source of transcriptional activation of oriλ, but the exact molecular mechanism of this phenomenon has not been resolved  observed in the clpP strain transformed with pKB2pO− plasmid, in comparison with the one bearing wild-type pKB2 . These effects, resulting most probably from the SOS response induction, could support the above-mentioned hypothesis, as replication forks collision is accompanied by frequent double-strand breaks and activation of the repair mechanisms . To sum up, these results disfavor over-initiation and suggest that, on the contrary, the formation of the λ replication initiation complex might be hampered in the absence of pO activity. In addition, it was demonstrated previously that the presence of the defective pO exerts adverse effect also on the replication of λ plasmid DNA, which was initiated by a replication complex inherited by one of the daughter copies . It was shown previously that a high level of activity of the rightward promoter caused drastic stimulation of multimer formation by λ plasmid, and its instability in bacterial population, even in the presence of the intact pO promoter , indicating that positioning of the promoter is important for its function in the replication control. One plausible explanation would involve interaction between the pO promoter and oriλ, most likely mediated by proteins bound to these sequences. Recently, we have shown that λO interacts directly with RNA polymerase . However, in the same set of experiments, the presence of the empty vector (used otherwise to oop expression) altered the ratio between the wild-type and mutated λ plasmids. On the other hand, no influence of the considerably increased oop RNA levels (obtained due to pcnB mutation and increased stability of this transcript) on the copy number of both wild-type and pO− λ plasmids was observed  [N301 4140 33 to M.G.] and the National Science Center (Poland) [2011/02/A/NZ1/00009 to G.W.]. Funding for open access charge: National Science Center (Poland) [2011/02/A/NZ1/00009 to G.W.].Conflict of interest statement. None declared.