The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA).Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI).The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the potential to improve national JEV surveillance and inform vaccination policies. The saturation of filter paper has potential use in the wider context of pathogen detection, including dried spots for detecting other analytes in CSF, and other body fluids. Japanese encephalitis virus infection is widespread in Asia, and primarily affects children in poor, rural areas. The virus spreads to the brain and spinal cord resulting in significant death and disability. Diagnosis requires testing for the immune response ‘antibody’ specific to Japanese encephalitis virus in the cerebrospinal fluid that surrounds the brain and spinal cord. However, in most areas where the infection occurs there are no laboratory facilities. In the absence of laboratory diagnosis, we have poor knowledge of the extent of the problem in these areas. Dried blood spots on filter paper have been used widely for many years as simple, cost-effective tools for transporting samples for testing. However, there have been few attempts at using dried cerebrospinal fluid spots. We developed a technique for using dried spots to store and test cerebrospinal fluid for antibodies to Japanese encephalitis virus. We compared the results of dried spots with routine neat cerebrospinal fluid in 132 patient samples, and demonstrated excellent agreement between the two tests. This novel method for saturating dried cerebrospinal fluid spots has the potential to enhance our knowledge of Japanese encephalitis virus epidemiology, and inform health policies where they are most needed. It could also be transferred for use in diagnosing other infectious diseases, including using other body fluid samples. The last few decades have seen a substantial growth of novel and complex diagnostic tests . This haStrikingly, there is a paucity of data on use of dried spots of other body fluids . There aAmongst CNS infections, Japanese encephalitis virus (JEV) is recognised as the most common causative pathogen in Asia. In the 24 endemic countries it is suggested that JEV causes 67,900 cases, and 20,000 deaths per year , 15]. I. I15]. IThe use of DCS in the diagnosis of JEV by JEV MAC-ELISA would have potential application in low-resource settings, for diagnostics, wider epidemiological studies on the aetiology of CNS infections and impact of vaccination. We aimed to optimise a DCS protocol for the detection of anti-JEV IgM using JEV MAC-ELISA and evaluate its performance in a pilot study of consecutive patient samples as compared to routine neat CSF.Patient CSF and serum samples were collected at three hospitals in Vientiane, the Friendship, Children and Setthathirat Hospitals, from 2009 to 2015 . CSF wasThe study was part of a study on the causes of CNS infections in Laos. Ethical clearance was granted by the Ethical Review Committee of the Faculty of Medical Sciences, National University of Lao, and the Oxford University Tropical Ethics Research Committee, Oxford, UK. Diagnostic testing was performed on anonymised and frozen CSF samples. LPs were performed as part of a routine diagnostic service if verbal (2003–2006) or written (2006–2011) consent was given by patients or their parents/guardian. Consent for all patients was documented on a form that had been approved by the ethics committee. The lead investigator has been trained in Good Clinical Practice, and has completed Human Tissues Training.The WHO recommended commercial JEV MAC-ELISA assay is the Inbios JE Detect  . This meTwo types of filter paper were chosen, and utilised in all experiments. The 903 Protein Saver Card (903)  has been used as DBS for neonatal screening since 1963 . It is mCSF-substitutes were prepared to compare the end-limit of detection of the DCS method to the routine method from neat sample, and because of the limited CSF volumes available. A fluid was needed with similar consistency to CSF, and low IgM titre. Two anti-JEV IgM positive serum samples (A and B respectively) were diluted in PBS to the limit of detection-the highest dilution found positive by JEV MAC-ELISA. This is referred to here as the end-limit dilution, and was 1:1600 and 1:3200 for samples A and B, respectively. It was expected that there would be some loss in sensitivity when testing from dried spots so two additional dilutions, more concentrated, were also prepared: x2 (1:800 and 1:1600 for A and B respectively) and x4 end-limit dilutions (1:400 and 1:800 for A and B respectively). All the CSF-substitutes, three dilutions  from the two sera A and B, were used in subsequent experiments. When directly used for ELISA testing, not spotted on paper, it is referred as neat CSF-substitute.The DCS protocol was adjusted to compare sample volumes, spotting techniques and filter paper. The general protocol is described here, with the variables elaborated below. The CSF-substitutes were spotted on 3MM and 903 filter paper, with the centre of the spot marked as an indent with a pin. These were air-dried for 4 hours and stored in individual zip-lock plastic bags with a teaspoon of silica granules and then stored overnight in an air-conditioned laboratory at 25–30°C. The elution process involved inserting punched or pre-cut filter paper in 1.5ml Eppendorf tubes, adding different volumes of PBS/ 0.05% Tween-20, agitating on a shaker for 1 hour at 180 revs/minute at room temperature, and refrigerating overnight at 4°C. The tubes were then centrifuged for 10mins at 3,000g at 20°C and 100μl undiluted supernatant was used to perform JEV MAC-ELISA, using the JEV Detect InBios kit. Neat CSF-substitute was tested, diluted 1/10, on the same plate as per the manufacturer’s instructions. Each plate was validated with kit controls, as well as a PBS spotted on filter paper as an additional negative control.A conventional DBS protocol was initially utilised, as per the in-house protocol for DBS and studies involving a wide-variety of analytes using DBS , 9]. 10. 109]. 1A modified protocol was used to increase the volume of CSF-substitute eluted using eight and ten 6mm punches in the minimum volume of PBS/0.05% Tween-20, 325μl, to provide the 275μl required eluate for subsequent JEV MAC-ELISA and potentially additional anti-dengue IgM and dengue NS1 assays. Different sample volumes were evaluated using the modified protocol with ten 6mm punches: 1) a single 100μl spot as previously described, 2) respotting a 100μl spot after completely air-drying a single spot, and 3) Two separate spots, with the innermost 5 punches taken from each spot.A pre-cut circle protocol was performed completely saturating a pre-cut filter paper circle. Increasing volumes of PBS were spotted on pre-cut filter paper placed on a 3x3cm clear plastic square. The largest volume of PBS the circle could hold without the fluid seeping off was defined as the saturating volume. This was 250μl spotted on a 1.49cm diameter circle, five times the volume of CSF-substitute per surface area when spotted on a large area of paper. This entire circle was eluted in 200μl PBS/0.05%Tween-20.The two patient samples at each of three dilutions of CSF-substitutes were subjected to the various protocols. Experiments were performed as duplicates tested on the same JEV MAC-ELISA plate, and the entire protocol was repeated on another day. In total there were 4 to 6 replicates of each experiment. The JEV MAC-ELISA result is a binary categorical outcome ‘positive’ and ‘negative’, and results of experiments are summarised as percentage of replicates (4–6) positive for each sample and as a total (62–65).Retrospective, consecutive analysis was performed on available CSF from all patients tested for JEV between February 2009 and July 2015. CSF was spotted on 903 paper as per the optimised ‘pre-cut circle’ protocol detailed above, and stored at 25–30°C for 30 days as an approximation of the maximum time for the paper to reach the laboratory if this was performed in the field. DCS and neat CSF were compared on the same plate. Investigators were blinded to the previous neat CSF JEV ELISA results.The sample size (n = 86) was calculated with values for sensitivity of Inbios JEV MAC-ELISA = 53%, expected positive agreement DCS with neat CSF = 75%, and expected negative agreement = 98%, programming formulae (as per the CDC report referenced by T. Lee) based on estimation of a kappa coefficient, into Microsoft Excel , 28]. R. R28]. RThe conventional DBS protocol applied to DCS did not produce any positive results at the end-limit dilutions . The modThe final protocol evaluated a pre-cut circle of filter paper saturated with the maximal volume of CSF. A 1.49cm diameter pre-cut circle could be loaded with 250μl CSF-substitute, and this permitted detection of anti-JEV IgM in 100% of replicates . In compCSF was received and tested for JEV using Inbios JEV MAC-ELISA from 248 patients admitted from February 2009 to July 2015. 132 patients had sufficient frozen CSF volumes, 10μl for neat CSF ELISA testing and 250μl to make the dried CSF spot following pre-cut circle protocol. After 30 days at room temperature (25–30°C) the DCS and corresponding neat CSF were tested by Inbios JEV MAC-ELISA. The positive agreement for dried CSF spots compared to neat CSF was 81.6% (65.7–92.3 95%CI), negative agreement 96.8% (91.0–99.3 95%CI), overall agreement 92.4% and kappa coefficient 0.81 (0.70–0.92 95%CI), There are still considerable global deficits in laboratory capacity for diagnosing CNS infections , 3]. Th. Th3]. TThe use of DBS for storing, transporting and detecting anti-JEV IgM and anti-dengue IgM have already been well-documented in previous studies, and this was not repeated here. , 31]–[3–[331]–3The experiments performed to optimise the DCS protocol highlighted the key difference between blood and CSF, with poor performance of the conventional DBS and modified protocols likely due to low IgM concentration in the final eluate, IgM concentrations are lower in CSF than in blood . This waIn contrast, the ‘pre-cut circle’ protocol worked very well. Saturation of filter paper could be achieved by cutting beforehand and then carefully loading with the maximum CSF volume the paper could hold. The volume of CSF that could be added to the pre-cut circle was five times that of a single CSF sample spotted on a sheet of filter paper. Prior cutting has been utilised in DBS , 40]. H. H40]. HThe retrospective study of 132 consecutive patients recruited from three hospitals demonstrated a high level of agreement between the optimised ‘pre-cut’ circle protocol and neat CSF, with positive agreement of 81.6% (65.7–92.3 95%CI), negative agreement of 96.8% (91.0–99.3 95%CI) and kappa coefficient 0.81 (0.70–0.92 95%CI). It is possible that kappa agreement overestimates the effect size, as the comparison involved paired samples, tested using the same diagnostic assay. Further, kappa agreement has been criticised as being dependent on disease prevalence, with a low prevalence seen in this study risking a false negative result . However10 of 132 (8%) CSF samples had discordant ELISA results between neat CSF and pre-cut saturated paper samples. Refreezing samples may have affected results. Unfortunately there was insufficient sample volumes to investigate this by the reference standard, PRNT.Cross-reactivity with JEV MAC-ELISA occurs with other Flavivirus antibodies, and in this region, testing for dengue is crucial. We had insufficient sample volumes to test for dengue antibodies as well as JEV antibodies in the DCS. A larger study is needed to evaluate the differential diagnosis of JEV from other flaviruses, performing the protocol in ‘full’ with 500μl CSF, on a 2.1cm diameter circle, eluting with 400μl PBS/0.05% Tween-20.Rules of thumb for the evaluation of any diagnostic test are the ‘ASSURED’ criteria, i.e. whether it is Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users . The curThis novel design has significant implications for use in the wider context of pathogen detection, including dried spots methods for other analytes in CSF, and other body fluids. There are no facilities for systematic, population based surveillance of CNS infections in Laos outside the capital, as is the case in many parts of the world . Even inS1 Table(DOCX)Click here for additional data file.


Commercial transplant tourism results in significant harm to both kidney donors and recipients. However, proponents of incentives for kidney donation assert that proper oversight of the process prevents these harms and also that transplant numbers can be safely increased so that the moral burden of poor end-stage kidney disease outcomes can be alleviated. In a moral dilemma analysis, the principle of preventing donor harm can be dissociated from the principles of providing benefits to the recipient and to society. It is plausible that an incentivized donor is fundamentally different from an uncompensated donor. Incentivized donors can experience harms unrelated to lack of regulation because their characteristics are determined by the incentive superimposed upon a poverty circumstance. Moreover, creating a system of incentivized donation without established national registries for capturing all long-term donor outcomes would be morally inconsistent, since without prior demonstration that donor outcomes are not income or wealth-dependent, a population of incentivized donors cannot be morally created in a clinical trial. Socioeconomic factors adversely affect outcome in other surgical populations, and interventions on income or wealth in these populations have not been studied. Coercion will be increased in families not affected by kidney disease, where knowledge of a new income source and not of a potential recipient is the incentive. In the case of elective surgery such as kidney donation, donor non-maleficence trumps donor autonomy, recipient beneficence, and beneficence to society when there is a conflict among these principles. Yet, we are still faced with the total moral burden of end-stage kidney disease, which belongs to the society that cannot provide enough donor kidneys. Acting according to one arm of the dilemma to prevent donor harm does not erase obligations towards the other, to provide recipient benefit. To resolve the moral burden, as moral agents, we must rearrange our institutions by increasing available donor organs from other sources. The shortage of donor kidneys creates a moral burden for society, but incentives for donation will only increase the total moral burden of end-stage kidney disease. Transplant tourism is widely condemned , but proThe moral dilemma is a useful tool that can be used to analyze many life situations. In a moral dilemma, one ought to do x and ought to do y, but one cannot do both simultaneously. When framed as a moral dilemma, on the one hand, we ought to safely increase total kidney transplant numbers, and on the other, we ought to prevent harm to incentivized kidney donors, but we cannot do both together. When properly framed in this manner, each dilemma arm must first be supported, and a resolution then proposed. There is no controversy about safely increasing total transplant numbers, which is the dilemma’s first arm. More contentious is the dilemma’s second arm, according to which regulated incentive systems cause donor harm, and which will therefore be discussed at length. Although poor outcomes in incentivized donors have been reported worldwide –5, propoThere are three parties involved in incentivized donation: donors, recipients, and society. In a moral analysis, the principle of preventing donor harm must be dissociated from the principle of providing benefits to recipients or to society. High-quality studies demonstrate safety in regulated uncompensated donation . UncompeIncentivized donors may be fundamentally different from other donors and therefore prone to different outcomes. Survey data from previously incentivized donors  remains Incentivized donors are likely to be in poverty, even in regulated systems. Society does not object to impoverished individuals autonomously assuming risky jobs . HoweverDonors from retrospective long-term donor follow-up studies  do not rWith sparse long-term data for incentivized donors, examining the relationship between income and outcomes in other surgical populations is immediately helpful. Socioeconomic factors adversely affect outcome in kidney , liver –15, and Significant financial transactions can occur in all donor-recipient relationships. Even without a reliable method for their capture, transplantation must continue. However, rather than being occasional instances, such transactions are institutionalized by incentivized donation and can only encourage their occurrence in all living donation. Some current living donation also possibly occurs by coercion. A medically suitable family member may be compelled to donate a kidney to a relative. Transplant programs attempt to identify such cases and prevent these transplants. With incentivized donation, a new form of coercion is being introduced, within families that are not affected by kidney disease. Knowledge of a new income source is now the cause of coercion, and so, there will be a new population of coerced donors. Transplant programs support reluctant donors to opt-out of surgery in support of the donor’s autonomy and to prevent long-term, non-surgical harm to the donor. In incentive-based systems, there will be opt-in reasons unrelated to poverty alleviation, some of which will be possibly illegal. Yet, moral consistency would require that the principle of autonomy preservation respect these reasons to donate.To summarize, the second arm of the moral dilemma, that we ought to prevent harm to incentivized kidney donors, is clearly supported by the lack of favorable outcomes in previously incentivized donors to promote equipoise, no clear relation of these outcomes to deficient regulation, lack of registries to capture all long-term donor outcomes, potential for increased poverty, relationship of wealth or income to outcome in other surgical populations, and increased coercion. Since incentives appear to strengthen and not resolve the moral dilemma, the moral burden from the shortage of available kidneys is increased, not decreased by a regulated system of incentivized donation.Since we cannot both increase total transplant numbers and prevent incentivized donor harm, how do we proceed? One method is specification. Specification works with norms that are not universal generalizations and allow us to articulate our reasons openly and publicly . SpecifiBeauchamp and Childress describe autonomy, beneficence, non-maleficence, and justice as the normative standards of conduct . When thThe total moral burden of end-stage kidney disease belongs to the society that cannot provide enough donor kidneys. Acting with good reasons according to one arm of the dilemma, to increase kidney transplant numbers, does not erase obligations towards the other, to prevent donor harm. The second-order principle in this situation is that as moral agents, we must arrange our institutions to minimize such conflicts . Increas


In 2012, it was estimated that 9180 people in the United States would die from melanoma and that more than 76,000 new cases would be diagnosed. Surgical resection is effective for early-stage melanoma, but outcomes are poor for patients with advanced disease. Expression of tumor-associated antigens by melanoma cells makes the disease a promising candidate for immunotherapy. The hematopoietic cytokine granulocyte–macrophage colony-stimulating factor (GM-CSF) has a variety of effects on the immune system including activation of T cells and maturation of dendritic cells, as well as an ability to promote humoral and cell-mediated responses. Given its immunobiology, there has been interest in strategies incorporating GM-CSF in the treatment of melanoma. Preclinical studies with GM-CSF have suggested that it has antitumor activity against melanoma and can enhance the activity of anti-melanoma vaccines. Numerous clinical studies have evaluated recombinant GM-CSF as a monotherapy, as adjuvant with or without cancer vaccines, or in combination with chemotherapy. Although there have been suggestions of clinical benefit in some studies, results have been inconsistent. More recently, novel approaches incorporating GM-CSF in the treatment of melanoma have been evaluated. These have included oncolytic immunotherapy with the GM-CSF–expressing engineered herpes simplex virus talimogene laherparepvec and administration of GM-CSF in combination with ipilimumab, both of which have improved patient outcomes in phase 3 studies. This review describes the diverse body of preclinical and clinical evidence regarding use of GM-CSF in the treatment of melanoma. In 2012, it was estimated that 9180 people in the United States would die from melanoma and more than 76,000 new cases would be diagnosed . GM. GM9]. GThe immune adjuvant properties of GM-CSF led to numerous preclinical studies assessing the ability of GM-CSF to inhibit tumor growth and/or mediate tumor regression. In a seminal study, a panel of recombinant retroviral vectors expressing various cytokines, co-stimulatory molecules, or adhesion molecules were used to infect murine B16 melanoma cells. Infected cells were then irradiated and injected subcutaneously into immune-competent hosts, followed by a subsequent challenge with wild-type B16 cells . The GM-+ T cells abrogated tumor regression.Exogenously administered GM-CSF has been shown to augment antitumor immunity Mice immunized with an HIV envelope peptide vaccine plus GM-CSF exhibited increased cytotoxic T lymphocyte (CTL) activity compared with vaccine alone or vaccine with interleukin-2 . StudiesGenetically modified vaccines can generate protective anti-melanoma immune responses in animal models . B16 tumGM-CSF) [Oncolytic immunotherapy with modified herpes simplex viruses (HSV) and vaccinia viruses expressing GM-CSF have also shown promise. These vectors have elicited tumor responses in mice when injected directly into tumor lesions -25. In aGM-CSF) . NotablyThe combination of GM-CSF–secreting tumor vaccines with other immunotherapies is another potentially promising approach. Antibodies that block the co-inhibitory T-cell molecules CTLA-4 and PD-1 plus GM-CSF have boosted immune responses against melanomas ,27-31. AGiven the evidence of antitumor activity in preclinical models of melanoma, there has been interest in using GM-CSF to improve outcomes in the clinical setting. Numerous studies have evaluated use of recombinant GM-CSF in completely resected stage III/IV melanoma patients. Data from these studies, however, have been inconsistent.P = 0.04 and P < 0.001, respectively) receiving GM-CSF (125 μg/m2 daily for 14 consecutive days in 28-day cycles up to 1 year) compared with historical controls [2 daily for 14 consecutive days in 28-day cycles) versus placebo in patients with completely resected stage IIIB/IIIC/IV or mucosal melanoma, there was a trend toward improvement in DFS although it did not reach statistical significance , and no improvement in overall survival  [P = 0.04) and a trend toward improved OS  was observed in stage IV patients (n = 258) [GM-CSF has been evaluated as an adjuvant, systemic monotherapy following regional lymphadenectomy, to prevent or to delay recurrence in high-risk stage III patients Table . In a phcontrols . A subsecontrols . The effcontrols . A rando = 0.78) . Howevern = 258) .Several small studies have evaluated GM-CSF administered as a monotherapy by direct injection into metastatic lesions Table . ClinicaP = 0.016) [A number of early-phase clinical studies have evaluated GM-CSF in conjunction with chemotherapy Table . These s= 0.016) .2/day for 21 days for use in myeloid reconstitution after autologous bone marrow transplantation [Given the significant body of evidence from preclinical studies ,20,21,70antation .In contrast to data from murine studies, the adjuvant effect of GM-CSF in human trials is inconsistent. In a study that evaluated coadministration of GM-CSF with multipeptide (including gp100 and tyrosinase peptides) melanoma vaccines incorporating GM-CSF and incomplete Freund’s adjuvant (IFA) in patients with advanced melanoma, there was a high T-cell response rate and a correlation between T-cell reactivity to the melanoma peptides and clinical outcome . GM-CSF Although results from these studies have suggested that administration of adjuvant GM-CSF might improve immune responses, none included control groups. Recently, clinical trials including controls have evaluated effects of GM-CSF administered locally at the vaccination site Table . In a ph2 for 14 days in 28-day cycles) increased circulating mature DC but did not increase MDSC in melanoma patients [Results from clinical studies evaluating GM-CSF as an adjuvant to melanoma vaccines suggest the biologic effects of GM-CSF are complex and can be influenced by numerous factors. GM-CSF administered with a heat shock protein vaccine has been implicated in the induction of myeloid-derived suppressor cells (MDSC) in melanoma patients . On the patients . It has patients . In a trpatients . Thus, ePreclinical studies have indicated an important role for GM-CSF in the tumor microenvironment and have suggested that increased expression of GM-CSF can inhibit tumor growth. However, administration of exogenous recombinant GM-CSF appears insufficient to mediate clinically meaningful improvements in outcomes in most instances. Consequently, there has been significant interest in novel treatment approaches incorporating GM-CSF.GM-CSF) [ICP34.5 and ICP47 viral genes, which renders the virus nonpathogenic in normal eukaryotic cells, promotes selective replication in tumor cells, and enhances immunogenicity [Among the most extensively evaluated agents is the attenuated, oncolytic, herpes simplex virus, type 1 (HSV-1)–encoding human GM-CSF talimogene laherparepvec  -88. The genicity  T-VEC haIn a phase 1 study, patients with subcutaneous or cutaneous metastases from breast, gastrointestinal, head and neck, or melanoma malignancies were treated with T-VEC. Clinically stable disease was noted in 3/26 patients administered intralesional T-VEC, with no patients having CR or PR during the study period . HoweverGM-CSF has also been combined with ipilimumab, an anti-CTLA-4 monoclonal antibody. In a multicenter, phase 2 study, patients with metastatic melanoma were randomized to treatment with ipilimumab plus GM-CSF (250 μg/d subcutaneously for 14 days in 21-day cycles) or ipilimumab alone . PatientSeveral other approaches utilizing GM-CSF are being developed. These include an oncolytic vaccinia virus encoding GM-CSF , autologGM-CSF has been studied extensively in murine models and in human clinical trials, alone and as adjuvant therapy for melanoma. There is evidence from numerous studies that GM-CSF can induce antitumor immunity when administered by a variety of different routes. Although there was initial enthusiasm for recombinant GM-CSF based on uncontrolled clinical trials in stage III/IV melanoma, therapeutic benefit has not been confirmed in larger, prospective, randomized trials. The adjuvant or combination use of GM-CSF has been more promising although results have been inconsistent and may depend on the potency of the immunotherapy regimen, GM-CSF dose, route and schedule of administration, and stage of disease. There is emerging evidence that GM-CSF may be a regulatory cytokine with the ability to promote both effector and regulatory/suppressor T cell populations. Thus, strategies that block suppressor T cell and myeloid-derived suppressor cell elements may enhance the antitumor activity of GM-CSF. GM-CSF has been particularly effective in studies of oncolytic HSV therapy of melanoma and in combination with ipilimumab. Further research into the basic biology of GM-CSF on effector and suppressor immune cells and expanded clinical studies of combination treatments will help define the full therapeutic potential of GM-CSF in treatment of melanoma.CTLA-4: Cytotoxic T-lymphocyte antigen 4; CR: Complete response; DC: Dendritic cells; DFS: Disease-free survival; GM-CSF: Granulocyte–macrophage colony-stimulating factor; HR: Hazard ratio; HSV: Herpes simplex viruses; IFA: Incomplete Freund’s adjuvant; KLH: Keyhole limpet hemocyanin; MDSC: Myeloid-derived suppressor cells; OS: Overall survival; PD-1: Programmed death 1; PR: Partial response; T-VEC: Talimogene laherparepvec.HLK serves as a paid consultant and advisory board member for Amgen Inc. CLS is a scientific advisory board member for Immatics Biotechnologies and Polynoma LLC and a funded investigator for GlaxoSmithKline in the field of cancer vaccines; all funds from these activities are paid to the University of Virginia, not to Dr. Slingluff personally. Dr. Slingluff is an inventor on several patents for peptides used in cancer vaccines; the patents are held by the University of Virginia Licensing and Ventures Group. CER and TH have no conflicts to declare.All authors participated in critical analysis of the literature and drafting of the manuscript. All authors read and approved the final manuscript.


N = 554 was surveyed. Data were analysed with correlation and multiple regression analyses and dependent t-tests. Over-reporting of handwashing was associated with factors assumed to be involved in (1) socially desirable responding, (2) encoding and recall of information, and (3) dissonance processes. The latter two factor groups explained over-reported handwashing beyond socially desirable responding. The alternative interview question—script-based covert recall—reduced over-reporting compared to conventional self-reports. Although the difficulties involved in measuring handwashing by self-reports and observations are widely known, the present study is the first to investigate the factors which explain over-reporting of handwashing. This research contributes to the limited evidence base on a highly important subject: how to evaluate handwashing interventions efficiently and accurately.Handwashing interventions are a priority in development and emergency aid programs. Evaluation of these interventions is essential to assess the effectiveness of programs; however, measuring handwashing is quite difficult. Although observations are considered valid, they are time-consuming and cost-ineffective; self-reports are highly efficient but considered invalid because desirable behaviour tends to be over-reported. Socially desirable responding has been claimed to be the main cause of inflated self-reports, but its underlying factors and mechanisms are understudied. The present study investigated socially desirable responding and additional potential explanatory factors for over-reported handwashing to identify indications for measures which mitigate over-reporting. Additionally, a script-based covert recall, an alternative interview question intended to mitigate recall errors and socially desirable responding, was developed and tested. Cross-sectional data collection was conducted in the Borena Zone, Ethiopia, through 2.5-hour observations and 1-hour interviews with the primary caregivers in households. A total sample of  Diarrheal diseases and acute respiratory infections are the main causes of death among children younger than 5 years old throughout the world  and are However, measuring handwashing behaviour can be quite difficult , 12, 13.Self-reported handwashing can be collected easily and efficiently through interviews , 12. Howand valid self-report measures of handwashing. Therefore, the main aim of the present study was to investigate socially desirable responding and additional potential explanatory factors for the bias between self-reported and observed handwashing behaviour. The research questions were whether socially desirable responding and other factors are associated with over-reported handwashing and whether additional factors beyond socially desirable responding explain over-reporting. In addition, we investigated whether an alternative self-report measure thought to reduce the influence of socially desirable responding and additional factors mitigates the response bias. In the following section we discuss potential sources of over-reported handwashing and deduce corresponding hypotheses.In sum, the measurement of handwashing behaviour is a major challenge for intervention evaluations as observations are considered valid but cost-ineffective and self-reports cost-effective but invalid , 13. AltA response might be biased as (1) the response was edited , (2) theH1: The higher the tendency to respond in a socially desirable manner is, the higher over-reporting of self-reported handwashing is.H2: The higher the need for conformity is, the higher over-reporting of self-reported handwashing is.H3: The higher the feeling of group attachment is, the higher over-reporting of self-reported handwashing is.Socially desirable responding is one of the most commonly studied forms of response editing and response biases . First, H4: The stronger the perceived social norms that favour handwashing are, the higher over-reporting of self-reported handwashing is.Second, socially desirable responding also depends on the sensitivity of a questionnaire’s topic, in other words, whether a clear social norm regarding the behaviour or attitude at hand exists . What isQ1: Is the presence of a spouse during the interview associated with over-reporting of self-reported handwashing?Q2: Is the presence of other adults during the interview associated with over-reporting of self-reported handwashing?Third, if a question is sensitive, the level of privacy or confidentiality offered in an interview influences socially desirable responding , 28. TheH5: A regular daily household routine is associated with less over-reporting of self-reported handwashing than an irregular daily household routine.H6: The more frequently tasks are interrupted, the higher over-reporting of self-reported handwashing is.Regarding inaccurate memory formation and recall errors, a first factor potentially distorting the encoded handwashing frequency is forgetting to wash hands at a certain moment. Compared to forgetting other health behaviours, forgetting to wash hands at a certain moment stands out as it might never become salient at any moment to individuals. Respondents might not realize how often they intended to but forgot to wash their hands; therefore, the encoded handwashing frequency might be biased against the actual amount and towards the intended amount. Regular daily routines facilitate and frequent task interruptions impede remembering and implementing intended behaviours , so we aH7: The more a person tends to over-estimate, the higher over-reporting of self-reported handwashing is.Regarding further recall errors, memory performance varies among people; some people perform better than others at a variety of memory tasks , 34. It H8: The higher handwashing-related health knowledge is, the higher over-reporting of self-reported handwashing is.H9: The higher cognitive rationalisation of neglected handwashing is, the lower over-reporting of self-reported handwashing is.H10: Health knowledge and rationalisation interact with the highest over-reporting found for high health knowledge and low rationalisation and with the lowest over-reporting found for low health knowledge and high rationalisation.Returning to information formation, a further factor that might increase encoding inaccuracy is cognitive dissonance, a state of psychological tension felt when one’s cognitions (e.g. knowledge or attitudes) and/or behaviour are dissonant . RegardiH11: Script-based covert recall produces less over-reporting than action-based prompted recall (i.e. a standard self-report measure).When factors that largely explain over-reporting are found, the response bias can be addressed by improving interview questions accordingly . To mitiTo sum up, the present study investigated (1) whether socially desirable responding, inaccuracies in encoding, recall errors and dissonance processes are associated with over-reporting of handwashing; (2) whether the latter processes explain over-reporting beyond socially desirable responding; and (3) whether an alternative self-report measure reduces over-reported handwashing. To address these research questions, a cross-sectional study that combined observational and self-reported data was conducted in Ethiopia.The study was conducted as part of a larger research project investigating handwashing in four Borena kebeles  in southern Ethiopia. The Borena Zone is a semi-arid region which suffers repeated droughts. Governmental and non-governmental organisations have implemented handwashing interventions in the Borena Zone as part of drought responses since 2006.Each of the four study kebeles consisted of approximately 30 hamlets, and only those that were reachable by car or a maximum 20-minute walk were included in the study. Within a hamlet, households were selected by the random-route method . The elin = 71 for the key time of wiping a child’s bottom).According to sample size estimation with G*Power 3.1 , a samplThe present study was interested in factors associated with over-reporting of handwashing and measures to mitigate it. This focus necessitated a comparison of observed and self-reported data, and to maximise comparability, a cross-sectional correlational design measuring all the variables at one point in time seemed appropriate.Data collection took place in February and March 2013 through 2.5-hour observations and 1-hour, oral, face-to-face interviews. Observations took place at dawn or around noon during lunch preparation and were followed by interviews. Data were collected by 14 local students and social workers, of whom two were female and 12 male. To ensure high quality in data collection, the team received extensive training in a 4-day workshop. The workshop included an introduction to different question types, demonstrations of how to ask the different question types, a discussion of what to do and what not to do during interviewing and observations  judging responses or behaviour, and lecturing the participant). In addition, the workshop offered a question-by-question discussion of the questionnaire and extensive practice in conducting the interview and the observational procedure through role playing. To ensure that data collection was conducted as in training, the first author, a local research collaborator and a postgraduate student supervised the team during data collection.http://www.apa.org/ethics/code/) and the World Medical Association (WMA) Declaration of Helsinki (http://www.wma.net/en/30publications/10policies/b3/). It was part of a larger research project on handwashing which received ethical approval from the Ethiopian National Research Ethics Review Committee and the Ethics Committee of the Philosophical Faculty of University of Zurich. Following approval from these ethics boards, oral informed consent was obtained from all study participants. Written consent could not be obtained because of the high illiteracy rate in the sample. When a selected household refused to participate in the study, the household members were thanked, and the research team members left immediately. The number of refusing households was recorded in a dedicated space in the questionnaire of the next consenting household.This study was conducted in strict compliance with the ethical principles of the American Psychological Association .The interviews were based on a structured questionnaire developed for this study. The items covered socio-demographic characteristics, self-reported handwashing (standard measure), potential explanatory factors and alternative self-report measures of handwashing. Most of the response options used Likert scales , which were transformed into a value range of 0–1 (or -1–1 for bipolar items) to facilitate interpretation. The questionnaire was prepared in English, translated into Afan Oromo and then retranslated into English to ensure the quality of the translation. A structured format was prepared for observations . The appbefore eating/after defecation?’ . Th10 times : after d10 times . This finstool = 24 and nfood = 17) as only a few participants were observed at all respective key times; therefore, internal consistency could not be conclusively assessed. Response scales ranged from 0 = 0% handwashing to 1 = 100% handwashing.The same key times measured by self-reports were observed. If a key event occurred, it was noted in a structured format, along with whether both hands were washed with water before or after the event separately and whether soap was used. During data processing, for each respondent and for each type of key event (e.g. food preparation) the percentage of times a participant washed both hands with soap at a key event out of all the times the key event occurred was calculated. To allow for direct comparison with self-reported data, observational data concerning faeces and food were averaged to form observed stool- and food-related handwashing, although internal consistency was rather low . However, these estimates were based on very small samples , the items were averaged according to the original short form C )actual effects on over-reporting in isolation from the influence that the factors  might have also had on actual behaviour and thus responding. Whether observed behaviour is the most valid measure for actual behaviour and, thus, the standard of comparison for other measures can be questioned. However, answering this question was not within the scope of the present study.The present study assessed over-reporting directly by subtracting observed from self-reported behaviour scores. Consequently, we could investigate the explanatory factors’ In the present study, care was taken to ensure the comparability of the measures used for observed and self-reported behaviour to the greatest extent possible. Self-reported and observed behaviour were measured on the same day, and comparable response scales in interviews and recording formats in observations were employed. While the worth of comparability seems rather basic, previous studies that compared self-reported and observed behaviour used answer scales and observation formats that were not comparable e.g. , 17, 19), 1917, 1Still, some factors limited comparability in the present study. Observations lasted for only 2.5 hours because the usual daily routines of primary caregivers in the studied region rarely left them at home for more than 3–4 hours at a time. Within 2.5 hours, repeated observation of one key time (e.g. food preparation) was rare. Accordingly, handwashing compliance at a specific key time was often determined by a single observation event. Self-reports, in contrast, asked for an integrated estimation of handwashing frequency at a key time. Future studies investigating over-reporting of handwashing should attempt to further increase the comparability between self-reported and observation measures.A further limitation of the present study is that some factors were tested even though the internal consistency of their measures was insufficient . The internal consistency of the observation measures was also not conclusively assessed. The internal consistency of these measures requires further investigation.Several of the assumed underlying mechanisms were not tested directly, in particular, dissonance processes and the influence of unnoticed forgetting of washing hands on over-reporting. In addition to the idea for future research regarding forgetting already mentioned, dissonance processes could be investigated through experimental research testing whether the induction of dissonance  increases over-reporting .Though many factors were considered in the present paper, several factors were not included even though they might affect over-reporting of handwashing . Although some of these are rather unlikely to be relevant given the response patterns found in the present study (e.g. acquiescence tendency), the factors should be investigated in subsequent studies.An additional aspect which is essential to measurement but was not considered in the present paper is reliability; a measure’s consistency or repeatability. Previous studies have questioned the inter-rater and retest reliability of observed handwashing in both household and hospital settings , 61, 62.The difficulties involved in measuring handwashing by self-reports and observations, including over-reporting of self-reported handwashing, are widely known. The present paper is the first to investigate explanatory factors for over-reporting of handwashing. This study also tested measures to mitigate over-reporting. This research has contributed to the limited evidence base for a complex phenomenon and a highly important subject: how to evaluate handwashing interventions efficiently and accurately. We hope that the present paper stimulates further research on over-reporting of handwashing and on alternative self-report measures to mitigate response bias.S1 FileM) and standard deviations (SD) of socio-demographic characteristics for inconsistent and consistent stool-related hand washers and related t-tests (Table A). Frequencies in socio-demographic characteristics for inconsistent and consistent stool-related hand washers and related χ2-tests (Table B). Means (M) and standard deviations (SD) of socio-demographic characteristics for inconsistent and consistent food-related hand washers and related t-tests (Table C). Frequencies in socio-demographic characteristics for inconsistent and consistent food-related hand washers and related χ2-tests (Table D). Means (M) and standard deviations (SD) of socio-demographic characteristics for consistent stool-related hand washers and consistent food-related hand washers and related t-tests (Table E). Frequencies in socio-demographic characteristics for consistent stool-related hand washers and consistent food-related hand washers and related χ2-tests (Table F).Means ((PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Patients treated with TMZ frequently receive midazolam for sedation prior to surgery and other interventions. Previous studies suggested both cytoprotective and apoptosis-inducing properties of midazolam. Therefore, the impact of midazolam on TMZ-induced cytotoxicity was investigated Human neuroblastoma cells were incubated with midazolam alone, as a pretreatment prior to incubation with TMZ or a coincubation of both. Cell viability and proliferation was analysed (XTT and BrdU assay) after 24 h and flowcytometric cell cycle analysis was performed after 24 and 48 h.25 1005 ± 197 μM; IC50 1676 ± 557 μM; P < 0.05) compared to incubation with TMZ alone  and reduced the antiproliferative effect of TMZ (1000 μM) by 43.9 % (P < 0.05). In contrast, cytotoxic effects of TMZ were increased  when midazolam pretreatment was followed by coincubation of midazolam and TMZ. Cell cycle analysis revealed increased fractions of cells in G2/M phase after TMZ treatment , irrespective of midazolam pretreatment.Midazolam alone increased cell viability at lower concentrations , whereas higher concentrations  reduced cell viability. Pretreatment with midazolam 6 h prior to TMZ incubation reduced cytotoxic effects (ICMidazolam causes a hormetic dose–response relationship in human neuroblastoma cells. Pretreatment with midazolam reduces the cytotoxic and antiproliferative effects of TMZ without interfering with G2/M cell cycle arrest. In contrast, subsequent midazolam coincubation increases overall cytotoxicity. O6 with subsequent impaired DNA-repair and G2/M arrest [In the past decade, the relevance of temozolomide (TMZ), a DNA-methylating agent, for the treatment of paediatric solid tumours, especially neuroblastomas, has been elucidated –6. ThereM arrest . ReductiMidazolam is one of the most common sedatives for paediatric premedication , 10 and 25, IC50 and IC75) after pretreatment and comedication with midazolam (16 μM) was investigated and cell cycle analysis was performed to elucidate the effect of midazolam on the primary mode-of-action of TMZ.We hypothesised, that midazolam at low concentrations increases cell viability, leading to a reduced toxicity of the anticancer agent TMZ by altering the cell cycle. A viability assay (XTT) was performed to evaluate the effect and toxicity of increasing concentrations of midazolam and TMZ. Additionally, a bromodeoxyuridine-based proliferation assay (BrdU) was used to estimate the effect of midazolam to the antiproliferative effect of temozolomide. The toxicity of TMZ . Ethanol was obtained from Carl Roth GmbH . Na2HPO4, DMSO, EDTA, propidium iodide, phenazine methosulfate, TMZ and staurosporine were purchased from Sigma-Aldrich . Preservative-free midazolam was obtained from ratiopharm GmbH . 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) sodium salt was obtained from AppliChem GmbH .Roswell Park Memorial Institute 1640 medium (RPMI) with and without phenol red and trypsin/EDTA (0.05 %/0.02 %) were purchased from PAN-Biotech . Penicillin and streptomycin were obtained from PAA Cell Culture Company . NaCl, KCl, KH−1 penicillin and 50 μg mL−1 streptomycin. Cells were cultured at 37 °C in a humidified 5 % carbon dioxide atmosphere.Human neuroblastoma cells (SHEP)  werCell viability was assessed using the XTT assay after the following treatments: 1: increasing concentrations of midazolam for 24 h ; 2: increasing concentrations of TMZ for 24 h ; 3: increasing concentrations of TMZ for 24 h after pretreatment with midazolam 16 μM for 6 h; 4: increasing concentrations of TMZ for 24 h after pretreatment with midazolam 16 μM for 6 h or pretreatment + coincubation for 24 h.Cell cycle analysis using the Nicoletti assay  was perf5 ml−1 per well were incubated overnight in a 96-well plate. Each well was washed with PBS to remove unattached cells and refilled with 200 μl colourless medium (RPMI without phenol red), supplemented with different concentrations of midazolam and TMZ alone or in combination. After 24 h, 100 μl cell culture supernatant were removed from each well and 50 μl of the dye solution were added, containing XTT (1 mg ml−1) and phenazine methosulfate (50 μM). After gentle mixing of the samples, the plate was incubated for 120 min at 37 ° C. Subsequently, samples were measured spectrophotometrically at 540 nm.The XTT assay was used for measurement of cell viability. Briefly, XTT -2H-tetrazolium-5-carboxanilide inner salt) is metabolised to a coloured formazan dye in vital cells by mitochondrial dehydrogenase. Due to the fact, that only vital cells are capable to metabolise XTT, the amount of produced formazan dye is indicative for the fraction of vital cells. To evaluate cell viability, 100 μl of cell suspension with a cell concentration of 104 ml−1 cells were incubated in each well of a 96-well plate overnight to ensure adherence and logarithmic growth. Subsequent incubation with the investigated substances was followed by fixing the cells and denaturation. Incubation with anti-BrdU-peroxidase conjugated specific antibodies to BrdU. After a washing step with 200 μl PBS and subsequent addition of 100 μl substrate solution containing tetramethylbenzidine, absorbance was measured at 370 nm and 492 nm (read-out absorbance = absorbance370nm - absorbance492nm).A colorimetric immunoassay was used to quantify cell proliferation. Shortly, during DNA synthesis, proliferating cells integrate the thymidine analogue 5-bromo-2’-deoxyuridine (BrdU) instead of thymidine. Following denaturation, a peroxidase-conjugated antibody binds to newly incorporated BrdU. After a specific substrate reaction with tetramethylbenzidine, spectrophotometrical measurement of absorbance indicates the extent of newly synthesised DNA as a parameter of proliferation. In detail, 200 μl of a cell suspension containing 1x10Cell staining with propidium iodide allows quantifying the amount of cellular DNA, which indicates the phase of the cell cycle. After permeabilisation and staining, the quantity of cellular DNA is analysed by flow cytometric analysis, using the intensity of fluorescence of the stained DNA. Modifications of the cell cycle by drugs like TMZ are indicated by a change of the flow cytometric patterns .5 cells ml−1 overnight to reach adherence. Medium was replaced and supplemented with the investigated substance. After the defined time period, supernatant was harvested and adherent cells were detached with trypsin/EDTA 0.05 %/ 0.02 % at 37 °C and added to the supernatant. After centrifugation , the supernatant was removed and cells were washed with 1 ml of PBS. Subsequently, cells were incubated with 250 μl Nicoletti buffer  and kept on ice until completion of flow cytometric analysis.For cell cycle analysis, 2 ml of cell suspension were incubated with a concentration of 0.5x1025, IC50 and IC75, respectively) and the zero equivalent point (ZEP) were obtained from nonlinear regression analysis for bell-shaped dose–response relationships. Statistical analysis was performed by means of Student’s t-Test or one-way analysis of variance (ANOVA) with Bonferroni post hoc test. P < 0.05 was considered significant.Results are expressed as means ± SD. All calculations were made with GraphPad Prism version 5.03 for Windows . The values for drug concentrations leading to 25 %, 50 % or 75 % reduction of cell viability , whereas higher concentrations  led to a profound decline of cell viability  or pretreatment followed by coincubation (6 h prior to TMZ incubation and subsequent coincubation for 24 h) with midazolam (16 μM) , indicating an amplification of the TMZ-induced cytotoxicity  cells was measured using the BrdU-assay after incubation with increasing concentrations of TMZ with and without midazolam. Pretreatment with midazolam 6 h prior to TMZ exposure significantly attenuated the antiproliferative effect of TMZ 1000 μM by 43.9 %  on the fraction of cells with a diploid DNA content indicating the G2/M phase. No difference was detected with or without pretreatment of midazolam 16 μM) after incubation for 24 h. After 48 h, the fraction of cells being in the G2/M phase was significantly increased after treatment with TMZ (100 μM), while pretreatment with midazolam (16 μM) did not modify this effect Figs.  and 6.Fi μM afterIn the present study, we investigated the dose-dependent effects of midazolam on cell viability in neuroblastoma (SHEP) cells and the impact of midazolam pretreatment and coapplication on the cytotoxicity and antiproliferative effects of TMZ. Furthermore, the effect on the main anti-cancer mechanism of TMZ, the G2/M arrest, was evaluated.50: 227 μM). Transition from protective to toxic effects of midazolam was estimated to occur at 79.4 μM. When administered as a pretreatment 6 h prior to TMZ exposure, midazolam at a stimulatory concentration of 16 μM significantly increased the IC25 and IC50 of TMZ, whereas the IC75 remained unaffected. Pretreatment followed by coincubation abolished this effect and lowered the IC75 of TMZ as a sign of additional toxicity. The antiproliferative effect of TMZ (1000 μM) was significantly attenuated by pretreatment with midazolam, whereas pretreatment followed by coincubation with midazolam had no impact. With view to cell cycle analysis, TMZ (100 μM) increased the fraction of cells in G2/M phase after 48 h. Pretreatment followed by coincubation with midazolam did not affect the G2/M arrest.We revealed that midazolam alone induced a significant increase of cell viability with 2, 4, 8 and 16 μM, whereas 128, 256 and 512 μM of midazolam reduced cell viability , peak levels, renal and hepatic elimination and tissue concentration. Nevertheless, this study reveales first evidence, that midazolam ameliorates the cytotoxic effects of TMZ in neuroblastoma cells in vitro. The time period of pretreatment with midazolam was 6 h in our study and therefore reflects an increased t1/2 of midazolam in children and infants compared to adults [1/2, a single time period of pretreatment might not be sufficient to investigate a so far unknown effect. Further evaluation is necessary to specify the pharmacokinetic impact on the cytoprotective effects of midazolam. One of the most frequently discussed criticisms of pharmacologic in vitro studies involves the concentrations of the applied substances, as they frequently differ from plasma or tissue concentrations used in clinical practice. In our study, we used a broad spectrum of concentrations of midazolam and temozolomide. The logarithmic increase of the applied concentrations enabled us to discriminate small effects of low concentrations of midazolam and the evaluation of the IC25, IC50 and IC75 of temozolomide. The concentration of midazolam (16 μM) used for pretreatment in our study was within the concentration range reached after premedication and continuous sedation (0.3 - 23 μM) as reported previously [in vitro and in vivo concentrations of midazolam remains somewhat artificial. Three concentrations of TMZ were tested with view to an expected G2/M-arrest. Solely 100 μM of TMZ induced this effect after 48 h. This concentration is comparable to plasma levels of patients treated with temozolomide (72 μM), but is 10-fold higher compared to levels of TMZ in the cerebrospinal fluid of these patients [To date, data focusing on a pharmacologic interference between the sedative midazolam and the chemotherapeutic agent TMZ were lacking. Since neuroblastoma tumour cells are a common therapeutic target of TMZ in clinical practice, we chose an o adults . As hepaeviously –21. Nevepatients . However50 of both agents, the range of stimulatory concentrations of midazolam (1.7-14 % of IC50) was comparable with analysed concentrations of TMZ, which did not induce any stimulatory effect. Taken together, there is no evidence that TMZ is involved in effects based on hormesis [25 and IC50 of TMZ, whereas pretreatment with subsequent coincubation amplified the toxicity at high concentrations (reduced IC75) of TMZ. Taken together, these findings suggest that the attenuating properties of low-dose midazolam for TMZ-induced cytotoxicity are dependent on the timing of exposure and the magnitude of the subsequent toxic stimulus.The observed increase of cell viability in a neuroblastoma cell line after incubation with low concentrations of midazolam is a counterintuitive finding. Sedatives like midazolam are known as potentially harmful agents especially for neuronal cells with apoptosis-inducing properties at high concentrations, as described above. Previously, Chong and colleagues had shown that midazolam is capable of protecting against reactive oxygen species (ROS) induced cell death in B35 neuroblastoma cells . They rehormesis . InteresFurther aspects of TMZ-induced toxicity were revealed by the proliferation assay (BrdU), which indicated dose-dependent inhibition of neuroblastoma cell proliferation as expected. Pretreatment with midazolam attenuated the antiproliferative effect of TMZ (1000 μM), whereas pretreatment with subsequent coincubation had no significant impact on cell proliferation. Thus, pretreatment with midazolam is capable to affect cell characteristics even followed by exposure to high TMZ concentrations.As the anticancer properties of TMZ are primarily related to the induction of a G2 arrest of the cells, we investigated the influence of TMZ with and without midazolam on the cell cycle. Surprisingly, 24 h of incubation with TMZ  did not lead to a significant increase of G2/M-positive cells. The detection of an effect on the fraction of G2/M-positive cells after incubation with TMZ might require an appropriate amount of cell division, which is only reached after 48 h. This hypothesis is based on the result of cell cycle analysis after 48 h, as TMZ (100 μM) increased the fraction of G2/M-positive cells significantly. Pretreatment with midazolam did not alter this result, suggesting a different mode-of-action for midazolam-induced cytoprotection. Although midazolam pretreatment does not seem to influence the specific anticancer mechanisms of TMZ, the increase of mitochondrial activity, as indicated by XTT assay analysis, may contribute to the cytoprotective properties of midazolam. This fits well to the detected hormetic effect of midazolam, as hormesis is generally understood not to be based on a single mechanistic pathway, but rather reflects a complex pattern of cellular reactions to an unspecific sublethal stimulus.in vitro study in neuroblastoma (SHEP) cells, midazolam induced a biphasic action with increased cell viability after incubation at low concentrations, whereas high concentrations led to a profound decline in cell viability. Pretreatment with low concentrations of midazolam attenuated the toxic effect of TMZ, whereas pretreatment followed by coincubation had no cytoprotective effect. High concentrations of TMZ abolished the cytoprotection of pretreatment with midazolam. Coincubation with midazolam aggravated the toxicity of high-dose TMZ. The antiproliferative effect of TMZ was attenuated by pretreatment with midazolam. The TMZ-induced G2/M arrest was detected after 48 h of incubation and was not attenuated by pretreatment with midazolam. Our findings may indicate the possibility of reduced anticancer effects of TMZ in patients pretreated with midazolam and therefore justify further investigations into the interaction of chemotherapeutic agents with frequently used comedications like midazolam and other agents with cytotoxic potential.In this


Haemophilus influenzae strain, which had been experimentally transformed by DNA from a diverged clinical isolate. Donor segments averaged 6.9 kb  and were clustered into recombination tracts of ~19.5 kb. Individual colonies had replaced from 0.1 to 3.2% of their chromosomes, and ~1/3 of all donor-specific single-nucleotide variants were present in at least one recombinant. We found that nucleotide divergence did not obviously limit the locations of recombination tracts, although there were small but significant reductions in divergence at recombination breakpoints. Although indels occasionally transformed as parts of longer recombination tracts, they were common at breakpoints, suggesting that indels typically block progression of strand exchange. Some colonies had recombination tracts in which variant positions contained mixtures of both donor and recipient alleles. These tracts were clustered around the origin of replication and were interpreted as the result of heteroduplex segregation in the original transformed cell. Finally, a pilot experiment demonstrated the utility of natural transformation for genetically dissecting natural phenotypic variation. We discuss our results in the context of the potential to merge experimental and population genetic approaches, giving a more holistic understanding of bacterial gene transfer.Naturally competent bacterial species actively take up environmental DNA and can incorporate it into their chromosomes by homologous recombination. This can bring genetic variation from environmental DNA to recipient chromosomes, often in multiple long “donor” segments. Here, we report the results of genome sequencing 96 colonies of a laboratory  Many bacterial species are naturally competent, able to take up DNA from their environment and incorporate it into their chromosome by homologous recombination . This naComparing DNA sequences from natural bacterial isolates has found that intraspecific recombination is often common, especially in species known to be naturally competent . To do tIn contrast to the recombination that repairs DNA double-stranded breaks , the donor DNAs in natural transformation are transported into the cytoplasm as single strands with their 3′-end leading through a conserved membrane pore (Rec2/ComEC), whereas the complementary strands are simultaneously degraded. If translocated single-stranded DNA (ssDNA) has high sequence similarity to a segment of the competent cell’s chromosome, it can undergo recombination once coated with the strand exchange ATPase RecA; otherwise, translocated DNAs also are degraded. Thus transformational recombination occurs without the formation or resolution of Holliday junctions, since the substrates are ssDNA donors and dsDNA recipients  Figure .Unless mismatch repair intervenes, the resulting heteroduplex recombination products  will simply segregate upon DNA replication to give rise to two homoduplex daughter molecules, one carrying donor alleles and the other unchanged from the recipient. This heteroduplex segregation can cause single cells transformed by more than one donor DNA molecule to give rise to genetically heterogeneous daughter cells . Such heFurther complicating the picture, previous studies of natural transformation have shown that contiguous runs of donor-specific variation (“donor segments”) often are clustered into complex mosaic patterns, suggesting that long DNA molecules frequently are disrupted after uptake. This could be due to either endonucleases acting on DNA prior to strand exchange or heteroduplex correction mechanisms acting after recombination products are formed , indicating that only a fraction of cells in a competent culture take up and recombine DNA . AlthougExperimental studies of natural transformation have typically focused on the extent of recombination around selectable antibiotic resistance markers, but the advent of inexpensive DNA sequencing allows investigation of its genome-wide effects  in 1 mL] at 37° for 30 min. Cultures were then treated with DNase I (1 µg/mL) for 10 min to degrade extracellular donor fragments, followed by 1:5 dilutions into the rich medium sBHI (Difco brain-heart infusion supplemented with 10μg/ml hemin and 2μg/ml NAD) and incubation at 37° for 80 min to allow expression of the NalR resistance allele. A late-log transformation was conducted in the same manner, except using a culture grown in sBHI that had reached stationary phase (OD600 = 1.2). methods . Compete methods  were pre methods  and incuRNalR derivative of the nontypeable clinical isolate 86−028NP  were >750 kb apart and hence always carried on different molecules in the donor DNA preparations.Donor DNA was either from strain RR3131, a NovR or NalR antibiotic-resistant colonies per CFU. For MIV cultures, observed NovRNalR cotransformation frequencies were compared with those expected if all cells were equally competent, (NovR/CFU) * , providing estimates of the fraction of competent cells in the culture if competence is a binary on/off cellular state  to measure transformation frequencies, calculated as Novg for 5 min to collect cell pellets for DNA extracts.To permit detection of heteroduplex segregation that had occurred within the original transformed cells, antibiotic-resistant colonies were (a) not restreaked prior to inoculation for sequence analysis and (b) picked from plates with <30 colonies to reduce the chance that individual colonies arose from more than one cell. Picked colonies were inoculated into 5-mL sBHI cultures overnight; 1 mL of each resulting culture was stored in 15% glyercol at −80°, and the remaining 4 mL was centrifuged at 4000g at 4° for 10 min to remove cell debris. Supernatants were precipitated in 1 vol of isopropanol, and pellets were then washed in 70% ethanol, resuspended in 500 mL of TE  + 20 µg/mL RNase A, and incubated for 1 hr at 37°. Resuspensions were extracted 2× in 1 vol phenol/chloroform/isoamyl alcohol (24:23:1). DNA was precipitated from the aqueous fractions by addition of 0.1 vol of 3M sodium acetate and 2 vols of 100% ethanol, followed by centrifugation, washing in 70% ethanol for 10 min, and resuspension in 50 µL TE. This typically yielded ~15−20 µg of pure DNA fragments. DNAs were checked for purity and yield by spectroscopy (NanoDrop 1000) and agarose gel electrophoresis, diluted to 100 ng/µL, and arrayed into a 96-well plate for sequencing. DNAs extracted from parental strains RR722, RR3131, and RR666 were included as controls.Cell pellets were resuspended in 500 µL of resuspension buffer , lysed by adding lysis buffer , then incubated at 65° for 60 min. Potassium acetate (pH 8) was added to a final concentration of 1.25M, and the mixtures were centrifuged at 10,000R and 36 NalR colonies grown from three independent MIV transformations by RR3131 donor DNA, eight NovR colonies grown from a late-log transformation, and 16 unselected NovS NalS colonies grown from MIV transformations. Because these last 16 genomic DNAs were expected to be mostly untransformed, they were pooled 1:1 in pairs prior to library construction, for a total of 91 DNA samples (File S2).A total of 99 genomic DNAs were extracted : three cFile S1. In brief, sequence reads from each DNA sample were aligned to both donor and recipient genome references, followed by re-alignment of reads with short indels  was used to evaluate goodness-of-fit (K-S test). Permutation tests were used to generate random distributions of donor segment locations in order to calculate the probability of overlapping donor segments between different clones. Differences between the size of breakpoint intervals and genome-wide SNP spacing were evaluated by K-S test. Fisher’s exact tests were used to compare transforming insertions to transforming deletions and to compare breakpoint classes with their expected distributions. Student’s t-tests  were used to evaluate differences in transformability between strains.Plotting, curve fitting, and linear modeling used the R statistical programming language, including the fitdistrplus package. The Kolmogorov-Smirnov statistic (Table S1.For one of the samples with two mixed segments (RR4074), allele-specific PCR was used to confirm that the original sequenced DNA sample contained distinct cell populations with either the donor or recipient versions of both segments. The stored strain was streaked and individual colonies containing each of the two genotypes were identified by colony PCR. Because RR4107 and RR4108 were sequenced as a pool, PCR was also used to confirm which clone contained the observed recombination tract. Primers used are in 600 = 0.2 were diluted 1/100 in sBHI containing 10 µg/mL MAP7 DNA, followed by incubation at 37° for 8 hr, dilution, and plating onto sBHI agar ± kanamycin (7 µg/mL). Candidate recombinants, including RR4108, were reassayed with the MIV starvation method. Radiolabeled DNA uptake assays were performed as described  were generated in three distinct strain backgrounds . Construction of the RR3131 comM::spec and RR4108 comM::spec strains used transformation by a 4.25-kb amplicon across the comM::spec allele generated from a previously characterized comM::spec deletion made in the RR722 background (RR3121)(comM-F and comM-R in Table S1). Mutants were selected on 200 µg/mL spectinomycin and confirmed by PCR and Sanger sequencing.Deletions of the comM also were cloned into the EcoRI site of the low-copy vector pSU20 , and electroporated into the RR722 comM::spec and RR4108 comM::spec strains. Complementation assays of the resulting strains were carried out with wild-type controls, measuring the KanR transformation frequencies from MIV-induced cultures. The gene map was made with the assistance of GenoPlotR  were purged of paired reads failing Illumina chastity filters or containing adaptor sequences, but unaligned reads were retained to allow realignment to the 86-028NP donor genome reference.Sequence data for each sample were deposited—to the NCBI short-read archive under project accession SRP036875—as BAM files reporting alignments to the Rd (KW20) genome reference, with filename prefixes according to Haemophilus influenzae recipient cultures of the lab strain Rd were transformed with donor genomic DNA from a derivative of the clinical isolate 86-028NP carrying two antibiotic resistance markers . The divergence between the strains provides a high density of genetic markers; the donor and recipient genomes differ at 2.34% of alignable nucleotide positions (at SNVs), and ~10% of each genome is absent from the other , typical for a pair of H. influenzae isolates . Median read depth per position was ~50-fold, and although variation in read depth along the genome was high, this variation was consistent between libraries . Short-read sequence datasets were processed using an improved and expanded version of our previous approach . The antibiotic resistance selection ensured that each sequenced clone had acquired at least one donor segment, precluding inadvertent resequencing of untransformed recipient chromosomes.Three of the four transformation experiments used the standard competence-induction protocol, in which exponentially growing cells are transferred to the starvation medium MIV to induce maximal competence, followed by incubation with donor genomic DNA . From eaFile S3). The total extent of recombination per clone was consistent with a lognormal distribution . Individual recombinant chromosomes contained on average 394 donor SNVs in 1−10 donor segments, collectively replacing between 2.7 and 58.5 kb of the recipient chromosome, or on average 1.13% of genome length. This overall extent of recombination was approximately twofold higher (mean 1.13% genome length) than that predicted from the transformation frequencies of the NovR and NalR markers alone , with the largest segment 43.1 kb long, or 2.4% of the genome length,. The exponential fit to the distribution of segment lengths was improved by excluding the eleven outliers that contained only a single SNV , suggesting that these arise by a distinct process. These and other very short donor segments are discussed below as potential remnants of donor-directed mismatch repair. These results with H. influenzae contrast with the shorter recombinant segments detected in Streptococcus pneumoniae and Helicobacter pylori transformants , when defined by their outermost donor-specific variants . Segment lengths followed an exponential distribution  , and three overlaps at only one interval  resulted in clones with extensive cotransformation of adjacent donor-specific variation .Local reductions in transformation around the selected sites often corresponded to positions of indels . Although this 100 kb cut-off is somewhat arbitrary, it is likely generous, since defining recombination tracts in this way gives a mean tract size of 19.5 kb (median 12.9 kb), and only 16 of the 130 tracts (collapsed from 216 segments) exceeded the size of the largest single uninterrupted donor segment . However, because the original taken up DNA fragments are unknown, it is important to note that some apparent clustering may instead result from coincidental transformation by two independent fragments. Clustering was determined using donor rather than recipient genome coordinates, since rearrangements between the two genomes otherwise caused the spurious appearance of clustering or independence of segments in several clones .As observed in previous work in several organisms, donor segments in individual recombinants were often clustered near other segments, suggesting that endonucleases disrupt long DNAs after uptake   had a single recombination tract spanning the selected Novl tracts . Notablyl tracts .Based on the observed number of tracts per recombinant, the line in R and NalR alleles transformed strain Rd about threefold more efficiently when the donor background was Rd than when the donor background was the diverged 86-028NP . The lower transformation frequency with DNA from a diverged background is presumably due to sequence heterology around the selected alleles, which could either reduce the probability of strand exchange or increase the probability of recipient-directed heteroduplex correction.As we previously observed, the Nov2 = 0.92), closely matching the genome-wide mean SNV density (superimposed solid lines in vs. a linear estimate of 18.1 ± 0.3 SNVs/kb in donor segments). The lack of effect of heterology was not an artifact of selection for the NovR and NalR alleles, since it held even when only donor segments >100 kb from the selected sites were considered (2 = 0.85). This result is consistent with recent observations in Streptococcus pneumoniae, as well as with the finding in H. influenzae that individual donor segments span regions of both high and low nucleotide divergence  often acted as blocks to recombination progression, they also sometimes transformed as parts of longer recombination tracts . Of the R and NalR variants , but a similar pattern was reported for S. pneumoniae transformants  . This difference was seen, even for subsets of the data that included only breakpoint intervals for unselected donor segments (>100 kb away) and when only unique breakpoint intervals were used .We defined donor segment intervals by the outermost variants of contiguous runs of donor-specific alleles, but the true recombination breakpoints must lie between these positions and their nearest flanking recipient-specific alleles. Because the lengths of breakpoint intervals are also measures of local sequence identity, we tested whether they were distinct from the genome-average variant spacing. The high sequence identity at breakpoints likely underestimates the effect of sequence divergence on recombination breakpoints, since many were likely to have been pre-determined by the donor fragment ends or by degradation of ssDNAs after uptake. The connection between high local identity at recombination breakpoints—yet no connection with limiting the locations of donor segments themselves—implies that initiation of strand exchange from the ends of RecA-coated donor DNA may require higher levels of sequence identity than the subsequent progression of strand exchange, consistent with biochemical analyses of RecA-mediated strand exchange .p-value << 0.001). Only part of this effect is due to recurrent breakpoints at the same SV . This enrichment could arise because strand exchange frequently terminates at SVs, or because heteroduplex molecules with mismatched SVs are more efficient targets for heteroduplex correction.In contrast, breakpoint intervals were ∼fourfold more likely to have a SV as the nearest recipient-specific variant : 10.9% o same SV , since eR or NalR allele . This was confirmed by allele-specific PCR from colonies streaked from the stored culture. In the other mixed colony  are also consistent with double translocation of a single donor DNA, in this case spanning an inversion breakpoint.More details were revealed by analysis of allele frequencies in the two colonies that contained clustered segments with a mixed genotype. In one of these colonies , the twod colony , the twoFor 11 donor segments only a single unambiguous donor-specific SNV is present; 7 of these are not clustered into longer recombination tracts. These likely represent genuine outcomes of transformation, rather than being artifacts . Two analyses suggest that the frequency such artifacts is expected to be very low. First, no new high quality variants  were found in the 72 recombinants, suggesting that new mutations are uncommon. Second, only 10 high-quality variants were identified by sequencing another lab strain, MAP7, an Rd derivative resistant to seven antibiotics .Because these singlet segments are clearly more abundant than predicted by the overall distribution of donor segments , they liR and NalR were ~10-fold higher than predicted by the product of their single marker transformation frequencies . This excess of double transformants (termed congression) is usually assumed to reflect the presence of noncompetent cells in the culture, and suggests that only ~10% of cells actively took up and recombined donor DNA into their chromosomes (Observed cotransformation frequencies of Novomosomes .File S3). The sizes of the two tracts fell within the range seen in selected clones (2.7 kb and 40.2 kb). The estimates in p-value = 0.01).To directly test whether congression measures % competence, we sequenced 16 unselected clones from the MIV transformations. Only 2 of 16 had been transformed, each carrying a single recombination tract, consistent with congression predictions of ~10% competent cells , File S3R/CFU of 1.15 × 10−5 , suggesting that transformation in late-log cultures results from a very small fraction of fully competent cells. This conclusion is also supported by co-transformation data from late-log cultures, which indicates ~0.1% competent cells (data not shown).Cultures grown to high density in rich medium (“late-log”) are ~100-fold less transformable than MIV-induced cultures . This identified a single NovSNalS recombinant clone . This recombinant has normal DNA uptake compared to Rd, so it must not have acquired alleles from the donor that affect either competence regulation or the outer membrane DNA uptake machinery: Following the method of .The recombinant contains a single 40.3 kb recombination tract carrying 483 donor-specific SNVs, spanning ~30 genes and deleting a 3.7 kb xylose ABC transporter operon specific to the recipient strain . At leascomM, is found within the RR4108 donor segment. Null mutants of comM have a 42-fold defect in the Rd strain background ; its protein product acts after DNA uptake to promote recombination, consistent with the normal uptake observed in the RR4108 recombinant , indicating nonsynonymous comM differences are not responsible for the phenotypic change.One known competence-regulated gene, comM is not responsible for the altered phenotype, the chromosomal comM locus is required for the difference between the strains to be observed: First, deleting comM from RR4108 reduced transformation to the same levels as in Rd . This might suggest that ComM activity contributes more to transformability in the recipient background, but the requirement for ComM was substantially stronger in the 86-028NP donor background . Second, the plasmids carrying the Rd and NP alleles of comM failed to complement a RR4108 comMΔ::spc mutant . This might indicate that variation in the RR4108 donor segment modulates comM activity in cis, but both promoter and terminator are invariant between the strains and the plasmid insert carrying the comM alleles was flanked by ±700 bp.Additional experiments complicated matters; these indicate that although genetic variation within comM locus and unidentified but linked natural variation.In summary, this pilot mapping experiment has used natural transformation to identify a genetic interval carrying natural variation that affects transformability itself. We found that the variation in transformability is a polygenic trait with differences affecting both pre- and postuptake steps, and we have begun unraveling complex interactions between the H. influenzae cells were incubated with DNA from a diverged clinical isolate, finding that the average cell’s daughters replaced ~1% of their chromosomes in multiple independent recombination events that spanned many genes. These data will help constrain parameters used for genome-wide inferences of historical recombination in natural populations, for example by providing a rate parameter to describe the exponential distribution of donor segment lengths , which could reflect their processing by distinct DNA-uptake mechanisms. Although the Gram-negative H. influenzae translocates large donor molecules from the periplasm through its inner membrane, S. pneumoniae and other Gram-positive bacteria process dsDNAs with membrane-bound nucleases before translocation through their single cell membrane  that contained natural variation with a 10-fold effect on transformability. To our knowledge, this is the first time transformation has been used to identify a bacterial quantitative trait locus, and importantly the causal variation is not accounted for by any of the known competence genes (