Extracellular ATP evoked (EC50 13.3 ± 1.4 μM) robust biphasic intracellular Ca2+ responses that showed a dependency on both metabotropic (P2Y) and ionotropic (P2X) receptors. qRT-PCR and immunocytochemistry revealed the expression of P2Y1, P2Y2, P2Y6, P2Y11, P2Y13, P2X1, P2X4, P2X5, and P2X7. Pharmacological analysis revealed contribution of only P2X4 and P2Y11 to the Ca2+ response evoked by maximal ATP concentrations (100 µM). This study reveals the contribution of P2X4 and P2Y11 receptor activation to ATP-evoked intracellular Ca2+ responses, and makes comparison with macrophage differentiated using granulocyte colony-stimulating factor (GM-CSF).Tissues differentially secrete multiple colony stimulating factors under conditions of homeostasis and inflammation, orientating recruited circulating monocytes to differentiate to macrophage with differing functional phenotypes. Here, we investigated ATP-evoked intracellular Ca CSF1 – macrophage colony-stimulating factor (M-CSF); CSF2 – granulocyte macrophage colony-stimulating factor (GM-CSF); and CSF3 – granulocyte colony-stimulating factor (G-CSF). While M-CSF is expressed in abundance by many tissues during homeostasis [The generation of monocyte-derived macrophage can be achieved by exposure to colony-stimulating factors (CSFs). CSF are secreted glycoproteins which can recognize and bind to receptors expressed on the surface of haemopoietic stem cells and stimulate proliferation and differentiation. CSFs are classically characterised in three types, as follows: eostasis ,5. M-CSFeostasis . M2-macreostasis ,8. M2-maeostasis . Converseostasis . M1-macreostasis . Hence, eostasis .2+ responses in M-CSF differentiated human monocyte-derived macrophage.In leukocytes, adenosine 5′-triphosphate (ATP) is a danger-associated molecular pattern (DAMP), released by damaged and inflamed tissues. In addition, ATP is physiologically released by healthy cells in response to stimulation, including mechanical distortion, hypoxia, and others . The bio2+ response that increased in magnitude with ATP concentration (2+ response (area under the curve) at maximal ATP concentrations were significantly larger at those observed previously for GM-CSF differentiated macrophage  . Furthermore, phospholipase C inhibition with U73122 significantly reduced ATP-evoked Ca2+ responses (2+ responses in M-CSF differentiated human macrophage result from the combined activation of metabotropic (P2Y) and ionotropic (P2X) receptors for extracellular ATP.ATP evoked Ca donors) A. ATP walar Ca2+ A and witesponses B, thoughesponses C. Taken 2+ responses, we first determined the expression of P2X and P2Y receptor subtype mRNA transcripts by quantitative RT-PCR. For P2Y receptors we explored both ATP and ADP activated receptor subtypes, as ADP can be liberated by the cell surface catabolism of ATP in macrophage [To investigate the molecular basis for ATP-evoked Cacrophage . Of the crophage    the ATP-evoked Ca2+ response (2+ response (N = 7) compared to 31.8 ± 9% (N = 4) for 5-BDBD. Both PSB-12062 and 5-BDBD inhibited the ivermectin potentiated response by 11.5 ± 4.5% (N = 6) and 16.3 ± 3.5% (N = 4), respectively. Interestingly, P2X1 antagonism with Ro0437626 caused a minor but significant increase in the response  . This daresponse A. Converresponse B,C. PSB-response A. In kee2+ response consisting of a rapid peak and return to baseline. ATP evokes intracellular Ca2+ responses in M-CSF and GM-CSF differentiated macrophage [2+ in M-CSF differentiated macrophage was approximately 3-fold greater in M-CSF differentiated macrophage. As with GM-CSF macrophage [2+response was due to activation of metabotropic P2Y receptors, revealed by U73122 sensitivity. Though not tested here, coupling of Ca2+ mobilisation to Orai1/STIM proteins are likely to be involved as well, as they have been shown to be important in GPCR-mediated Ca2+ signals in macrophage and microglia [2+ response in GM-CSF differentiated macrophage [Monocyte-derived macrophage differentiated using GM-CSF or M-CSF serve as a well-established tool to study primary tissue-specific macrophages . In thiscrophage  with simcrophage  and modecrophage  the majoicroglia ,24. P2Y1crophage , does nocrophage , howevercrophage . ADP is crophage . Differe2+ responses is greater in M-CSF differentiated macrophage than that observed in GM-CSF differentiated macrophage, based upon U73122 sensitivity. These findings suggest that monocytes that differentiate to macrophage in a M-CSF containing environment are equally sensitive to ATP as GM-CSF differentiated macrophage, but respond with a greater intracellular Ca2+ response, and P2X receptors contribute more to this response. In keeping with our previous observations in human primary macrophage [2+ response at maximal ATP contributions. Others have observed P2X7 receptor contribution with the agonist BzATP, or at supramaximal ATP concentrations in the presence of unphysiologically low divalent cation concentrations [A comparison of M-CSF differentiated macrophage in this study and GM-CSF differentiated macrophage in our previous work  suggestscrophage , P2X7 dotrations ,29,30. Atrations .2+ responses is greater in GM-CSF compared to M-CSF macrophage. In GM-CSF differentiated macrophage, activation of the P2X4 receptor elicits secretion of the pro-inflammatory chemokine CXCL5 [Monocyte-derived macrophages differentiated with colony stimulating factors GM-CSF or M-CSF are good in vitro models for the study of primary human macrophage . Though ne CXCL5 . P2X4 an2+ responses, though the molecular basis for this differs. In M-CSF differentiated macrophage, both P2X and P2Y receptors contribute to the response with P2X4 and P2Y11 receptors playing a dominant role. This study provides novel information regarding the molecular basis of ATP-evoked Ca2+ signals in human primary macrophage differentiated by M-CSF.In summary, our data reveal that human macrophage differentiated from monocytes exposed to different colony stimulating factors both respond to ATP by generating intracellular Cag for 10 min to isolate plasma. Whole blood was diluted with RPMI medium in a 1:1 ratio and layered onto a Histopaque-1077  density gradient before centrifuging at 1000× g for 25 min. Mononuclear cells were collected by gently transferring the opaque interface into a fresh centrifuge tube. Cells were washed in RPMI and sedimented at 300× g for 10 min. PBMCs were washed in PBS followed by resuspension in RPMI .Human whole blood was obtained from healthy volunteers through the Faculty of Medicine and Health Sciences Research Ethics Committee , University of East Anglia. Whole blood was centrifuged at 1000× 6 cells/cm2 and incubated at 37 °C in a humidified 5% CO2 incubator for 2 h. Following incubation, adhered monocytes were washed thoroughly with PBS. Cells were cultured in RPMI-1640 with L-glutamine, penicillin-streptomycin, and 2.5% (v/v) heat-inactivated autologous serum. Monocytes were differentiated to macrophage by addition of 10 ng/mL recombinant human macrophage colony stimulating factor (M-CSF)  or recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech) for 6 days.PBMCs were seeded in RPMI at a density of 1.5 × 10mRNA quantification was performed using methodology and Taqman primer sets for P2 receptor subtypes, as previously described .w/v) paraformaldehyde for 15 min. Cells were permeabilised for 10 min with 0.25% (v/v) Triton X-100 followed by blocking with 1% (w/v) bovine serum albumin. Cells were incubated overnight with rabbit polyclonal primary antibodies  for 10 min at room temperature prior to staining. Cells were incubated with either a mouse monoclonal anti-CD14 antibody conjugated to phycoerythrin (PE), or a mouse PE-conjugated isotype control (Biolegend). Flow cytometry was performed using a Cytoflex Instrument .2, 1.2; CaCl2, 1.5; D-glucose, 8; HEPES, 10; and pH 7.4. For experiments performed in the absence of extracellular Ca2+, CaCl2 was omitted from SBS and replaced with 2 mM EGTA. Macrophage resuspended in SBS were loaded with calcium indicator using 2 µM Fura-2 AM for 1 h at 37 °C. After washing in SBS, 20,000 macrophage per well were seeded in a 96-well plate and allowed to adhere for 1 h at 37 °C. Intracellular Ca2+ measurements were made using a Flexstation III instrument (Molecular Devices). Fluorescence emission at 510 nm was collected every 2 s, and fluorescence using excitation wavelengths of 340 and 380 nm were used to quantify changes in intracellular Ca2+, represented as F-ration following subtraction of baseline.Assays were performed in salt-buffered saline (SBS) containing the following (mM): NaCl, 130; KCl, 5; MgClATP, PSB-12062, and 5-BDBD were procured from Sigma Aldrich. U73122, MRS2500, ARC-118925, MRS2578, NF340, A438079, Ro0437626, MRS2211, and ivermectin were procured from Tocris. Receptor antagonists, inhibitors, and ivermectin were applied to cells 30 min before ATP challenge.t-test. Concentration-response relationships were fitted using a Hill Equation, as follows:k = the Michaelis constant and n = the number of cooperative sites.All data were analysed using Origin Pro 9.1 software . Data is represented as mean ± standard error of the mean (SEM). N is defined as the number of biological replicates (number of donors). Data was tested for normality using a Shapiro–Wilk test, followed by hypothesis testing by


Measurement of high-sensitivity troponin T (hs-TnT) has become an essential step in the diagnosis of acute myocardial infarction. This high-sensitivity method allows quantifying the concentration of troponin T in blood of healthy subjects with a lower inaccuracy compared to previous reagent generations. However, the presence of certain compounds in the sample may interfere with the result. We present a patient who had repeatedly high concentrations of hs-TnT in the serum sample that did not agreed with the signs and symptoms. In addition, ultrasensitive troponin I concentration was undetectable.To investigate the presence of an interfering compound, different analysis were carried out. In order to discard macro complexes in the sample, the serum was precipitated with polyethylene glycol. In addition, the serum was incubated with Scantibodies Heterophilic Blocking Tube, which can block heterophilic antibodies. Finally, a size exclusion chromatography of the sample was performed by the manufacturer.The interfering substance was allocated into fractions with proteins of 150kDa, corresponding to high molecular weight proteins like immunoglobulin G (IgG). This compound was responsible for the falsely elevated hs-TnT results and it affected only the high-sensitivity methods.The detected interfering compound was probably an IgG. This type of interference must be kept in mind in front of discordant results, even if they are extremely rare. Therefore, interdisciplinary cooperation between clinicians, laboratory and manufacturer is essential. It is useful in particular to detect earlier troponin changes after the acute event. Since other cardiac and no cardiac pathologies can slightly increase the troponin concentration the recent measurement improvement reduces the specificity for the diagnosis of AMI (The use of the new high-sensitivity troponin (hs-Tn) tests has changed the approach to the diagnosis of the acute myocardial infarction (AMI). These methods of high sensitivity are characterized by the improvement of the detection limit, which is defined as the lowest concentration of analyte detectable by analytical method  of 51 ng/L (99th percentile = 14 ng/L), creatine kinase (CK) within the reference range and preserved renal function. Exercise test was within normality range and thus acute process by ischemia, pericarditis or myocarditis was discarded.Blood tests were repeated 4 months later and an increase of hs-TnT up to 98 ng/L was found, while CK and other parameters were within normal ranges. A second echocardiography was performed and no pathological alterations were detected.A new blood sample was collected one month later and high concentration of hs-TnT (52 ng/L) was observed again. Given the absence of compatible symptoms, a falsely positive result caused by interference was suspected.All patient samples were drawn in gel separator tubes without anticoagulant . Tubes were left at room temperature for 15 minutes approximately to allow blood coagulation and then centrifuged at 2030xg for 10 minutes. Both analysers for measuring the concentration of hs-TnT available in our laboratory, Cobas e801 and Cobas e601 , employ electrochemiluminescence immunoassays (ECLIA) and use a sandwich method with two mouse monoclonal antibodies (capture and detection), which are specifically directed against epitopes 125-131 and 136-147 of human cardiac troponin T  was processed during the study period for both analysers. The coefficient of variation (CV) obtained during this period was 3.0% for Cobas e601 and 3.5% for Cobas e801.e.g. even among various hs-TnI assays)  on the Architect-i System platform , using the hs-TnI STAT reagent. However, it is important to take into account that the interference susceptibility might vary among the different measurement systems .In order to confirm the results, another visit was scheduled 3 months later in which all tests were repeated and the concentration of various autoantibodies by indirect immunofluorescence assay  was measured . In addiFinally, the serum sample was sent to the manufacturer to further investigate the potential interference. The sample was fractionated by size exclusion chromatography to detect the molecular size that could cause interference with the hs-TnT test. The principle of this technique includes a different elution speed depending on the molecular weight. Therefore, since the molecular weight of troponin T is known (~36 kDa), it is possible to infer if the fraction in which troponin T is expected is also showing the reactivity with the hs-TnT assay.2 = 0.99).According to the literature on interference in immunoassays additional studies were performed, as summarized in Size exclusion chromatography results suggest that the interference can be allocated to fractions with proteins of 150 kDa, corresponding to high molecular weight proteins like immunoglobulin G (IgG). Quoting the manufacturer report: “the observed hs-TnT values are falsely elevated in the sample. The interference is caused by a high molecular weight compound, presumably IgG”. Independent studies concluded that such type of interference are rare and are covered by disclaimers in the method sheet .We report the case of a 12-years-old boy with high concentration of hs-TnT without evidence of myocardial injury. As a consequence of this discordance, a falsely positive result was suspected.After checking the correct calibration status of the analyser and repeating the hs-TnT measurements, possible pre-analytical causes that could alter the results were studied. The presence of fibrin clots or micro particles was ruled out after inspection of the sample and besides that, the haemolysis, icterus and lipemia indices were found to be under the interference level  and hs-TnI (< 10 ng/L). Troponin T and Troponin I are both elevated in case of myocardial injury, so an undetectable concentration of hs-TnI meant that there was no real injury and the elevation of hs-TnT was caused by an interfering compound.In addition, the presence of macro complexes was investigated but after not observing different results in the sample before and after PEG 6000 addition, such type of interferent was excluded . Its incidence in the population lies between 0.2 - 40% and it includes two specific circumstances: the so-called “true” HA, which are multispecific and low affinity antibodies, and human anti-animal antibodies (HAAA), which are high affinity antibodies against specific animal immunoglobulins (HAMA)  in the measurement procedure of Cobas e601, which reduces the analysis time from 18 minutes to 9, by reducing the number of incubations from two to one. It might be possible that an extended incubation time could favour the union of the IgG to the antibodies, and therefore enhancing the interference, justifying the higher concentration of hs-TnT observed in the method that uses two incubations instead of one, but further validation was not possible. Nevertheless, such single results should not be over interpreted.In addition, a former less sensitive method (Troponin T STAT) provided no evidence of troponin elevation in the same patient sample (< 10 ng/L), which indicates that the high-sensitivity methods might be more susceptible to such type of interference. Irrespectively of the used immunoassay type, either one or two step assays, the frequency of such HAMA interference has been reported between 0.1 - 3.1% and may involve both Troponin I and Troponin T tests (Nowadays, troponin measurements are indispensable part of AMI definition and related guidelines for their interpretation are urgently required in daily clinical practice. When a high concentration of troponin is not compatible with the clinic signs and symptoms of the patient, the presence of an interfering factor in the sample should always be suspected.In the case reported here, falsely elevated results of hs-TnT in two different analysers might have been caused by an IgG antibody that could not be removed after incubation with HBT. At low analyte concentration, this effect was observed only for the high-sensitivity methods of troponin T. In addition, the concentration of hs-TnT was lower when STAT technology was used, which could be explained by the lower incubation time. Even if this type of interference is disclaimed by the manufacturer, the present case described the track to substantiate such hypothesis.Interference by HA is a problem that may affect the immunoassays, especially after the monoclonal antibody-based therapy use in recent years (


An accurate and timely diagnosis for Alzheimer’s disease (AD) is important, both for care and research. The current diagnostic criteria allow the use of CSF biomarkers to provide pathophysiological support for the diagnosis of AD. How these criteria should be operationalized by clinicians is unclear. Tools that guide in selecting patients in which CSF biomarkers have clinical utility are needed. We evaluated computerized decision support to select patients for CSF biomarker determination.We included 535 subjects  from three clinical cohorts. Positive (AD like) and negative  CSF biomarker profiles were simulated to estimate whether knowledge of CSF biomarkers would impact (confidence in) diagnosis. We applied these simulated CSF values and combined them with demographic, neuropsychology and MRI data to initiate CSF testing (computerized decision support approach). We compared proportion of CSF measurements and patients diagnosed with sufficient confidence (probability of correct class ≥0.80) based on an algorithm with scenarios without CSF , CSF according to the appropriate use criteria (AUC) and CSF for all patients.The computerized decision support approach recommended CSF testing in 140 (26%) patients, which yielded a diagnosis with sufficient confidence in 379 (71%) of all patients. This approach was more efficient than CSF in none  diagnosed), CSF selected based on AUC (295 (55%) CSF, 350 (65%) diagnosed) or CSF in all  diagnosed).We used a computerized decision support with simulated CSF results in controls and patients with different types of dementia. This approach can support clinicians in making a balanced decision in ordering additional biomarker testing. Computer-supported prediction restricts CSF testing to only 26% of cases, without compromising diagnostic accuracy. An accurate and timely clinical diagnosis of Alzheimer’s disease (AD) is important both for care and research . Over thDiagnostic guidelines do not specify which patients should receive CSF testing however, and as a result there is a large practice variation . RecentlData-driven support tools could be helpful to answer the question ‘for which patient should CSF biomarkers be measured?’ before embarking on actual testing. By using simulated positive (AD like) or negative  CSF biomarker profiles for a specific patient added to their priori clinical information, such a tool could inform the clinician on how the knowledge of CSF biomarkers would impact (confidence in a) diagnosis .The Disease state index (DSI) classifier is a clinical decision support system (CDSS) which supports the differential diagnosis of several types of dementia by combining and weighing all available information, including cognitive tests, automated MRI features and CSF biomarkers . This muIn this work, we took the functionality of our CDSS as an input to predict who would benefit from CSF testing. More specifically, we tested whether the CDSS may help to answer the following question: if a clinician already has information on neuropsychological tests and MRI brain, would additional CSF testing contribute significantly to a more accurate diagnosis? We thus aimed to evaluate how a computer-based tool can help in selecting those patients by using a data-driven approach and simulated CSF outcome.We included 535 subjects from three memory clinics, as part of the PredictND project : 463 subAll subjects had received a standardized work-up, including medical history, physical, neurological and neuropsychological assessment, MRI, laboratory tests and CSF. Subjects were diagnosed as SCD when the cognitive complaints could not be confirmed by cognitive testing and criteria for mild cognitive impairment (MCI) or dementia were not met. Probable AD was diagnosed using the core clinical criteria of the NIA-AA for AD dementia . ProbablCognitive functions were assessed with a brief standardized test battery including widely used tests. We used the Mini-Mental State Examination (MMSE) for global cognitive functioning . For memCSF beta amyloid 1–42 (AB42), total tau and tau phosphorylated at threonine 181 (p-tau) were measured with commercially available ELISA tests  locally according to standard procedures. Raw data values were used for analysis. Analysis showed comparable values between the centers and hence no further correction was performed.Apolipoprotein-E (APOE) genotyping were performed locally in each center. Patients were dichotomized into APOE e4 carriers (hetero- and homozygous) and non-carriers. APOE data were available in 472 (88%) subjects.® cMRI quantification tool: 1) computed medial temporal lobe atrophy (cMTA), 2) computed global cortical atrophy (cGCA), 3) AD similarity scale, 4) Anterior Posterior index and 5) white matter hyperintensities (WMH). First, we automatically computed the MTA and GCA scores , but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” :[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The study showed that using a computerized decision support as a tool to guide in selecting patients who need to be detected CSF is helpful in clinical operations. The computerized decision support approach reduced the patients who need CSF testing and classified varied types of dementia with increased sufficient confidence. The work has the potential clinical utility value.I still have some questions for this work.1. In this study, I noticed the controls were individuals with subjective cognitive decline. As far as I know, SCD is defined as the population in higher risk for future cognitive impairment and may have already exist abnormal CSF alteration. Thus, I would like to ask why to chose SCD as controls.2. The description of imaging markers is too general. In this study, T1 images were analysed to extracted quantitative indicators. However, the procedures were not clear. For example,VBM was conducted by which software and whether to eliminate the effect of volume of cranial cavity. Please specify the processes which would help others to verified the results.3. In the study, CSF biomarkers included beta amyloid 1-42 (AB42), total tau and tau phosphorylated at threonine 181 (p-tau), which were all AD-related markers. Thus, whether to test these CSF markers would help improve the accuracy of diagnosis in VaD and FTD?Reviewer #2: 1) Line 131, the reference associated with RAVLT needs to be revised.2) Line 134, in [21] the paper presents different verbal fluency tests, why the choice of animals ? and did the author try with other categories to see if this leads to different findings ?3) Line 157, incorrect citation [9], please provide the original references, I suppose the author needs to cite this paper in his/her case; Lötjönen J., Wolz R., Koikkalainen J., Thurfjell L., Waldemar G., Soininen H., Rueckert D. Fast and robust multi-atlas segmentation of brain magnetic resonance images. NeuroImage. 2010;49(3):2352–23654) Line 247, in table 1, not all the characteristics are available for all patients, how did the author deal with missing information ? How confident is he/she about their effect on the results.5) Line 265, it is unclear to me how the author has simulated the CSF values.6) Line 276, table2 , I would appreciate if the author interprets and explains why scenario A had higher rate than scenario D.7) According to the figure in the supplementary notes, the higher DSI/delta_DSI the closer PCC to 1. Hence, the higher PCC the better for the diagnosis, how does the author explain that for values larger than 0.8 the accuracy of diagnosis decreased (line 335).**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  8 Nov 2019Amsterdam, 31 October 2019Regarding PONE-D-19-25997Dear dr Han Zhang, Please find uploaded our revised manuscript for publication in PLOS ONE as part of the special collection on ‘Early diagnosis and treatment of Alzheimer’s disease’. We thank the editor and reviewers for their careful reading, thoughtful comments, and recommendation for revision. Please find below our responses to the comments in a point-by-point fashion. We have highlighted changes (via track&change) in response to the reviewers’ comments in the manuscript and supplemental data. We hope the rebuttal adequately addresses the points raised during the review process, Kind regards, also on behalf of the co-authors, Hanneke Rhodius- Meester===========================================================================Journal Requirements:http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at Reply: We have updated our manuscript and file naming according to your requirements.2. Please describe in your methods section how capacity to provide consent was determined for the participants in this study. Please also state whether your ethics committee or IRB approved this consent procedure. If it was not necessary to assess the capacity to provide consent of your participants please outline why this was not required in this caseReply: We added information on the consent procedure to the paragraph ‘subjects’ in the methods section (L124-126). http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions.3. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For information on unacceptable data access restrictions, please see In your revised cover letter, please address the following prompts:a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail  and who has imposed them . Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent.team@predictnd.eu. We hope you understand the restrictions we unfortunately have to deal with.Reply: We address this in our revised cover letter by adding a paragraph on data access. For this study we requested data from the PredictND consortium. The data-sharing agreement of the PredictND consortium however allows us use of data for this specific project only. The data underlying the results presented in the study are available upon request from the PredictND-board; 4. Thank you for stating the following in the Financial Disclosure section:'The author(s) received no specific funding for this work.'We note that one or more of the authors are employed by a commercial company: Combinostics Ltd and VTT Technical Research Centre of Finland Ltd.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.Please also include the following statement within your amended Funding Statement.“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.2. Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” Reviewer #1: The study showed that using a computerized decision support as a tool to guide in selecting patients who need to be detected CSF is helpful in clinical operations. The computerized decision support approach reduced the patients who need CSF testing and classified varied types of dementia with increased sufficient confidence. The work has the potential clinical utility value.I still have some questions for this work.1. In this study, I noticed the controls were individuals with subjective cognitive decline. As far as I know, SCD is defined as the population in higher risk for future cognitive impairment and may have already exist abnormal CSF alteration. Thus, I would like to ask why to choose SCD as controls.Reply: Thank you for this observation; the use of SCD patients as controls can be seen as a topic of discussion, which we added . SCD might be an enriched population, and we think that the reviewers’ concern is that the ability of our computerized decision support approach to select the correct patient for CSF testing would be underestimated. If we used ‘healthy controls’ the tool might even perform better. However, we feel that using SCD patients in this computerized decision support approach is actually a strength of our study. In fact, clinical practice is not about comparing AD patients with healthy controls, but on the differential diagnosis. As SCD patients do seek help for their complaints, they also reflect daily clinical practice, where clinical decision support systems should work. Finally, biomarker status in healthy controls is often unknown, whereas a proportion of these ‘healthy’ controls will be biomarker positive. 2. The description of imaging markers is too general. In this study, T1 images were analyzed to extracted quantitative indicators. However, the procedures were not clear. For example,VBM was conducted by which software and whether to eliminate the effect of volume of cranial cavity. Please specify the processes which would help others to verified the results.Reply: We apologize that the description on imaging markers was unclear to you. As described in the second line of this section, the processes were conducted by the cNeuro® cMRI quantification tool. To enhance readability, and because our core message is not on the imaging markers, we have chosen to show limited details on these markers. These processes of deriving these markers have been described elaborately in several papers, to which we refer in the text. All markers were corrected for age, sex and head size, we added this information to the methods section (L169-170). 3. In the study, CSF biomarkers included beta amyloid 1-42 (AB42), total tau and tau phosphorylated at threonine 181 (p-tau), which were all AD-related markers. Thus, whether to test these CSF markers would help improve the accuracy of diagnosis in VaD and FTD?Reply: You are of course correct in stating that the chosen CSF biomarkers are all AD-related markers. In clinical practice these are the CSF biomarkers that clinicians can use for detecting AD pathology during life; CSF biomarkers for FTD and VaD unfortunately have not been developed yet. Nonetheless, CSF biomarkers for AD may still be useful in the differential diagnosis of FTD and VaD. In clinical practice the clinician is faced with a differential diagnostic dilemma’s for all his/her patients. It is thus not about ‘controls versus AD’, but about ‘this type of dementia or that type of dementia’. A tool that supports clinicians in ordering CSF biomarkers in daily practice, should thus be able to cope with this differential diagnostic dilemma. Especially since there can be a broad overlap in clinical symptoms in AD, FTD and VaD. Negative AD-biomarkers increase the likelihood of another underlying pathology, and may in turn help the clinician with this differential diagnostic dilemma. Our computerized decision approach aids for all diagnostic groups, as can be seen in table 3, pointing out both patients in which CSF is not needed and patient in which CSF is useful. For this group with different types of dementia, reflecting clinical practice, we need CSF in only 1 out of 4 patients, without compromising diagnostic accuracy. Reviewer #2: 1) Line 131, the reference associated with RAVLT needs to be revised.Reply: We apologize for this mistake and added the correct reference (L132).2) Line 134, in [21] the paper presents different verbal fluency tests, why the choice of animals ? and did the author try with other categories to see if this leads to different findings ?Reply: Thank you for this interesting suggestion. One can definitely argue for example letter fluency, and the difference between letter and animal fluency, can be very indicative. In our dataset (which we used retrospectively) we only had information on animal fluency, not on other categories. However we were able to construct a brief standardized test battery that contains widely used tests, and is thus representative for clinical practice, where our tool should work. 3) Line 157, incorrect citation [9], please provide the original references, I suppose the author needs to cite this paper in his/her case; Lötjönen J., Wolz R., Koikkalainen J., Thurfjell L., Waldemar G., Soininen H., Rueckert D. Fast and robust multi-atlas segmentation of brain magnetic resonance images. NeuroImage. 2010;49(3):2352–2365Reply: We added this reference to the revised manuscript (L158). 4) Line 247, in table 1, not all the characteristics are available for all patients, how did the author deal with missing information ? How confident is he/she about their effect on the results.Reply: Indeed, information on APOEe4 status and several cognitive tests is missing in a small proportion of the patients. Due to the design of DSI, there is no need to impute data or exclude cases with incomplete data, as only available data are used. Furthermore, we repeated our analysis with whole cases only, and found no relevant change. We added a remark on this to the paragraph on the DSI in the Methods section and included a reference to Tolonen Front Aging Neurosciene 2018 (ref 10), were this is explained in detail (L187-188).5) Line 265, it is unclear to me how the author has simulated the CSF values.Reply: We simulated the CSF values by taking age- and sex-normalized median AB42, tau and p-tau values of the AD group  and by taking age- and sex-normalized median AB42, tau and p-tau values of the SCD group . This age- and sex normalization is done in the same way as for the MRI markers (see ref 31). We described this in the paragraph on ‘Simulated CSF to guide clinical decision making’ in the Methods section and added reference 31 to the text (L216). Hopefully this clarifies the used method.6) Line 276, table2 , I would appreciate if the author interprets and explains why scenario A had higher rate than scenario D.Reply: Thank you for the opportunity to elaborate on this interesting finding. Scenario A results in a higher proportion of patients diagnosed with sufficient probability as compared to the other scenario’s (including scenario D), while proportion of performed CSF is much lower. This shows exactly why tools like develop in our study are needed; CSF testing is clearly only useful when it increases the confidence in the diagnosis. One can imagine that borderline CSF values or in case of multiple pathologies, adding CSF values only confuses and leads to lower proportion of diagnosed patients. This can also be seen in f.e. group 2 and group 4 (as shown in Table 3) were CSF did not help. We added a sentence on this to the section above Table 2 (L276-278).7) According to the figure in the supplementary notes, the higher DSI/delta_DSI the closer PCC to 1. Hence, the higher PCC the better for the diagnosis, how does the author explain that for values larger than 0.8 the accuracy of diagnosis decreased (line 335).Reply: You are correct in assuming that the PCC correlates to the accuracy of the diagnosis. As can be seen in S1 Fig, the accuracy (as indicated by the black line) increases with increasing PCC, also for values larger than 0.8. Setting the cut-off for a ‘certain’ diagnosis higher, results in less patients diagnosed. This can be seen in Fig 2: the proportion of diagnosed patients decreases with increasing PCC, since fewer patients while have a very high PCC leading to a smaller proportion of patients. Please also note that in this line we refer to the number of patients diagnosed, and not to the accuracy. We hope this explains both Fig 2 and S1 Fig. ________________________________________6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #2: NoAttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 6 Dec 2019Selection of memory clinic patients for CSF biomarker assessment can be restricted to a quarter of cases by using computerized decision support, without compromising diagnostic accuracyPONE-D-19-25997R1Dear Dr. Rhodius-Meester,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The manuuscript has been revised thoughtfully.The application of this tool in CSF biomarkers in clinic is valuable. 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IDH1/IDH2) are important drivers of cancer, including central nervous system (CNS) tumors. Therefore, we hypothesized that the abnormal metabolic state of CNS cancer cells leads to abnormal levels of metabolites in the CSF, and different CNS cancer types are associated with specific changes in the levels of CSF metabolites. To test this hypothesis, we used mass spectrometry to analyze 129 distinct metabolites in CSF samples from patients without a history of cancer (n = 8) and with a variety of CNS tumor types (n = 23) . Unsupervised hierarchical clustering analysis shows tumor-specific metabolic signatures that facilitate differentiation of tumor type from CSF analysis. We identified differences in the abundance of 43 metabolites between CSF from control patients and the CSF of patients with primary or metastatic CNS tumors. Pathway analysis revealed alterations in various metabolic pathways  between IDH-mutant and IDH-wildtype gliomas. Moreover, patients with IDH-mutant gliomas demonstrated higher levels of D-2-hydroxyglutarate in the CSF, in comparison to patients with other tumor types, or controls. This study demonstrates that analysis of CSF metabolites can be a clinically useful tool for diagnosing and monitoring patients with primary or metastatic CNS tumors.Cancer cells have altered cellular metabolism. Mutations in genes associated with key metabolic pathways  contains supplementary material, which is available to authorized users. Alterations in cellular metabolism are a critical part of cancer cell biology . StudiesIDH1/IDH2), are important cancer drivers  [IDH1/IDH2 mutations are associated with the production of an oncogenic metabolite, D-2-hydroxyglutarate (D-2-HG), which appears to be a critical aspect of tumor development [IDH1/IDH2 mutant cells and culture media [IDH1/IDH2 as a critical part of the diagnosis of infiltrating gliomas [PTEN, PI3K) have known effects in metabolic pathways. For example, activation of the PI3K/AKT/mTOR pathway leads to increased translation of the hypoxia inducible factor 1α (HIF1α), increased glucose uptake, and increased uptake of essential amino acids [Mutations in genes involved in important metabolic pathways, such as isocitrate dehydrogenase 1 and 2 (ukemias) . IDH1/IDelopment , 21. Incre media . The sur gliomas . Also, gno acids . Similarno acids .The tools currently utilized for the diagnosis and monitoring of patients with CNS tumors include CNS imaging, evaluation of tumor cells in the cerebrospinal fluid (CSF-cytology) and brain biopsies. However, CNS imaging studies lack specificity, CSF-cytology has extremely poor sensitivity, and brain biopsies are an invasive procedure. Therefore, there is a critical need for more specific and less invasive methods for diagnosing and monitoring patients with CNS tumors. In particular, minimally invasive methods that inform aspects of CNS tumor biology that influence treatment decisions. Although it is recognized that metabolic alterations are common in cancer cells, it remains unclear to what extent the analysis of cellular metabolites in biofluids can be utilized in the clinical management of cancer patients. Several studies have demonstrated differences in circulating metabolites in the blood of patients with a variety of cancer types , 20. HowConsidering the preliminary evidence showing alterations in metabolites in CNS tumors , 12 we dn = 7), IDH-mutant , metastatic lung cancer (n = 7) or metastatic breast cancer (n = 5) to the CNS were included (Table n = 8). All gliomas were sequenced for mutations in IDH1 or IDH2. Contrast-enhanced brain MR with optimum 2D/3D images matching the CSF collection date were available for 23 of the 31 patients. MRI scans were interpreted as positive or negative for tumor by a neuroradiologist. Patient characteristics are included in Table The study was approved by the institutional review board (IRB). All patients provided informed consent for participation of their samples in research. CSF was collected via lumbar puncture (LP) or intraventricular catheter . Samples from patients with glioblastoma IDH-WT . We have identified 129 metabolites. AUC (area under the receiver operating characteristic curve) as well as its 95% confidence interval was evaluated by the “DeLong” method with “pROC” package in R (Version 3.4.2) computing environment. The data was log2-transformed and normalized with internal standards on a per-sample, per-method basis.Targeted Metabolic profiling by LC-MS Single Reaction Monitoring (SRM) was used to characterize metabolites. We measured the metabolites using three different chromatographic methods  from 31 patients; 13 males and 18 females with age ranging from 20 to 77 years old were included. CSF-cytology results were available for 22/31 patients. One out of three (1/3) available CSF-cytology results from patients with an IDH-mutant glioma was reported as “atypical cells present”, 2/5 CSF-cytology results from patients with metastatic breast cancer were reported as “suspicious” or “consistent” with metastatic carcinoma and 1/7 CSF-cytology results from patients with metastatic lung cancer were reported as “atypical cells present”. CSF-cytology results were available for 3/7 patients with IDH-WT gliomas, the 3 samples were reported as negative for tumor cells. CSF-cytology results for 4/8 control patients were available and the result was negative for tumor cells. In total, there were 14 instances in which CSF samples were reported as negative for tumor cells, but the MRI results demonstrated the presence of tumor involving the CNS (Table CSF (p < 0.25). By mapping the 43 altered metabolites into known metabolic pathways, we identified several pathways significantly affected, including glycine, arginine, choline, nitrogen metabolism and glycolysis  in the CSF of individuals with no cancer history. Differences in the abundance of 43 metabolites were found between CSF from control patients and CSF from patients with a history of a primary or metastatic CNS tumor.  and CSF collected from an intraventricular catheter . The was done by fitting a linear mixed effect model and conducting the likelihood ratio test. To account for the multiplicity, the g method . An adjuOM Table .Our data shows that it is possible to discriminate CSF from patients with CNS tumors from CSF obtained from patients with non-neoplastic conditions. There are 14 CSF samples that were reported as negative for tumor by CSF-cytology, even when tumor was detected on the MRI, highlighting the limited sensitivity of CSF-cytology for detecting CNS malignancies, and the need for more sensitive and specific methodology to evaluate patients with CNS tumors. Only 1/12 (~ 8%) patients with a metastatic CNS tumor had a positive CSF-cytology result. Our results demonstrate that analysis of CSF metabolites could help identify patients with primary or metastatic CNS tumors, even in the absence of detectable tumor cells in the CSF.Metabolites in nitrogen metabolism and aminoacyl-tRNA biosynthesis were elevated in the CSF of patients with CNS tumors. Also, glycine, serine threonine, alanine, aspartate and glutamate were present at significantly different levels in the CSF of patients with tumor when compared to controls. These data show that the metabolism of non-essential aminoacids is altered in the CSF of patients with brain tumors and elevations of these amino acids in the CSF could indicate a neoplastic process. Acetylcarnitine and shikimate were elevated in the CSF of patients with IDH-WT-gliomas in comparison to control CSF, an observation that is consistent with a previous report that analyzed CSF from 10 patients with gliomas .It is not surprising that several of the altered metabolites are involved in the TCA cycle. Malic acid and succinate were particularly elevated in IDH-mutant gliomas, consistent with dysregulation of the TCA cycle in these tumors. These data indicate that TCA metabolite alterations in the CSF of patients with IDH-mutant gliomas could be helpful in distinguishing IDH-mutant from IDH-WT tumors. Also, our data shows higher D-2-HG levels in the CSF of patients with IDH-mutant gliomas, consistent with prior reports , 10, 18.To our knowledge, this study is one of the most comprehensive analysis of CSF metabolites in patients with different types of primary or metastatic CNS tumors , 16, 23.Additional file 1:Supplementary methods. (PDF 167 kb)Additional file 2:Figure S1. Pathway analysis was performed using ingenuity pathway analysis (IPA) through overlap statistics. In this study, metabolite with raw p value less than 0.05 and absolute fold change larger than 1.5 was considered as significant metabolites. Enrichment in the pathway was evaluated by Fisher’s Exact test. The pathways with adjusted p value using Benjamini and Hochberg method less than 0.01 were reported and generated in barplot. (TIFF 57607 kb)Additional file 3:Figure S2. Heat map of unsupervised hierarchical clustering of samples. Unsupervised hierarchical clustering using the complete agglomeration method was used for metabolite and sample clustering. (TIFF 4103 kb)


A systematic review of the literature was conducted in order to identify clinical studies evaluating the effectiveness of oral midazolam for sedation in pediatric patients in the context of premedication before anesthesia or during diagnostic/treatment procedures. The percentage of responders (response rate) after single administration of oral midazolam was evaluated and compared versus placebo in a subset of placebo‐controlled studies. The range of oral midazolam doses providing effective sedation in the different pediatric age subsets was analyzed in order to assess optimum dosing strategies. A total of 25 pediatric clinical studies, utilizing a variety of measures of sedation effectiveness, were selected. These studies included a total of 1472 patients (aged 4 months‐18 years) treated with midazolam (0.25‐1.5 mg/kg) and 138 patients treated with placebo. The response rates [95% confidence interval] with oral midazolam ranged from 36.7%  to 97.8% , while with placebo response rates ranged from 4.0%  to 41.0% . When considering the 4 placebo‐controlled studies, the odds ratios [95% confidence interval] for the comparison of midazolam vs. placebo ranged from 13.4  to 25.9 . The analysis of subgroups by context of sedation showed response rates [95% confidence interval] with oral midazolam ranging from 36.7%  to 97.0%  for anesthetic premedication and from 56.1%  to 97.8%  for medical procedures. The efficacy of midazolam for pediatric minimal/moderate sedation from a dose of 0.25 mg/kg and above was demonstrated. The probability of occurrence of adverse events and over‐sedation increases with increasing doses.One of the most widely used options for minimal/moderate sedation in pediatric patients is oral midazolam, as it presents an alternative to less well‐accepted routes of administration ( A recent Cochrane review evaluated midazolam for sedation before procedures and discussed data about the effectiveness of midazolam in adults and pediatric patients (by any route) in comparison with other medications using different outcome measures.The major aims of the present review were to summarize and analyze the available literature data related to minimal/moderate sedation with oral midazolam in pediatric patients, both prior to anesthesia and during minor procedures, and to evaluate oral midazolam doses providing effective minimal or moderate sedation in different pediatric age subsets and contexts of sedation.22.1eg, MEDLINE search ("midazolam"[MeSH Terms] OR "midazolam"[All Fields]) AND “sedation”[All Fields] AND ("child"[MeSH Terms] OR "child"[All Fields] OR "children"[All Fields]) AND ("pediatrics"[MeSH Terms] OR "pediatrics"[All Fields] OR "pediatric"[All Fields])) and by manual searches from further evaluation of key review articles and bibliographies of articles. All studies published or in press between January 1988 and March 2016, and written in English, were considered.A systematic review of the literature was conducted in order to identify clinical studies evaluating the use of oral midazolam in pediatric patients in the context either of premedication before anesthesia for surgical procedures or during diagnostic or treatment procedures. Articles were identified from electronic resources including PubMed and ScienceDirect  was reported, were considered. All studies evaluating moderate sedation using discrete sedation scales were selected.The outcome used for comparison purposes was the proportion of patients considered adequately sedated for the specific intervention (response rate), as detailed in each study. Studies that used placebo, other drugs, different oral midazolam doses, different oral midazolam preparations, or other routes of administration as comparators were acceptable. Information about comparators used in the different studies was extracted. Observations regarding vital signs and reported adverse events were summarized.2.3As shown in Figure A summary description of the individual clinical efficacy studies for both contexts of sedation, including efficacy results per treatment (midazolam and placebo) and dose, is shown in Table The response rates (95% CI) and odds ratios  were calculated for the individual studies and depicted graphically. Statistical analyses were carried out using SAS v 9.4 under PC windows.3The relevant literature on the use of midazolam in minimal/moderate sedation for children was reviewed. Variability in the assessment of sedation was observed between studies. However, there was a clear superiority of midazolam when compared to placebo in the range of doses evaluated.3.1Sedation was measured using a variety of discrete sedation scales, with a sedation success criterion defined for each study. The scales used in the studies for the evaluation of sedation in the present review were mainly 3‐ to 5‐point scales, and two studies were identified that utilized the Observer's Assessment of Alertness/Sedation (OAA/S) scale. It was, however, not possible to find a group of studies with perfectly consistent and relevant success criteria. Also, it was noted that the sedation levels “drowsy” or “awake but calm” could have slightly different meanings depending on the studies and that there could be a level of overlap of terminology between studies. The difficulty in finding a homogeneous group of studies in terms of sedation outcomes, together with the low quality of evidence of most of the studies , precluded a meta‐analysis with a direct comparison of the effectiveness of midazolam at various doses, despite the amount of available data.Nevertheless, the objective of this review was to report the data collected on the experience with oral midazolam in producing effective minimal/moderate sedation, and, as such, the different assessment methods were described as part of the reporting of results. However, conclusions could only be drawn from the results of individual studies. The difficulty in comparing the results from the different studies highlights the need to use validated scales for the evaluation of sedation., effective sedation defined as a score of 17 or less in the 20‐point scale).The review of the selected articles indicated that three types of studies could be distinguished, according to the type of success criterion: (a) those using nonrestrictive criteria ; (b) those using more restrictive criteria ; and (c) those using a criterion based on a sedation threshold using a validated scale, the Observer's Assessment of Alertness/Sedation (OAA/S) scale , and the rating can be more easily repeated in a short period of time  to evaluate the evolution of sedation from midazolam administration up to the start of the procedure or anesthesia induction. Rating is more subjective with 3‐ to 5‐point scales, and the OAA/S scale is more reliable for measuring sedation in clinical studies;, time of evaluation of sedation) and population characteristics , could also contribute to this heterogeneity.The variability of the rating scales used clearly contributes to the heterogeneity in the response rates observed in the different studies. The observed heterogeneity could be further explained by the context of sedation, and the age of the patients and the dose, but other elements of the design or the methodologies used in the different studies  reported response rates in the context of anesthetic premedication (including 3 placebo‐controlled studies) and only 6 trials (N = 258) reported response rates in the context of medical procedures (including 1 placebo‐controlled study). The studies presented high variability, with response rates [95% confidence interval] in anesthetic premedication ranging from 36.7%  to 97.0%  and from 56.1% [43.1%‐68.4] to 97.8%  in medical procedures . There was no apparent difference in terms of response rates between both contexts of sedation.Only six of the studies fulfilled the selection criteria for sedation before procedures, and therefore, limited information could be gathered from studies performed in this indication. The effectiveness of midazolam in children undergoing procedures, such as dental treatment, has already been reported in a previous review.3.3Only 4 trials compared midazolam (N = 176) to placebo N = 138). The number of responders with midazolam and with placebo in the individual studies is shown in Figure 8. The nuThe “placebo‐effect” observed may be explained by the fact that calm children were considered as responders in most of the placebo‐controlled studies, regardless of their level of nervousness at baseline. Indeed, epidemiological data suggest that while 60% of children are anxious prior to undergoing surgery or a medical procedure, the rest may remain calm before an intervention.The OR [95% CI] ranged from 13.4  to 16.5 ) for studies in premedication before anesthesia.3.4The dose most frequently used in the trials, irrespective of the context of sedation, was 0.5 mg/kg. Among all trials, 5 reported response rates at 0.25‐0.3 mg/kg (N = 236), 22 at 0.5 mg/kg (N = 811), 3 at 0.75 mg/kg (N = 55), 5 at 1.0 mg/kg (N = 234), and 1 at 1.5 mg/kg (N = 136). Six of the trials presented dose comparisons. In most cases, statistically significantly higher response rates were shown at doses of 1.0 or 1.5 mg/kg as compared to doses of 0.25 or 0.5 mg/kg. However, no statistically significant differences were shown between 0.25 and 0.5 mg/kg doses or between 0.75 and 1.0 mg/kg doses.Some reports conclude that small doses of midazolam (0.25‐0.5 mg/kg) are highly effective and that little advantage is gained by increasing the dose (0.75‐1.5 mg/kg). Among them is a very well‐designed study that enrolled 397 patients and used a 5‐point scale to demonstrate satisfactory sedation at 3 midazolam doses , where response rates of 93.2% (123/132), 97.7% (129/132), and 100% (133/133), respectively, were observed.In noncooperative children, midazolam was shown to be efficacious for dental procedures, both when used as premedication before general anesthesia (0.5‐1.0 mg/kg)3.5The age of the patients included in the different studies is shown in Table When assessed individually, the single study (N = 397) evaluating the efficacy of midazolam in different age groups indicated that, although more children in the youngest group were agitated at baseline, the response rates with midazolam according to sedation criteria were not significantly different across ages, with overall response rates of 97, 96, and 98% for children 6 months to <2 years, 2 to <6 years, and 6 to <16 years, respectively.3.6Descriptive and/or quantitative safety information from the selected studies has been summarized in Table It was noted that some of the adverse events reported are observed when oral midazolam is used in combination with other drugs  and can therefore not be attributed to midazolam treatment exclusively.3.7Some of the studies were performed using commercial oral syrups of midazolam , but the majority used liquid extemporaneous preparations made from existing parenteral formulations, generally mixed with syrups or flavorings to improve palatability . Unfortunately, these extemporaneous preparations suffer from a lack of standardization, particularly with regard to pH, concentration, and ingredients. In one of the studies,3.8Certain studies used other drugs  as comparators , and others compared oral midazolam with alternative routes of administration , as summarized in Table Among the studies reviewed, some compared oral midazolam 0.5 mg/kg to oral ketamine 5‐6 mg/kg and demonstrated that both presented similar efficacy.One of the studies indicated that midazolam 0.5 mg/kg and intranasal administration of dexmedetomidine 2 µg/kg were comparable in terms of response rates, although intranasal dexmedetomidine could be more useful when the intervention requires sleep induction.When compared to other alternative noninvasive routes, midazolam (0.5 mg/kg) administered orally produced equivalent response rates as rectal (0.5 mg/kg), intranasal (0.3 mg/kg), and sublingual (0.3 mg/kg) midazolam. Intranasal presented the most rapid onset of action but was less well‐tolerated.Most of these alternative medications are used off‐label in children.4Oral midazolam is an efficacious medication with an adequate safety profile for use in minimal or moderate sedation in children from 4 months to 18 years old at doses from 0.25 to 1.5 mg/kg. The incidence of adverse events, such as paradoxical reactions and respiratory events, and the risk of over‐sedation increase with increasing doses. Oral midazolam compares well with other alternative noninvasive medications. The need to use validated scales for the evaluation of sedation in future studies is highlighted.MA Manso, C. Guittet and LA Granier are employees of Advicenne and hold stock options or shares in the company.


Pre-clinical models and clinical trials demonstrate that targeting the action of the cytokine, granulocyte macrophage-colony stimulating factor (GM-CSF), can be efficacious in inflammation/autoimmunity reinforcing the importance of understanding how GM-CSF functions; a significant GM-CSF-responding cell in this context is likely to be the monocyte. This article summarizes critically the literature on the downstream cellular pathways regulating GM-CSF interaction with monocytes (and macrophages), highlighting some contentious issues, and conclusions surrounding this biology. It also suggests future directions which could be undertaken so as to more fully understand this aspect of GM-CSF biology. Given the focus of this collection of articles on monocytes, the following discussion in general will be limited to this population or to its more mature progeny, the macrophage, even though GM-CSF biology is broader than this. Howeveror cells . While nor cells , 7. Thisor cells . Recentlor cells . There ior cells .Seeing that this Review resides within a collection of articles on monocytes its content will generally be focussed on this population and its tissue counterpart, the macrophage, even though GM-CSF biology is broader involving other responding cell types such as neutrophils and eosinophils.Based mainly in data using GM-CSF gene deficient mice or neutralizing monoclonal antibody (mAb) in models of autoimmunity and chronic inflammation, it is apparent that GM-CSF can be a key driver of tissue inflammation and its associated pain. Examples include arthritis, EAE, cardiovascular disease, and lung disease. The data summarizing these findings have been reviewed recently –14 althoGiven the potentially wide range of GM-CSF biology involving multiple cellular sources and responding myeloid cell types , human cin vivo (The GM-CSF receptor (GM-CSFR) is a type I cytokine receptor comprising in a multimeric complex a binding (α) subunit and a signaling (β) subunit, the latter shared with interleukin 3 (IL-3) and interleukin-5 (IL-5) receptors. These pathways have been linked to key residues in the intracellular regions of GM-CSFR using mainly receptor mutants expressed in cell lines –18. Key in vivo . The GM-in vivo  and PPARin vivo . The debin vivo –26, is din vivo has been described that disables their inflammatory functions and promotes their conversion into suppressor cells  appear to be explained by dose-dependent and sequential activation by GM-CSF of specific signaling pathways downstream of the activated receptor , 27. Foror cells : this twor cells . Monocytathology —cells geBoth hemopoietic [e.g., T and B lymphocytes , 32–35 aTo help understand the chronicity of certain inflammatory/autoimmune responses, a “CSF network” hypothesis was originally proposed in which there is an interdependent co-regulation of proinflammatory cytokines, such as IL-1 and TNF, with GM-CSF as part of a positive feedback “loop” involving communication between monocytes/macrophages and neighboring cell populations, such as fibroblasts, endothelial cells etc. –5, 46; tin vivo, significant cytokine secretion usually requires another stimulus, such as lipopolysaccharide  also need further analysis. Mouse CD103+ DCs  from different lymphoid and non-lymphoid tissues have distinct functional activities and there has been disagreement about the contribution of GM-CSF to their development in vivo (in vivo are guided by their tissue microenvironment (It is debated as to whether GM-CSF can give rise to monocyte-derived DCs (MoDCs) o or not , 59–61 eectively , 62–64.  subsets —their re in vivo , 66, 67  in vivo . Obviousironment .in vivo relationships of the in vivo generated populations from mouse bone marrow and human monocyte cultures are not fully defined.Mouse populations generated by GM-CSF from bone marrow precursors are heterogeneous with cells having both DC and macrophage features being observed—such features include surface markers, morphology, motility, antigen presentation, T cell activation, cytokine production, and gene expression profiles , 70–73; + Ly6C+ monocytes require GM-CSF to lead to a pathogenic signature for EAE progression characterized by the induction of genes linked to inflammasome function, phagocytosis and chemotaxis, i.e., they become pathogenic DCs  differ substantially  and disp stimuli , 90, strhi Trem4neg mouse monocytes can differentiate into Zbtb46+ MoDCs in response to GM-CSF and IL-4 in an IRF4 dependent manner . Interestingly, CCL17 may not necessarily be functioning as a chemokine in its regulation of inflammatory pain and arthritic pain/disease  whether GM-CSF controls MoDC development in vivo, (iii) the nature of GM-CSF-induced cell polarization, (iv) whether IRF4- or IRF5- dependent pathways are more important for GM-CSF-dependent biology, (v) when and how endogenous GM-CSF can act systemically in addition to locally in tissues, and (vi) how relevant are the effects of systemically administered GM-CSF to the actions of endogenous GM-CSF.It would appear from the above that GM-CSF-dependent inflammatory pathways in monocytes (and macrophages) are likely to be critical for the purported role of GM-CSF in inflammation, autoimmunity and host defense. In addition to attempting to summarize the relevant literature on this topic I have tried to highlight some of the contentious issues which are currently being debated. Such issues, which I have endeavored to represent diagrammatically , are:  additional clinical trials targeting GM-CSF action and that of other putative downstream mediators, such as CCL17, (ii) human monocyte/macrophage studies, (iii) cellular metabolic responses to GM-CSF, and (iv) the significance of GM-CSF → IRF4 signaling.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Furthermore, alcoholics were less likely to complain of headache (p < 0.001) and nausea/vomiting (p = 0.005) and had lower concentration of glucose in cerebrospinal fluid (CSF) (p = 0.025). In the multiple logistic regression analysis, alcoholism was associated with lower GCS (p = 0.036), presence of seizures (p = 0.041), male gender (p = 0.042), and absence of nausea/vomiting (p = 0.040). Furthermore, alcoholism (p = 0.031), lower GCS score (p = 0.001), and higher blood urea concentration (p = 0.018) were independently associated with worse outcome measured by GOS. Compared with non-alcoholics, chronic alcohol abusers are more likely to present with seizures, altered mental status, and higher SOFA score and have an increased risk of unfavorable outcome. In multivariate analysis, seizures and low GCS were independently associated with alcoholism, while alcoholism was independently associated with worse outcome.The aim of the study was to determine the effect of chronic alcohol abuse on the course and outcome of bacterial meningitis (BM). We analyzed records of patients with BM who were hospitalized between January 2010 and December 2017 in the largest neuroinfection center in Poland. Out of 340 analyzed patients, 45 (13.2%) were alcoholics. Compared with non-alcoholics, alcoholics were more likely to present with seizures (The online version of this article (10.1007/s10096-019-03661-5) contains supplementary material, which is available to authorized users. In Poland, where the registration of bacterial meningitis (BM) cases is mandatory, the annual incidence of BM in the years 2010–2017 ranged between 1.97/100,000 and 2.5/100,000  which isWe evaluated records of adult (≥ 18 years old) patients with community-acquired BM who were admitted to the Hospital for Infectious Diseases in Warsaw from January 1, 2010, to December 31, 2017. The diagnosis of BM was based on fulfilling at least one of the following criteria: positive cerebrospinal fluid (CSF) culture, positive CSF Gram staining, and typical CSF findings (pleocytosis ≥ 100 cells/μL with ≥ 90% neutrophils and decrease of CSF glucose level < 2.2 mmol/L). Patients with CSF findings typical for BM but negative blood and CSF culture and negative microscopic CSF examination were considered to have BM of unknown etiology. Diagnosis of tuberculous meningitis was based on at least one of the following: positive culture, positive nucleic acid amplification, and positive Ehrlich-Ziehl-Neelsen staining of CSF. Glasgow Coma Scale (GCS) and Sequential Organ Failure Assessment (SOFA) scores were calculated at admission, while Glasgow Outcome Score (GOS) was assessed at the time of discharge from hospital.Initial antimicrobial treatment followed current guidelines . PatientAlcoholism was diagnosed according to the World Health Organization (WHO) criteria . QuestioU test was used to compare continuous variables, and the chi-square test was used to evaluate nominal variables. Logistic regression was used to calculate adjusted odds ratios and to determine variables independently associated with alcoholism and those independently influencing outcome reflected by GOS. Since GOS is not a nominal but continuous variable, we used the general linear model to calculate the coefficients for this analysis. Statistical analyses were performed using program R version 3.5.2 [The Mann-Whitney on 3.5.2 .The final analysis included 340 patients with bacterial meningitis . Among this group, 45 (13.2%) patients were considered alcoholics .p < 0.001) (Table p < 0.001) and nausea/vomiting . Furthermore, they scored higher on the SOFA  and lower on the GCS . Alcoholic patients were also more likely to require intensive care unit (ICU) admission (48.9% vs 37.6%), and their mortality was higher (24.4% vs 15.4%), but these differences did not reach statistical significance. The clinical outcome reflected by the GOS was significantly worse among alcoholics  and lower concentration of glucose in CSF , male gender , the presence of seizures , and the absence of nausea/vomiting . Furthermore, alcoholism, lower GCS score, and higher urea blood concentration were independently associated with worse prognosis as assessed by GOS . While high incidence of seizures among alcohol abusers presenting with BM is a well-known phenomenon, the numbers in previous reports were lower and ranged from 13 to 18% [Compared with non-alcoholics, alcohol abusers were less likely to present with fever, headache, and nausea. The absence of such typical symptoms of BM  might ca3 to 18% , 12. FurThe presence of seizures and other signs and symptoms associated with the alcohol withdrawal syndrome or even alcohol intoxication itself could also negatively affect consciousness level: in our study, alcoholic patients scored significantly lower on the GCS scale at admission compared with non-alcoholics. These findings are compatible with previous studies of van Veen et al.  and WeisWhile alcohol abuse was not an independent predictor for mortality in multivariate analysis, it was associated with worse outcome as measured by the GOS. These results are similar to those reported by Wiesfelt et al.  who alsod-dimer levels and CSF glucose levels, no differences were found in the results of routine tests between alcoholic and non-alcoholic patients. High blood d-dimer levels are common in chronic alcohol abusers and could be the effect of hemostatic system activation related to oxidative stress [In addition to alcohol, low GCS score and high blood urea levels were independently associated with worse outcome Table . The infe stress . Interese stress .Streptococcus pneumoniae was the most common causative microorganism (19.4%) in our patients


In the original article, there was an error. “G719A” was erroneously written as “G791A.”Case Report section, paragraph four:A correction has been made the “After discharge, the patient continued to take afatinib (30 mg po qd). Headache and dizziness improved substantially, suggesting effective treatment. Follow-up on 25th July 2018 (day 98) demonstrated reduced pulmonary lesions  and improved supratentorial ventricular dilation. On 27th July 2018 (day 100), lumbar puncture showed a CSF pressure of 15 mmHg and CSF CEA levels of 2,111 ng/mL. Intrathecal chemotherapy was performed using methotrexate (8 mg) and dexamethasone (1 mL) in saline (9 mL). CSF and peripheral blood samples were collected for repeat genetic testing; the pressure of the ventriculoperitoneal shunt device was adjusted to 1.0, with afatinib dose increased to 40 mg qd. The main adverse effects during afatinib therapy were face and trunk rashes, requiring no treatment. On 10th August 2018 (day 114), NGS results for the CSF revealed an EGFR G719A mutation with a frequency of 22.2% (around 60% less than pre-afatinib therapy) and a very low frequency (0.6%) of NRAS G12V mutation, which is associated with drug resistance (Table 1). EGFR G719A mutation was undetectable in peripheral blood, although EGFR G873R mutation was identified at low frequency (Table 1). On 20th November 2018 (day 216), peripheral blood CEA level was 93 ng/mL; CSF CEA level was 1,590 ng/mL , with a CSF pressure of 6 mmHg, indicating maintenance of the response to afatinib therapy. NGS results for the CSF revealed an EGFR G719A mutation frequency of 23.1%, comparable to that measured on 10th August 2018, while liquid biopsy of peripheral blood indicated increased frequency of EGFR G873R mutation to 2.2% (Table 1). The patient reported intermittent headache likely from meningeal metastasis. However, the patient had good clinical condition . At last follow-up , the patient remained in good condition. Although the patient reported mild rash, diarrhea, and oral mucositis, no serious adverse reactions to afatinib had occurred. Thus, the patient had progression-free survival (PFS) > 7 months.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.