Successful implantation requires a receptive endometrium and a good quality egg. The challenges a physician encounters with regard to this in assisted reproductive technology are obtaining good quality embryo, achieving optimal endometrial thickness (EMT), and subsequently implantation, which is denotive of a receptive endometrium. Granulocyte colony-stimulating factor (G-CSF) has been observed to be a biomarker of oocyte quality and has been shown to enhance EMT and implantation because of its immunological effects. A systematic search for all relevant articles on G-CSF in follicular fluid and its therapeutic benefit in thin endometrium and recurrent implantation failure was performed, and peer-reviewed, full-text articles related to humans were included in the study. As a tool to determine the potentiality of oocyte, G-CSF shows promise with its predictability increasing in combination with morphological embryo scoring or interleukin 15. For the thin endometrium, G-CSF is especially useful in patients who are refractory to other treatment modalities. In recurrent implantation failure (RIF), G-CSF showed potential in a subset of patients with immunological deficiency lacking killer cell immunoglobulin-like receptor genes. This review highlights the various forms of usage of G-CSF and the effectiveness of G-CSF in infertility. G-CSF equips embryologists with a tool to determine the potentiality of oocyte and physicians with therapy for thin endometrium and RIF, especially since the available treatment options are ineffective. During the first cycle of ICSI, although good follicular growth was observed, endometrial thickness (EMT) was 4 mm despite the use of estradiol and vasodilators. At the patient’s insistence, embryo transfer (ET) was performed, but it did not yield a positive result. For the second cycle, she opted for frozen embryo transfer (FET) to improve her chances of having a thicker endometrium. This time, even with proper estrogen priming of the endometrium, the thickness remained below 7 mm. The patient pressed for another option to improve her endometrium as she could not afford another ICSI cycle. Is granulocyte colony-stimulating factor (G-CSF) the answer?Globally, 8% to 12% of reproductive-age couples suffer from infertility [Successful implantation depends on the embryo quality, receptive endometrium, and excellent ET technique . EndometWhile thin EMT seems to be a determinant of endometrial receptivity, successful implantation is the test. The immunological etiology of many causes of recurrent implantation failure (RIF) explains why G-CSF could be useful in patients with RIF. While many researchers have studied the effect of G-CSF on thin endometrium, its benefit in patients with recurrent pregnancy loss and RIF is insufficiently proven . Wurfel Moreover, G-CSF is also regarded as a biomarker of oocyte quality. Immunology plays a crucial role in implantation failure; hence, increased G-CSF in the follicular fluid is associated with an increased implantation rate -18. GiveDespite all the advances in IVF, most clinicians are still unable to resolve problems on refractory endometrium and RIF. Various existing modalities proposed over the years have shown inconsistent results. G-CSF has shown promising results, but considering it as an absolute value in treating such problems remains uncertain. This article reviews all the published studies on the effectiveness of G-CSF for treating thin endometrium and RIF and as a diagnostic tool of oocyte quality. We examined whether G-CSF is associated with any substantial improvement in clinical pregnancy in patients with thin endometrium and RIF and if increased G-CSF values are associated with embryos with improved quality.MethodsSearch Method and StrategyA computerized, systematic search was performed on April 17, 2019, using combinations of the following search keywords: granulocyte colony-stimulating factor, female infertility, thin endometrium, recurrent implantation failure, granulocyte colony-stimulating factor and female infertility, granulocyte colony-stimulating factor and implantation failure, granulocyte colony-stimulating factor and follicular fluid, granulocyte colony-stimulating factor, and thin endometrium. The MeSH keywords used were female infertility, granulocyte colony-stimulating factor, and granulocyte colony-stimulating factor with female infertility  showed potential in a particular subset, those lacking KIR genes . We haveDiscussionImplantation is a vital step during reproduction; the steps involved are apposition, attachment, and then the invasion of trophoblasts . A cascaUterine receptivity can be subdivided into three phases: prereceptive phase, receptive phase, and the nonreceptive phase. The receptive phase is in the midluteal phase, approximately 7-10 days after ovulation. If a competent embryo engages with the endometrium during this transient receptive phase, implantation ensues; if not, implantation fails because synchronization was not attained . TrophobG-CSF is a glycoprotein synthesized by mononuclear cells , fibroblasts, endometrial cells, and natural killer (NK) cells . LocallyIn the reproductive tract Figure , G-CSF i G-CSF Quantification in Follicular FluidOne of the techniques to improve the probability of obtaining a live birth is culture prolongation through the blastocyst stage. However, with this method, we run the risk of ending up with no embryos to transfer on day 5 . AnotherIn the study of Ledee et al., 132 individual follicles exhibited significant differences in implantation rates between embryos with low (<20 pg/ml) and high (>24 pg/ml) G-CSF levels  . In combMost studies demonstrated that the effectiveness of G-CSF as a marker of successful implantation is possible, especially because it has a reasonable sensitivity rate. Compared with emerging techniques of genomic analysis of cumulus cells, G-CSF is simplistic and economical ,35. HoweG-CSF for Thin EndometriumEMT is often regarded as a marker of endometrial receptivity. Though the usage of endometrial volume and Doppler sonography of the uterine and subendometrial blood flow as a predictive marker has gained traction in the recent years, EMT determination is still the most extensively used method in clinical practice -7. In a The endometrium has two layers, namely, the basal layer, which consists of large spiral arteries but remains intact during the menstrual cycle, and the functional layer, which is supplied by a thin capillary network that grows during the menstrual cycle and is entirely shed during menstruation. Following ovulation, the blood flow to the functional layer is reduced due to vasoconstriction of the spiral arteries. The resulting reduced oxygen tension aids implantation . When thThe influence of G-CSF on a thin endometrium was studied originally by Gleicher et al. in a case series where an intrauterine infusion of G-CSF resulted in an increased EMT within 48-72 hours of infusion and a 100% pregnancy rate . While KPlease see Table However, other studies recorded no increase in EMT ,42. In aG-CSF being a relatively new therapeutic method needs more RCTs to determine the ideal dosage, frequency, and day of administration. Additionally, RCTs are required to verify if G-CSF is indeed useful as a stand-alone tool or only when combined with other modalities, such as vasodilators, to improve the chances of implantation. The smaller number of currently available studies and the small sample size, along with the different study designs, made the findings difficult to interpret. Nonetheless, G-CSF shows promising results in its ability to significantly increase the EMT within 48-72 h and presents a trend toward higher implantation and pregnancy rates.G-CSF in Recurrent Implantation FailureUterine NK (uNK) cell modulation is the cause of recurrent miscarriage, given that it is regarded as a biosensor of foreign antigens. Extravillous cytotrophoblast cells manifest nonclassical human leukocyte antigens, which interact with killer cell immunoglobulin-like receptors (KIRs) located in uNK cells. Consequently, uNK cell cytotoxicity is hindered, and angiogenic cytokine production increases .Wurfel et al. conducted a study, but the inclusion was not only based on the history of repeated implantation failures; only patients lacking 3 KIR receptors were included. The incidence of this finding was extremely high, amounting to 78%. They showed a pregnancy rate of 42% for D2 transfer and 73.8% for D5. However, abortion rates were also high. They conducted a further pilot study wherein G-CSF was administered in patients with repeated IVF failures but did not lack the KIR gene, and the pregnancy rate was only 10% . HoweverG-CSF has many uses in diverse forms in relation to reproductive health. It may be utilized as a biomarker to determine oocyte quality and may enhance the EMT in thin endometrium and RIF. Consequently, pregnancy rates and live birth rates improve. From an immunological perspective, the role it plays in the abovementioned indications can be explained. However, particular areas of ambiguity exist; examples are the dosages and modes of administration and continuity of administration or even the idea if the effects of G-CSF were simply additive or possibly synergistic combined with other modules.LimitationsWhen interpreting the conclusions, certain limitations should be considered. The included studies range from experimental studies to observational studies, including RCTs, nonrandomized experimental studies, and cohort studies. The heterogeneity of the studies made an accurate comparison of results impossible. Limitations also included the small sample size, along with different study designs, in many of the studies because G-CSF application is a relatively new concept. In addition, various researchers who tried out different dosages, modes of administration, and days of administration of G-CSF contributed to a weak comparison. The high cost of using G-CSF might have been responsible for the small sample size in the studies. For the review, we relied heavily on old literature for an explanation of the immunological processes, possibly indicating a gap in research that needs to be filled. Lastly, particular pertinent literature could not be accessed as a student because of an existing paywall.Ascertaining the G-CSF in the follicular fluid in each oocyte has its advantages such as noninvasiveness and sensitivity. The opportunity to choose only the most potential embryos averts the need for multiple IVF and the accompanying financial and psychological burdens. In combination with morphological embryo scoring at day 2, its predictability improves, thereby realizing the full potential of this tool. However, some disparity was observed in reaching conclusions regarding its usage in patients with PCOS. The expansion of the endometrium at approximately 48 hours in some patients is what makes G-CSF an excellent therapeutic tool. Insight into whether the same effects would be seen if G-CSF was used alone or its best usage would be achieved when it is combined with aspirin, sildenafil, or endometrial scratch. Considering that endometrial volume and Doppler sonography of uterine and subendometrial blood flow are better determinants of endometrial function or even endometrial receptivity array, more studies are needed to assess these endometrial markers with the use of G-CSF. G-CSF in RIF shows its effectiveness only in a subset of patients with immunological deficiency lacking KIR genes. This review highlights the various forms of the usage and effectiveness of G-CSF in infertility while emphasizing on the multiple gaps in the literature and further recommendations for research.


Background: Blood biomarkers may aid in recruitment to clinical trials of Alzheimer’s disease (AD) modifying therapeutics by triaging potential trials participants for amyloid positron emission tomography (PET) or cerebrospinal fluid (CSF) Aβ and tau tests.Objective: To discover a plasma proteomic signature associated with CSF and PET measures of AD pathology.Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics were performed in plasma from participants with subjective cognitive decline (SCD), mild cognitive impairment (MCI), and AD, recruited to the Amsterdam Dementia Cohort, stratified by CSF Tau/Aβ42 (n = 50). Technical replication and independent validation were performed by immunoassay in plasma from SCD, MCI, and AD participants recruited to the Amsterdam Dementia Cohort with CSF measures (n = 100), MCI participants enrolled in the GE067-005 study with [18F]-Flutemetamol PET amyloid measures (n = 173), and AD, MCI and cognitively healthy participants from the EMIF 500 study with CSF Aβ42 measurements (n = 494).Results: 25 discovery proteins were nominally associated with CSF Tau/Aβ42 (P < 0.05) with associations of ficolin-2 (FCN2), apolipoprotein C-IV and fibrinogen β chain confirmed by immunoassay (P < 0.05). In the GE067-005 cohort, FCN2 was nominally associated with PET amyloid (P < 0.05) replicating the association with CSF Tau/Aβ42. There were nominally significant associations of complement component 3 with PET amyloid, and apolipoprotein(a), apolipoprotein A-I, ceruloplasmin, and PPY with MCI conversion to AD . In the EMIF 500 cohort FCN2 was trending toward a significant relationship with CSF Aβ42 (P ≈ 0.05), while both A1AT and clusterin were nominally significantly associated with CSF Aβ42 (both P < 0.05).Conclusion: Associations of plasma proteins with multiple measures of AD pathology and progression are demonstrated. To our knowledge this is the first study to report an association of FCN2 with AD pathology. Further testing of the proteins in larger independent cohorts will be important. The accumulation of amyloid-beta (Aβ) plaques followed by the deposition of hyper-phosphorylated tau protein in neurofibrillary tangles, central to Alzheimer’s disease (AD) neuropathology, is thought to develop around 20 to 30 years in advance of clinical symptom onset . Given tTo date the best characterized and most frequently used biomarkers relating to amyloid and tau pathology are PET imaging measures of brain amyloid deposition and cerebrospinal fluid (CSF) measures of Aβ, total tau (tTau) and phospho-tau (pTau) . HoweverMany studies have investigated blood-based proteomic biomarkers to distinguish Alzheimer’s disease cases from cognitively healthy elderly controls as reviewed . HoweverAPOE genotype, could predict MCI conversion to AD with 87% accuracy  visiting the Alzheimer Center Amsterdam as previously described . In brie42 and tau measures and, using an extreme phenotype approach, were stratified as very low CSF pathology  to very high CSF pathology  (Table x = (373 + 0.82[tau])/[Aβ42] (Plasma samples were selected on the basis of CSF Aβ5) Table . Calcula)/[Aβ42] .APOE) genotype data  and subjects who remained MCI over this time period . Participants were assessed clinically every 6 months until conversion, dropout or completion of the 3-year follow-up. [18F]Flutemetamol PET amyloid imaging data were available for all subjects, who were categorized as having either an “abnormal” PET amyloid scan  or a “normal” PET amyloid scan . Visual image interpretation of the PET scan was performed by five independent trained readers who were blinded to the participants’ clinical history and diagnosis. Scan interpretation was based on the majority classification from these five independent readers. The details of image interpretation are published elsewhere , prevalence of diabetes and years of education. Whole blood was collected in EDTA tubes and processed for plasma , Hospital Sant Pau, Barcelona (n = 154) and the Alzheimer Unit of Fondazione Ca Granda, IRCCS Ospedale Maggiore Policlinico, Milan (n = 88). Diagnoses were made according to standard criteria (APOE genotype data). CSF Aβ42, tTau and pTau cut-off values for assigning high and low categories were provided by each individual cohort; Perugia, Aβ42 = 800 pg/mL, tTau = 300 pg/mL, pTau = 60 pg/mL  is a precompetitive, public-private Innovative Medicines Initiative funded platform facilitating access to cohort studies and real world observational data from across Europe. We used EMIF to identify three cohorts with samples suitable for validation studies. Plasma samples from AD, MCI and cognitively healthy participants (CTL), for whom a measure of CSF Aβcriteria  and gene60 pg/mL ; Barcelo61 pg/mL ; and Mil61 pg/mL . CSF Aβ43 (version 2.3) to determine peptide identifications. Processing of the Mascot output data files was performed in R, and included median ratio normalization, calculation of peptide ratios and subsequent protein scores using median and mean roll-up methods of peptide ratios as described  coupled with tandem mass tagging (TMT). The data acquisition and preprocessing pipelines are described in detail elsewhere . In brieescribed . Where tTechnical replication was performed on proteins in the same 50 samples that underwent LC-MS/MS based proteomics with an additional 50 samples selected from the Amsterdam Dementia Cohort Table . The cri10 transformed prior to statistical analysis and age, sex, APOE 𝜀4 allele presence, plate batch and sample storage duration were included as covariates in regression models.Proteins were measured by commercially available single-analyte enzyme-linked immunosorbent assays (ELISAs) according to the manufacturer’s instructions (Supplementary Table 11C] Pittsburgh compound B (PiB) PET amyloid . For both discovery and replication phase studies, proteomic data were analyzed using the Mann Whitney A generalized linear regression model (GLM) was used to adjust the data for covariates. Machine learning  was performed in R on the GLM adjusted data. The minimal protein set with optimal AUC characteristics for prediction of amyloid status were assessed by Support Vector Machines combined with LASSO, as a variable selection method, and performance was assessed using 100 repeats of 10-fold cross validation.42 pathology were identified through gene enrichment analysis on the results of the LC-MS/MS analysis. For this analysis only protein molecular weight isoforms detected in 80% or more of the TMT6plexs and for which gene IDs were available were included. The p-values derived from the univariate analysis of the protein molecular weight isoforms were used to estimate a single p-value per protein by applying Fisher’s method as described here. For each protein the sum of logarithms of the p-values of all the molecular weight isoforms were calculated. The chi-squared distribution was then used to derive the protein p-value from this sum of logarithms.Differential regulation of pathways associated with CSF Tau/Aβp-values were log transformed and then the proteins known to interact with these proteins were identified by STRING  with gene-lists corresponding to pathways , diseasIn order to identify a biomarker that might predict brain amyloid pathology we performed a proteomic study in three phases in three independent sample collections. First, we used mass-spectrometry based discovery in the Amsterdam Dementia Cohort including a technical replication phase using immunocapture. Then we used immunocapture to replicate these and previous findings in a cohort derived from a clinical trial of a radiotracer for detection of brain amyloid (GE067-005 study) and finally an independent validation phase study, again using immunocapture methods in an independent set of samples collated from three separate cohorts sourced using the EMIF. The study workflow is illustrated in Figure 42  and FGB was positively correlated with CSF pTau by spearman rank correlation .Apolipoprotein C-IV was nominally significantly increased in the high CSF Tau/Aβp-values were calculated for 77 proteins from 233 protein MW isoform p-values for inclusion in gene enrichment analysis. Using STRING this list was expanded to include proteins for which there is direct experimental evidence of an interaction, giving a total of 769 proteins. When comparing this protein set to the Reactome database, three pathways were significant after FDR correction for multiple comparisons; HDL-mediated lipid transport (q = 0.010), lipoprotein metabolism (q = 0.035), and lipid digestion, mobilization, and transport (q = 0.035) (Table q = 0.034), brain diseases (q = 0.034), and metabolic bone disorder (q = 0.034) . From 37 proteins measured, three proteins were excluded from analysis due to technical failure of the assays . Intra-assay CV was <12% for all other assays, and batch variation was included as a covariate in regression analysis to control for any inter-assay differences.Using immunocapture, we then performed a validation phase study in an independent cohort using a different end point measure of AD pathology  formed the minimal protein panel for classifying amyloid positivity and achieved modest accuracy . In the AD group, only RANTES was associated with CSF Aβ42, as shown by both logistic regression . In the high CSF tTau pathology group, clusterin and complement C4B were nominally significantly increased  and nominally significant reduction of C4  in association in the high CSF pTau group . The minimal protein set with optimal AUC characteristics for prediction of CSF Aβ42 amyloid was assessed by SVM combined with LASSO and performance was assessed using 100 repeats of 10-fold cross validation. Five proteins  formed the minimal protein panel for classifying amyloid positivity and achieved modest accuracy  and using independent or orthogonal protein assay technologies (mass spectrometry and ELISA). Moreover, the association of FCN2 with CSF Aβomarkers  it mightTo our knowledge this is the first study to identify and validate FCN2 as a biomarker of AD pathology. Ficolins and mannose-binding lectins (MBL) are both activators of the lectin complement pathway  and CSF 18F]-flutemetamol PET amyloid from low [18F]-flutemetamol PET amyloid subjects, the accuracy of the 6-protein classifier was only modest (AUC = 0.69). Given that the proteins measured in this study included those that were previously identified as markers of other AD related measures -flutemetamol PET amyloid and four proteins; Apo(a), ApoA-I, Ceruloplasmin and PPY with MCI conversion to AD, building upon previous findings of their relationship with AD and amyloid pathology. These results would suggest a biologically relevant role for these proteins in AD. Further analysis of the potential of these proteins as a biomarker of AD pathology and progression, in combination with other proteins or multimodal measures, and in larger independent cohorts will be essential. Such a blood-based biomarker could be of value as a triaging tool for PET and CSF based tests and hence aid in recruitment to clinical trials of disease modifying treatments.In conclusion, in this study we have identified a number of proteins that are associated with CSF AβThe datasets for this manuscript will be made available by the authors to qualified researchers upon reasonable request. Requests to access the datasets should be directed to the corresponding author.This study was carried out in accordance with the recommendations of METC of VU University Medical Center for the Amsterdam Dementia Cohort, and the medical ethics committee at each site for the GE067-005 and EMIF 500 cohorts . The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Twenty patients were divided into two groups. Patients in intermittent infusion group received vancomycin as a loading dose of 25 mg/kg over two hours, followed by 25 mg/kg over two hours every 12 h. In the Continuous Infusion group, patients received vancomycin as a loading dose of 25 mg/kg over two hours, followed by 50 mg/kg/day by continuous infusion. In the intermittent infusion group, mean ± SD of serum trough, peak and CSF concentrations were 17.49 ± 2.46 mg/L, 41.33 ± 2.73 mg/L, and 4.83 ± 1.05 mg/L, respectively. Mean of CSF/trough% ratio was 27.39 ± 2.43%. A positive linear correlation was found between the serum trough levels and CSF levels . In continuous infusion group, mean ± SD of serum and CSF concentrations were 24.76 ± 2.02 mg/L and 6.20 ± 1.31 mg/L respectively. Mean ± SD of CSF/serum% ratio was 24.84% ± 3.54%. The serum and CSF levels revealed positive linear correlation . The mean of CSF concentration in CI group was 6.20 ± 1.31 mg/L which was significantly higher than II group . CSF/serum ratio did not show any significant difference between the two groups. Continuous infusion of vancomycin makes it possible to achieve faster and constant target level in serum but did not have any significant effect on the penetration (CSF/Serum ratio) of vancomycin in to the CNS.Poor penetration of vancomycin into Central Nervous System (CNS) can lead to treatment failure. The aim of this study was to evaluate and compare CSF concentration and serum pharmacokinetics of high dose vancomycin by continuous infusion  There are few studies regarding pharmacodynamics of continuous infusion of vancomycin in human models. Unfortunately, most of these studies have inconclusive findings in determining parameters which predicting patients’ outcomes. The most limitation of these studies is low sample size of patients with a variety of infection types. The present study was accomplished to evaluate and compare CSF concentration and serum pharmacokinetics of high dose vancomycin by continuous infusion vs. intermittent infusion in post neurosurgical meningitis patients.Among hospital associated infections, post-neurosurgical meningitis is quite important because of high morbidity and mortality rates. The prevalence rate of post neurosurgical meningitis has been reported from 0.3% to 1.5% in many countries. Hence empiric antimicrobial therapy must be administered promptly . In addiPatientse.g., headache, loss of consciousness, fever) plus either positive CSF culture or the presence of at least two criteria of CSF proteins more than 100 mg/dL, glucose concentration less than 50 mg/dL or simultaneously obtained serum glucose value less than 50%, CSF lactate more than 36 mg/dL and elevated White Blood Cells (WBC), usually more than 500 cells/μL  of Loghman Hakim Hospital, Tehran, Iran between January 2015 and January 2017. The Medical Ethics Committee of Shahid Beheshti University of Medical Sciences  approved the study. The protocol of this clinical trial had been documented in «Iranian Registry of Clinical Trials» (IRCT) with number: IRCT2016071114693N6. Written informed consent form was obtained from responsible caregivers. Twenty patients aged between 18 and 65 years with diagnosis of post-neurosurgical meningitis, were enrolled in the study. These patients were all of eligible cases in this two years period. Also, the sample size of this study was similar to other previous studies , 16, 17.cells/μL .Exclusion criteria were defined as: pregnancy, chronic kidney disease (creatinine clearance less than 50 mL/min) before treatment, occurring of acute kidney injury (based on AKIN criteria) during treatment , vancomyDosing RegimenAll patients had received vancomycin as antibiotic prophylaxis two hours before surgery at a dose of 1 g over an hour. In the II group, patients received vancomycin as a loading dose of 25 mg/kg over two hours, followed by 25 mg/kg over two hours every 12 h by intermittent infusion as maintenance dose and meropenem was administered 2 g over 30 min every 8 h. In the CI group, the patients received vancomycin as a loading dose of 25 mg/kg over two hours, followed by 50 mg/kg/day by continuous infusion as maintenance dose and meropenem was administered as same as II group. Additionally, in both groups, dexamethasone was administered 6 mg every 6 h intravenously for 2 days, with the first dose given just before or concomitant with the first dose of antimicrobial therapy.Serum SamplingIn II group, to determine vancomycin serum concentration, the serum samples were obtained at the baseline before starting vancomycin then to find out trough and peak concentrations of vancomycin, the serum samples were obtained 30 min before and an hour after each maintenance doses, respectively. After achieving steady state concentration, on the days 4 and 8 concomitant with serum trough samples (trough 4 and 8), CSF samples (CSF 1 and 2) were obtained to determine CSF concentration of vancomycin.Determination and identification of vancomycin serum concentration in CI group was exactly the same as the II group. At first, serum samples were obtained at the baseline before starting vancomycin then in accordance with II group, the serum samples were obtained daily. Like the II group, after achieving steady state concentration, on the days 4 and 8 concomitant with serum samples (serum samples 4 and 8), CSF samples (CSF 1 and 2) were obtained to determine CSF concentration of vancomycin. Additionally, for determining pharmacokinetic parameters of vancomycin, the serial serum samples were obtained at 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after starting the last dose of vancomycin in both groups.Assessment of Vancomycin Level Vancomycin concentration, as a primary outcome, was measured by Fluorescent Polarization Immunoassay (FPIA) method by Cobas Integra apparatus . Prior to the measurement, the method was validated in terms of linearity, precision, and accuracy.Pharmacokinetic Analysise) was calculated by least squares regression of serum concentration–time data points in the terminal log-linear region of the curve. The elimination half-life (t1/2) was calculated as 0.693 divided by Ke. The area under the serum concentration-versus time curve (AUC) was calculated using the trapezoidal rule with extrapolation to infinity. Clearance (Cl) was calculated by dividing dose over AUC.Pharmacokinetic parameters were calculated by using non-compartmental method. Elimination rate constant  was calculated then patients with GFR less than 50 mL/min were excluded from the study. CSF concentration and CSF/serum concentration ratio was calculated and compared between two groups to evaluate penetration of vancomycin into the CSF.Statistical Analysist-test was used for parametric variables and mann-whitney U test was used for non-parametric variables. P-value less than 0.05 was considered as significance level. The correlation between serum and CSF concentration of vancomycin was examined by Pearson’s correlation coefficient. Data were analyzed using SPSS software version 21 .The descriptive statistics presented and Kolmogorov-Smirnov test used to determination the normality distribution. Consequently, the independed During a 2-year period, 69 patients with diagnosis of post-neurosurgical meningitis were screened, 28 patients were excluded according to exclusion criteria and 41 patients were enrolled in the study. During follow of study, 21 patients were excluded as described in P = 0.773). The sex ratio  also was 1 in II group and 2/3 in CI group. The baseline laboratory data of participants were summarized in The mean age ± SD of patients in II group and CI group was 49 ± 7.25 and 48 ± 8.02, respectively. The age was not significantly different between two groups (P < 0.001). The mean ± SD value for CSF-to-trough concentration ratio was 27.39% ± 2.43%.In the II group, after 4 to 5 doses, the steady state serum concentration was achieved. At steady state, mean ± standard division (SD) serum trough and peak concentrations were 17.49 ± 2.46 mg/L and 41.33 ± 2.73 mg/L respectively and mean ± SD of CSF concentration was 4.83 ± 1.05 mg/L. A positive linear correlation was found between the serum trough levels and CSF levels . The mean ± SD value for CSF-to-serum concentration ratio was 24.84% ± 3.54%. As shown in P = 0.019). However, the CSF/serum ratio did not show any significant difference between the two studied groups. Results of vancomycin concentration in serum and CSF in both groups are summarized in In CI group, after 3 doses, steady state serum concentration was achieved. At steady state, mean ± SD serum concentration of samples 4 and 8 and overall average serum concentration, were 24.37 ± 1.92 mg/L, 25.15 ±2.24 mg/L, and 24.76 ± 2.02 mg/L, respectively. Mean ± SD of CSF concentrations was 6.20 ± 1.31 mg/L. The serum and CSF levels revealed positive linear correlation  and in the CI group two patients had positive CSF culture (Methicillin Resistant Staphylococcus Aurous (MRSA) and Pseudomonas Aeruginosa). Biochemical analyses in both groups were associated with trend of treatment. All patients recovered and in spite of high dose of vancomycin in both groups, the therapy was well tolerated. In 5 patients, GFR fell down and were excluded from the study according to exclusion criteria; these patients have received medications (like aminoglycosides) that increased risk of nephrotoxicity.To evaluate the efficacy and response to the treatment, the CSF cultures and CSF samples were analyzed. The most of cultures were negative, in the II group just one patient had positive CSF culture  may be more likely reasons, also their measurement method by High Performance Liquid Chromatography (HPLC) was different from the other studies  in 13 mechanically ventilated patients with different types of infections. CSF/Serum ratio in the meningitis patients was 48% vs. 18% in the non-meningitis patients. They concluded that vancomycin penetration in meningitis group is higher than non-meningitis group. Although they used high doses of vancomycin, they didn′t compare their data with intermittent infusion method and number of patients with meningitis were low (7 patients)  of vancomycin in the CSF was obtained by administration of continuous infusion of high-dose vancomycin. The mean serum concentration of vancomycin was 25.2 mg/L (ranging between 14.2 mg/L and 39.0 mg/L)  . A clini.0 mg/L)  .Similar.0 mg/L) . These s.0 mg/L) , but our.0 mg/L)  .A meta-.0 mg/L) . Clinica.0 mg/L) . Continu.0 mg/L) . But a rfections . In the Continuous infusion of vancomycin makes it possible to achieve faster and constant target level in serum but did not have any significant effect on the penetration (CSF/Serum ratio) of vancomycin, although lead to higher CSF concentration of vancomycin. It seems that higher CSF concentration by continuous infusion of vancomycin is the result of constant and higher serum concentration. High-dose, continuous infusion of vancomycin may be considered in patients not responding to standard dosing methods and impairment of vancomycin penetration into CSF could be overcome by increasing the dosage of vancomycin.


Presumed autoimmune diseases affecting the central nervous system (CNS) of dogs are common. In people, antibodies against neuronal cell surface antigens that are associated with a wide variety of neurological syndromes have been identified. The presence of cerebrospinal fluid (CSF) autoantibodies that target neuronal cell surface proteins has not been reported in dogs with neurologic disorders.Autoantibodies to neuronal cell surface antigens can be found in the CSF of dogs with inflammatory CNS disease. Our aim was to determine whether 6 neuronal cell surface autoantibodies were present in the CSF of dogs diagnosed with inflammatory and noninflammatory CNS disease.Client‐owned dogs with CNS disease and complete diagnostic evaluation including magnetic resonance imaging and CSF analysis were included. One healthy dog was included as a negative control.Cerebrospinal fluid was tested for 6 antigenic targets with a commercially available indirect immunofluorescence assay test kit.d‐aspartate receptor 1 (NMDAR1) antibodies were detected in 3 dogs . All 3 dogs responded to treatment of meningoencephalomyelitis of unknown etiology (MUE).There were 32 dogs with neurological disease, 19 diagnosed with inflammatory disease , 10 with noninflammatory disease , 2 with no diagnosis, and 1 with neoplasia and meningoencephalitis. Anti‐N‐methyl‐Further evaluation of the prevalence and clinical relevance of CSF and serum antibodies to neuronal cell surface antigens is warranted. Defining antigenic targets associated with encephalitis in dogs might allow diagnostic categorization of MUE antemortem. AEautoimmune encephalitisAMPAα‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acidCASPRcontactin‐associated proteinCNScentral nervous systemCSFcerebrospinal fluidDPPXdipeptidyl aminopeptidase‐like proteinBGABAgamma‐aminobutyric acid type BGMEgranulomatous meningoencephalomyelitisIFimmunofluorescenceIFAimmunofluorescent assayLGI1leucine‐rich glioma‐inactivated protein 1MRImagnetic resonance imagingMUEmeningoencephalomyelitis of unknown etiologyNMDAN‐methyl‐D‐aspartateNMDARN‐methyl‐D‐aspartate receptorNMDARENMDA receptor encephalitisPBSphosphate‐buffered salinePBSTphosphate‐buffered saline containing 0.2% Tween‐20VGKCvoltage‐gated potassium channelVGCCvoltage‐gated calcium channelPCRpolymerase chain reaction1Meningoencephalomyelitis is a common cause of neurological disease in dogs, with small breed, young adult dogs particularly at risk.d‐aspartate receptor encephalitis (NMDARE).In people, the description of antibodies to neuronal cell surface or synaptic proteins has revolutionized the understanding of neurologic disorders in recent years.In veterinary medicine, antibodies against glial fibrillary acidic protein (GFAP), an intracellular astrocytic protein, have been identified in the CSF of dogs with granulomatous meningoencephalomyelitis (GME) and necrotizing meningoencephalitis.22.1BR1/R2), LGI1, contactin‐associated protein receptor 2 (CASPR2), and dipeptidyl aminopeptidase‐like protein (DPPX)A BLASTp analysis using the following human proteins as a query in the National Center for Biotechnology Information database was performed to identify canine homologs: glutamate receptor ionotropic NMDA1 receptor (NMDAR1/GRIN1), alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionic acid receptors 1 and 2 (AMPAR1/R2), gamma‐aminobutyric acid type B receptors 1 and 2 . Additional serology was performed in certain cases at the discretion of the clinician. Cerebrospinal fluid banked from a healthy, neurologically normal beagle dog taking part in a separate research study served as the negative control. All research samples were acquired in accordance with guidelines and approval of the North Carolina State University Institutional Animal Care and Use Committee (protocol number: 17‐053‐O).Cerebrospinal fluid samples were selected from the Neurology Service CSF bank at North Carolina State University College of Veterinary Medicine. These samples were obtained from dogs undergoing diagnostic evaluation for CNS disorders. Samples were obtained from the cerebellomedullary or lumbar cistern and submitted for routine analysis . Residual CSF then was stored at −80°C within 24 hours of sampling according to the immunofluorescence assay (IFA) manufacturer's directions. Samples were selected from dogs with neurological signs localizing to the brain or spinal cord that also had brain or spinal cord MRI and CSF analysis. Clinical details of all dogs were extracted from their medical records and the final diagnosis and treatment were recorded. All dogs diagnosed with MUE also had negative infectious disease testing, which included negative polymerase chain reaction (PCR) of blood and CSF for canine distemper virus, West Nile virus, 2.3BR1, and GABABR2 receptors, along with other protein targets: CASPR2, LGI1, and DPPX. To minimize background signal in samples from dogs with severe inflammatory conditions, CSF samples with protein concentration >50 mg/dL were diluted to a final protein concentration of 50 mg/dL with ×1 phosphate‐buffered saline (PBS) containing 0.2% Tween‐20 (PBST). Cerebrospinal fluid samples were applied to the kit's proprietary “biochip” according to the manufacturer's instructions. An anti‐human NMDAR1 antibody, supplied by the manufacturer, was used as a positive control. A sample from a healthy beagle dog was included in each technical replicate as a negative control. After incubation, samples were washed with ×1 PBST and secondary antibodies were applied according to manufacturer's instructions. Secondary antibodies provided by the manufacturer included a fluorescein‐labeled goat anti‐dog IgG  applied to all canine samples and a fluorescein‐labeled goat anti‐human antibody  was used for the positive control. Biochips were incubated and washed with PBST. Fluorescent and differential interference contrast (DIC) images were acquired with a Leica DM5000B fluorescent microscope at ×20. Each reaction field was captured in 4 quadrants using the same imaging technique. Images were reviewed for the presence of immunofluorescent cells. Manufacturer's instructions note that not all cells are transfected with the antigen, thus the presence of immunofluorescence (IF) is not expected of all cells within a field. The characteristics of cellular IF differ among the antigens and a full description of expected staining patterns was provided by the manufacturer and used to determine the presence of positive staining. Samples from dogs that had positive results were retested, with positive and negative controls, to ensure the results were repeatable.Autoimmune Encephalitis Mosaic 6 testing kit  is an indirect IFA used to screen for autoantibodies against 6 targets associated with autoimmune encephalitic diseases of humans.33.1BR1/R2, LGI1, CASPR2, and DPPX, high similarities (94‐99%) were observed between the human and putative canine homologs . At the time of writing, the dog was alive and neurologically normal 22 months after diagnosis and was being treated with 0.33 mg/kg of prednisone PO q24h. Decrease in prednisone below this dose resulted in neurologic deterioration. Dog 10 had normal MRI results but CSF pleocytosis  starting at 1 mg/kg/day while also treating the tooth root abscesses with clindamycin 15.6 mg/kg PO q12h for 21 days  and minocycline 10 mg/kg PO q12h for 21 days . Clinical signs initially resolved but recurred as the prednisone dose was tapered. The dose was increased back to 1 mg/kg/day and then tapered again over a 3‐month period and signs resolved.4We screened CSF from 32 dogs diagnosed with a variety of neurologic disorders with a commercially available IFA kit that detects antibodies to 6 different neuronal cell surface or synaptic targets associated with AE in humans. Cerebrospinal fluid samples from 3 dogs were positive by IF indicating that antibodies against NMDAR were present, whereas all other antibody targets were negative in all dogs. Two of the 3 dogs that were positive for anti‐NMDAR antibodies were diagnosed with MUE and responded to immunosuppression. A specific diagnosis was not established in the third dog, but this dog responded to a combination of antibiotics and prednisone treatment. Our results suggest that autoantibodies against NMDAR deserve further investigation for their role in neurologic signs in dogs with encephalitis.B  of flu‐like clinical signs that progress to abnormal behavior or cognitive dysfunction, speech abnormalities, seizures, movement disorders (including dyskinesias and rigidity), decreased level of consciousness, and autonomic dysfunction. A fatal catatonic state can occur, but appropriate treatment with immunosuppression results in a favorable outcome.N‐methly The 3 dogs with CSF positive for anti‐NMDAR1 antibodies underwent extensive diagnostic evaluation. The first dog had a history and multifocal neurological signs typical of encephalitis as well as both multifocal MRI changes and CSF pleocytosis. The second dog had severe cerebellar signs, consistent with the atypical presentation reported in people more recently,Our study had several limitations. Although high homology of all protein targets among species allowed for confidence in results, the commercially available kit used antigen targets designed for humans specifically and we did not confirm our findings by use of another assay. This project was developed to screen CSF banked from dogs with CNS dysfunction of a variety of causes for the presence of CSF antibodies against neuronal surface targets. Although the panel of antigenic targets was negative in dogs with epilepsy and degenerative conditions, the number of dogs with these conditions was limited and a much larger study of the prevalence of these antibodies in the CSF and serum of dogs with neurologic disease as well as the clinical relevance of these antibodies is warranted in light of our results. In addition, CSF and serum samples from a much larger cohort of neurologically normal dogs of a range of ages should be evaluated. Finally, the retrospective nature of our study means that dogs with CSF positive for antibodies were not followed prospectively and the presence of antibodies in serum was not evaluated.Our results need confirmation by an additional assay or assays, but suggest that anti‐NMDAR1 antibodies can be found in the CSF of dogs with encephalitis. Although their pathologic role is unclear, these findings suggest that neuronal surface protein antibodies might be a cause of AE, and a much wider search for autoantibodies is warranted. Indeed, it is possible that dogs with idiopathic syndromes such as idiopathic cerebellitis  or so‐called “fly biting” might have antibodies to an identifiable antigenic target in their CSF. Identification of these autoantibodies may allow better antemortem categorization of MUE as specific types of AE and improve our ability to provide a prognosis. A much larger prospective study evaluating a wide range of neuronal cell surface or synaptic antigenic targets is warranted.Authors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.This study was conducted in accordance with guidelines and approval of the North Carolina State University IACUC (protocol number: 17‐053‐O).Authors declare human ethics approval was not needed for this study.Data S1: Supporting Information, tables.Click here for additional data file.Figure S1 Immunofluorescent assay results for the presence of CSF antibodies against AMPA receptors 1 and 2 (A), GABA receptors 1 and 2 (B), CASPR2 (C), LGI1 (D) and DPPX (E). Cellular immunostaining is uniformly absent in these samples although there is some artifactual staining of debris in D and EClick here for additional data file.


As compared to controls, schizophrenic and schizoaffective patients displayed significantly poorer ability to copy three-dimensional figures  and to orient in space on a road-map test; group differences in copying two-dimensional figures and on objects' recognition against a background noise were not apparent. In the schizophrenia/schizoaffective group, more mistakes on the hidden line elimination cube was associated with greater body mass index and greater severity of nicotine dependence measured via the Fagerstrom Test of Nicotine Dependence. The above findings replicate those of prior reports and extend them to the tasks that do not involve motivational and attentional confounds. Furthermore, the present data support the hypothesis that subtle cerebral cortical abnormalities detected with specific NSSs tests may be related to some aspects of metabolic and motivational function in patients with schizophrenia/schizoaffective disorder.Neurological soft signs (NSSs) are highly prevalent among patients with schizophrenia, but their pathophysiological significance remains unclear. The present study employed perceptual-motor and visuospatial processing tests that have not yet been attempted in this patient population. Patients with schizophrenia or schizoaffective disorder ( Obesity and smoking are identified by the World Health Organization as leading preventable causes of mortality in the industrialized worldOne of the most consistent findings in clinical studies of schizophrenia is the heightened prevalence of temporally stable , 8 markeIn a previous paper, we reported, using easily administered tasks, which impose few cognitive demands and thus not substantially affected by performance confounds , 21 thatAlthough only one of many legitimate ways that neurological compromise might be operationalized, NSSs' generalized rather than localized nature renders them a relevant investigational target in patients with schizophrenia wherein the list of brain regions that have been found to differ on various comparisons stretches from cerebellum to prefrontal cortex , 26. CliAccordingly, we administered to patients with schizophrenia or schizoaffective disorder visuospatial and sensory integration tasks that were carefully chosen from a comprehensive NSSs assessment battery due to their sensitivity and discriminative ability in addicted- , 21 and n = 34) or schizoaffective disorder (n = 8) were recruited via advertisement to participate in a double blind placebo-controlled clinical trial the results of which are reported elsewhere (n = 10) had no psychiatric history as determined by SCID; their data were previously reported  for a minimum of 2 months. The total scores on the Scale for the Assessment of Negative Symptoms  , on the The three tests were administered in sequential order during one session. The CFT comprised seven figures that the participants were instructed to copy exactly as they appeared with a pen and to not attempt to erase any mistakes made Figure . The figThe DROT consisted of six images of basic household objects, including a key, shovel, pitcher, eyeglasses, hammer, and kettle. The recognition of each object was complicated by the addition of background “noise” which consisted of a field of black dots. The six images were presented in two separate sets. The first set of images were in high density  while the second set of the same images were placed in low density . Participants were instructed to identify each object and could manipulate the page freely.In the Road Mad Test (RMT) participants were presented with a map of an imaginary town with a route consisting of 32 intersections. Following a brief practice trial to familiarize the participants to the task, each participant was instructed to mentally drive the route, and to sequentially determine whether they were to turn left or right at each intersection to follow the route. The researcher followed the route with a pencil and marked R or L based on the participant's verbal response at each intersection. Throughout the duration of the test, the map remained in a fixed position in front of the subjects without a possibility to move it.The three tests were scored by a dually trained Board-Certified psychiatrist and neurologist (TVG) who was blind to the subjects' diagnoses and specifically trained in the administration and scoring of these tests. A four-point scale was used for each figure in the CFT. A score of zero (0) was given for perfect or near perfect reproduction; 1 was given for slight distortion or rotation; 2 was given for moderate distortion or rotation, severe scaling mistakes, or loss of three-dimensionality; and 3 was given for severe distortion or an unrecognizable image. The number of failed identifications or of wrong turns were respectively scored for the DROT and the RMT.t-tests or Fisher's exact tests as appropriate. Because most of the CFT, DROT, and RMT data were ordinal and not normally distributed, they were summarized as both median and mean ± standard deviation (SD). The univariate nonparametric Mann-Whitney U test was used to compare groups. The non-parametric Spearman correlation coefficient was used for correlation analyses. Significance was defined as p < 0.05, with more abnormalities predicted in the schizophrenia/schizoaffective group.Demographic variables were analyzed by Student's 2 for healthy controls) and endorsed 4.6 ± 2.5 out of 17 items on the developmental history questionnaire ; among schizophrenic/schizoaffective subjects 33 reported smoking ; all healthy control subjects were non-smokers.Table Table p > 0.13) correlated significantly with BMI . No relationships were observed between SANS, PANSS and BPRS scores on those for the Necker- and hidden elimination cubes and RMT (p > 0.16).Performance on the hidden elimination cube , but not two-dimensional figures and impaired left-right orientation in the patients' group. The constructional apraxia uncovered by the CFT was not secondary to visual agnosia as evidenced by the DROT findings. Although the employed tasks did not constitute real life challenges, our findings may have clinical significance in patients with schizophrenia because difficulties with perceptual  and spatper se. Thus, this independent replication strongly supports the validity of schizophrenia-related constructional and visuospatial impairments as defined by the easily administrated paper-and-pencil tasks  or result from each other. Accordingly, these findings may provide a foundation for further, more rigorously designed projects. Such projects may include prospective design with a longitudinal NSSs assessment  in relation to food and nicotine intake, metabolic status and clinical course of the illness.This pilot study has several additional limitations, including the small sample size, explorative methodology, and the sole reliance on the visuospatial and sensory integration tasks selected from the 45-item battery of NSSs tests available for neuropsychiatric research . ConsequNonetheless, the presented findings add to the substantial body of evidence implicating NSSs in the course of schizophrenia/schizoaffective disorder by suggesting that the relationship is relatively specific to the traits defined by copying Necker- and hidden line elimination cube as well as the left-right orientation and do not generalize to other forms of constructional apraxia or visual agnosia. More research is needed to determine which aspects of NSSs are most strongly associated with schizophrenic neuropsychopathology and to compare them to other syndromes with dopaminergic etiology, such as chemical and behavioral addictions as well as depression and post-traumatic stress disorder . FurtherIE and TG conceived and designed the experiments. IE performed the experiments. IE, JB-N, and TV analyzed the data. TG, DB, and JB-N contributed reagents, materials, and analysis tools. TV, TG, IE, JB-N, and DB wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Analgosedation is a cornerstone therapy for mechanically ventilated patients in intensive care units (ICU). To avoid inadequate sedation and its complications, monitoring of analgosedation is of great importance. The aim of this study was to investigate whether monitoring of analgosedative drug concentrations (midazolam and sufentanil) might be beneficial to optimize analgosedation and whether drug serum concentrations correlate with the results of subjective  and objective  index) monitoring procedures.ρ).Forty-nine intubated, ventilated, and analgosedated critically ill patients treated in ICU were clinically evaluated concerning the depth of sedation using RASS Score, Ramsay Score, and BIS index twice a day. Serum concentrations of midazolam and sufentanil were determined in blood samples drawn at the same time. Clinical and laboratory data were statistically analyzed for correlations using the Spearman’s rank correlation coefficient rho (ρ = − 0.467) and Ramsay Scores (ρ = 0.476). Serum concentrations of sufentanil correlated weakly with RASS (ρ = − 0.312) and Ramsay Scores (ρ = 0.295). Correlations between BIS index and serum concentrations of midazolam (ρ = − 0.252) and sufentanil (ρ = − 0.166) were low.Average age of the population was 57.8 ± 16.0 years, 61% of the patients were males. Most frequent causes for ICU treatments were sepsis (22%), pneumonia (22%), or a combination of both (25%). Serum concentrations of midazolam correlated weakly with RASS  contains supplementary material, which is available to authorized users. Critically ill patients on intensive care units require regimens of analgosedation for several reasons such as mechanical ventilation. Finding the optimal treatment and sedation depth is often challenging because of multimorbidity and polypharmacy. High interindividual variability concerning pharmacokinetics as well as insufficient monitoring can lead to inadequate dosage of drugs, which might increase morbidity and mortality. Low states of analgosedation can cause hypercatabolism, immunosuppression, hypercoagulopathy, awareness and increased sympathetic activity, or inadvertent extubation, whereas deep sedation can be responsible for extended mechanical ventilation, higher risk of nosocomial pneumonia, increasing costs, and neuropsychological dysfunction –6. HenceTo improve individual treatment, clinical scores such as the Richmond Agitation-Sedation Scale (RASS) Score  and RamsGold standard for the assessment of sedation depth is the RASS Score in combination with physiological parameters like heart rate, blood pressure, mimic, gesture, lacrimation, and perspiration , 6. ReliThe BIS index is a unitless value ranging from 0 to 100, a value of 100 representing an adequate awake condition  as well as objective monitoring procedures (BIS-monitoring) were investigated in this study. The aim of this study was to clarify whether or not therapeutic drug monitoring is useful to assess the sedation depth in intensive care patients.This study was performed between December 2012 and December 2014 in cooperation with the ICU of the Department of Internal Medicine and the Center of Pharmacology, University Hospital of Cologne. Intubated, artificially ventilated, and analgosedated intensive care patients, who agreed to this study by themselves or through legal representatives before intubation, were included. Exclusion criteria were age < 18 years, missing patient’s consent, history of alcohol or drug abuse, history of neurological or psychiatric conditions, polytraumatization, conditions after CPR, and suspicion of hypoxic brain damage. RASS Score, Ramsay Score, BIS index, and serum concentrations of analgosedatives were measured twice a day (7:00 a.m. and 7:00 p.m.). Overall, 49 patients were included in the study, and 538 data points were determined. The maximal period under consideration was 10 days. Sepsis was defined according to the criteria by Bone et al. ./kg/h i.v.) and sufentanil (infusion rate of 0.1–1.0 μg/kg/h i.v.). According to the clinical presentation and sedation depth, the infusion rates were adapted.After induction with fentanyl, etomidate and rocuronium orotracheal intubation was performed. Analgosedation was then maintained with midazolam  or “sedated” . “Awake” patients were assessed regarding the reaction while the observer was entering the room. If the patient was considered to be “sedated,” further evaluation was made using a fixed protocol in order to cause eye-opening or a change in facial expression: observer entering room, verbal contact, light physical contact, severe physical contact by shaking patient’s shoulder, induction of light pain by pinching the back of patient’s hand, and induction of severe pain by rubbing patient’s sternum.A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative serum concentration measurements of four analgosedatives  frequently used in intensive care medicine has been previously developed and validated according to ICH Guidelines Q2 (R1) . This teρ). The value of ρ was interpreted as follows: 0 ≤ |ρ| < 0.1—no or very weak correlation; 0.1 ≤ |ρ| < 0.5—weak correlation; 0.5 ≤ |ρ| < 0.8—moderate correlation; 0.8 ≤ |ρ| ≤ 1—strong correlation. Box plots were used for graphic illustration.Statistical analysis and graphic design were performed using IBM SPSS Statistics version 22. Correlations were analyzed using Spearman’s rank correlation coefficient rho . A Ramsay Score of 6 was observed in patients with a median midazolam level of 594 ng/ml, and high intersubject variability was seen. Similar results were described by Glass et al. using the Observers’ Assessment of Alertness/Sedation Scale  [Serum concentrations of sufentanil had only a weak correlation with RASS and Ramsay Scores, whereas serum concentrations of midazolam showed a better but still weak correlation. Bremer et al.  investig= 0.746) .In this study, serum midazolam levels correlated weakly with RASS and Ramsay Scores. A Ramsay Score of 6 was associated with a median midazolam concentration clearly above 1000 ng/ml, whereas the other patients showed median midazolam concentrations lower than that.Bremer et al.  describeComedication with opioids could inhibit midazolam metabolism . MoreoveIn this study, 57% of the patients suffered at least from sepsis or acute renal failure, which led to high intersubject variability Figs.  and 2b aEthuin et al.  analyzed−1) and a large dose midazolam group (0.3 mg kg−1). After remifentanil, midazolam, and vecuronium were administered, intubation was performed, and eight blood samples were collected within 1 h before the operation. Although midazolam plasma concentrations were significantly higher in the large dose group, the authors found no differences concerning BIS index between the two groups. This indicates that there is no correlation between BIS index and serum concentrations of midazolam. In this study, BIS index and midazolam serum concentrations showed only a weak correlation (ρ = − 0.252), which stands in line with the results of Miyake et al. [Miyake et al.  investige et al. . A limite et al.  observede et al. . Severale et al. , 34. Miye et al.  found a e et al.  describee et al.  also foue et al.  showed tConclusively, BIS index will reach its limits—especially as a primary monitoring of sedation depth—when the effect of midazolam is monitored because it does not further decrease although the patient is clinically sedated and plasma concentrations are higher than needed for adequate sedation.Glass et al.  showed tThis study was a single-center study with a relatively small population of 49 intubated patients. Moreover, BIS index values are very susceptible. For example, endotracheal or oral suctioning, body hygiene procedures, passive movements, and physical contact are able to influence BIS index without changing the sedation depth necessarily , 40. To Delirium may influence the assessment of sedation. However, we did not screen our patients for delirium since Haenggi et al.  reportedCorrelations between drug serum concentrations (midazolam and sufentanil) and RASS Score, Ramsay Score, or BIS index were only weak, the results for midazolam being slightly better than those for sufentanil. This might be due to the intersubject variability, polypharmacy with drug-drug interactions, and complex metabolism, which can be altered especially in critically ill patients. Therefore, individual course of disease and patients’ comorbidity have to be taken into account. Therapeutic drug monitoring is not beneficial to determine the depth of sedation in ICU patients. Analgosedation of patients in ICUs should therefore be guided by subjective monitoring procedures.Additional file 1:Richmond Agitation Sedation Scale . (PDF 50Additional file 2:Ramsay Sedation Scale . (PDF 44Additional file 3:Monitoring the depth of sedation with BIS-monitoring . (PDF 44


Citrus changshan-huyou + Citrus unshiu) is a new graft chimera originating from the junction where a Citrus changshan-huyou (“C”) scion was top-grafted onto a stock Satsuma mandarin ‘Owari’ . The chimera was named OCC because the cell layer constitutions were O for Layer 1(L1) and C for L2 and L3. In this study, profiles of primary metabolites, volatiles and carotenoids derived from different tissues in OCC and the two donors were investigated, with the aim of determining the relationship between the layer donors and metabolites.Chimeras synthesized artificially by grafting are crucial to the breeding of perennial woody plants. ‘Hongrou Huyou’  in each tissue was identified in the three phenotypes. According to principal component analysis (PCA), overall, the metabolites in the peel of the chimera were derived from donor C, whereas those in the juice sac of the chimera came from donor O.The comparison of the metabolite profiles showed that the amount and composition of metabolites were different between the peels and the juice sacs, as well as between OCC and each of the two donors. The absence or presence of specific metabolites (such as the carotenoids violaxanthin and The profiles of primary metabolites, volatiles and carotenoids derived from the peels and juice sacs of OCC and the two donors were systematically compared. The content and composition of metabolites were different between the tissues and between OCC and the each of the two donors. A clear donor dominant pattern of metabolite inheritance was observed in the different tissues of OCC and was basically consistent with the layer origin; the peel of the chimera was derived from C, and the juice sacs of the chimera came from O. These profiles provide potential chemical markers for genotype differentiation, citrus breeding assessment, and donor selection during artificial chimera synthesis. Citrus sinensis) and Satsuma mandarin. The stomatal density and the flesh aroma of the chimera fruits in their studies were not consistent with those of the source donor; the chimera fruits showed combined characteristics of both donors [Plant chimeras are plants composed of cells with more than two genotypes. According to the theory of ‘Tunica-Corpus’, the shoot apical meristem (SAM) of dicotyledonous plants is composed of three cell layers, namely, L1, L2 and L3, from the outermost layer  and ‘Honh donors , 5. SincC. sinensis), mandarins (C. reticulata), pummelos (C. grandis) and grapefruits (C. paradisi) [d-Limonene is a dominant volatile in citrus and specifically protects against breast and rectal cancer [β-ring moieties are precursors of vitamin A, which are highly beneficial to chronic disease and cancer prevention [Citrus fruits are highly valued for their nutrient components, and many studies have investigated the metabolites in oranges (aradisi) –9. Primaaradisi) . For exaaradisi) . Volatill cancer . Linalool cancer , and real cancer . Carotenl cancer . Some caevention . Carotenevention –19, and Citrus changshan-huyou + C. unshiu) was originated from the junction of the scion ‘Changshan Huyou’  and the stock ‘Owari’ Satsuma mandarin . This chimera remained yellow-skinned, similar to C, but gained the dark orange juice sacs observed in O  were the lowest in OCP.Eighteen primary substances, listed in Table Interestingly, 3 compounds showed some hereditary differences in OCJ . Among them, oxalic acid and rhamnose were undetectable only in OCJ, which caused obvious discrepancies between OCC and its donors. However, sorbose was undetectable in both OCJ and its layer source donor O.d-limonene was the dominant compound, accounting for 88.65, 81.23 and 80.77% of the total volatiles in OP, OCP and CP, respectively. After d-limonene, the main common compounds in the three cultivars were γ-terpinene, β-myrcene and α-pinene.With regard to the volatiles in the peels of the three cultivars, 36 substances are listed in Table d-limonene, γ-terpinene, germacrene D, β-myrcene and α-pinene (sorted from high to low concentrations), but the relative concentrations of the main volatiles in OP were divergent . This was mainly because the content of germacrene D was significantly higher in OCP and CP than in OP, strongly suggesting that germacrene D mainly originated from CP and that OP had less impact on the development of OCP. Third, it is worth noting that 2,4-di-t-butylphenol was truly unique and was only undetected in OP; but possessed by both OCC and C.The results showed that OCP had a stronger correlation with CP than with OP. First, according to the statistical analysis, 25 volatiles were not significantly different between OCP and CP, but only 16 volatiles were not significantly different between OCP and OP. This finding indicated that CP had the dominant position in the regulation of the chemical profiles in OCP and that more chemical traits in OCP were inherited from CP than from OP. Second, the main volatiles in OCP were completely consistent with those in CP, including E)-3-hexen-1-ol and 3-hexenal were significantly increased in OCP compared with the two donors.In addition, most of the volatiles in OCP were either inclined to one donor or maintained some degree between the two donors. However, only  was performed to compare the different tissues in OCC and the two donors.In the PC1 direction of the score map, there was a clear distinction between donor O and the other genotypes (OCC and donor C) in primary metabolites Fig. -1, volatIn the juice sacs, donor C was clearly distinguished from OCC and donor O in the primary metabolites Fig. -1, volatStudies focused on phenotypes, fruit qualities and genome compositions –5 have cβ-cryptoxanthin content of juice [cis-violaxanthin and β-cryptoxanthin in juice [β-cryptoxanthin in the flavedo and juice sac were successfully used to differentiate among 39 citrus genotypes [β-cryptoxanthin in both the flavedo and juice sac. Oranges are rich in violaxanthin in both the flavedo and juice sacs. Pummelo was separated from oranges and mandarins, as it lacks β-cryptoxanthin and violaxanthin [β-cryptoxanthin. In this study, the level of β-cryptoxanthin in OCP was as low as that in CP, while OCJ accumulated much more β-cryptoxanthin than did CJ. Likewise, a previous study on the citrus chimera Ekuliku revealed that its juice sac was developed from the L1 donor Nankan (C. unshiu), and the peel was developed from the L2 and L3 donor Hamlin (C. sinensis) [Brassica chimeras was only controlled by red cabbage and was reproducible and directional in progenies [In citrus chimeras, the juice sacs develop from the L1 cell layer, and the peels are derived from the L2 cell layers . Severalof juice . Similarin juice . Furtherenotypes . Herein,axanthin . In thisaxanthin –29. Notarogenies .CitPds, CitZds and CitCrt) upstream of the carotenoid biosynthesis pathway were reported to be expressed at low levels in a somatic hybrid between C. reticulata and C. limon, which were biased towards parent lemon, resulting in low carotenoid content in the hybrid [C. sinensis) and rough lemon (C. jambhiri) showed a similar carotenoid content to that of rough lemon, whose expression patterns of the lycopene ε-cyclase gene (LCYE) and the zeaxanthin epoxidase gene (ZEP) were more similar to those of rough lemon [Metabolites are first biosynthesized in vivo. Three key genes  in OCC were specific to the layer source donors; some metabolites deviated far from the profile observed in both donors . This observation was similar to two citrus hybrids that exhibited 56 of the 113 volatile profiles in hybrids that were significantly higher or lower than in parents . In thisα-ylangene was exclusively detected in the juice sac of the chimera OCC  within a plant [MYB transcription factors on chromosome 2 [VvpI gene expression in L1, in L2 or in both cell layers, leading to phenotypic variation (fleshless) in progeny [Interestingly, a volatile named CC Table . α-ylangn grapes . Similarn grapes . Therefo a plant , 38. In  a plant . Some stmosome 2 –42. Fern progeny . In a pe progeny .γ-terpinene) were partly different from the layer donor and displayed possible “recombination” between layers. Recently, small RNAs and DNA methylation have been considered to be involved in stock-scion interactions to describe genetic variations in graft chimeras. For instance, researchers have found that some conserved miRNAs were differentially expressed in graft chimera (Brassica juncea + B. oleracea) progeny rTTT  and donor plant TTT (B. juncea), which may contribute to the changes in the expression of their target genes [Brassica juncea and B. oleracea, sequencing analysis revealed that DNA methylation affects flowering time- and gibberellin response-related gene expression and may lead to phenotypic variations in progenies [To date, there is limited knowledge available regarding the inheritance pattern of chemical compounds in plant chimeras. The donor bias was a compelling issue in the artificial synthesis of chimeras and in plant breeding. Arguments on the relationship between the chimeric phenotype and the traits of grafting donors have been proposed. It seemed that the stock donor Satsuma mandarin likely acted as the inner layer (L1) donor, with a focus on carotenoid synthesis , 5, 45. et genes . Furtherrogenies . TherefoThe gene expression pattern and accumulation of primary metabolites, volatiles and carotenoids derived from the peels and juice sacs of OCC and the two donors were systematically investigated and compared. The content and composition of metabolites were different among the genotypes and the tissues. Metabolites specifically present or absent in certain tissues (α-carotene and phytoene) were identified in three genotypes. A clear donor dominant pattern of metabolite inheritance was observed in the different tissues of OCC, indicating that the metabolites in the juice sacs of the chimera were similar to those from the L1 donor O and that those in the peels of the chimera were similar to the L2/L3 donor C. These profiles provide potential chemical markers for genotype differentiation and citrus breeding assessment; moreover, they provide information for donor selection during artificial chimera synthesis.Poncirus trifoliata in 2005 and maintained stable morphologies for 12 years under regular management. Three individual trees were selected for each genotype, and 10 fruits with uniform size, peel color and location on the tree were harvested from each tree at the full ripening stage. Peels including the epidermis, flavedo and albedo were separated carefully and quickly from the juice sacs of each genotype by girdling. The peels and juice sacs obtained from one tree were separately blended and ground into powder in liquid nitrogen. Finally, the samples were preserved at − 80 °C for subsequent research.The OCC was generated by top-grafting of the scion C and the stock O in 2001, however, it was found recently in our bud mutation investigation in an orchard in Changshan County of Zhejiang Province (China). Recently, OCC was identified to be a grafting chimera in our analysis of the morphological and DNA characteristics of the chimera and the two donors (unpublished data). For commercial production, OCC and the donors (O and C) were separately grafted onto The primary and volatile substances were evaluated using a modification of the procedure originally developed . To deteFor volatile analysis, the samples were freeze-dried with a vacuum freeze-drier  and fully ground in liquid nitrogen. A 0.2 g sample of powder was poured into a centrifuge tube (2 ml volume), which was homogenized with 500 μl of double distilled water (DDW) and 500 μl of MTBE (containing 0.02 μl/ml methyl pelargonate), followed by gentle shaking. The samples were vibrated using an ultrasonic bath  maintained at 4 °C for 40 min and were centrifuged at 12000×g for 10 min at 4 °C. The supernatants (200 μl) were then transferred into another tube. Finally, 1 μl of sample was injected with a syringe and filtered through a 0.22 μm membrane  for gas chromatography-mass spectrometry (GC-MS).The compounds were identified by using TRACE GC Ultra GC coupled with a DSQ II mass spectrometer  with a TRACE TR-5 MS column . With pure helium as a carrier gas, the peels  and juice sacs of the samples were assayed at 1.0 ml/min with a split ratio of 50:1 and 1:1, respectively. The concentrations of the primary and volatile substances were calculated as μg/g FW. Three replications were used for each sample.http://www.massbank.jp/) and Metlin (https://metlin.scripps.edu/index.pCF) supported the identification of tentative metabolite substances; for some other compounds, we obtained information from the published literature.The public databases Massbank  and B  as the mobile phase, was used to determine the carotenoid contents. The flow rate was fixed at 1 ml/min. The following gradients were used: 0 min, (95:5); 0–10 min, A-B (95:5); 10–19 min, A-B (86:14); 19–29 min, A-B (75:25); 29–54 min, A-B (50:50); 54–66 min, A-B (26:74); and 67 min, A-B (95:5). The volume of the above gradient solvent was 20 μl, and the test adopted an external standard method for quantitation. All carotenoid extraction, saponification and other assays described above were conducted under low light levels or in the dark.p < 0.05) and are shown in the tables with lowercase letters  between cultivars. Undetectable substances are marked with “nd” in all metabolite profile tables. Correlation analysis was carried out by Pearson’s test, and significant differences were marked with “**” (p < 0.01). Principal component analysis was carried out by SIMCA 14.1.The concentration of each chemical compound is shown as the mean ± standard deviation of three replicates. Statistical analysis was performed using SPSS 19.0 software . Significant differences were calculated using one-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test at the 5% level (


Soluble aggregates of amyloid-β (Aβ) have been associated with neuronal and synaptic loss in Alzheimer’s disease (AD). However, despite significant recent progress, the mechanisms by which these aggregated species contribute to disease progression are not fully determined. As the analysis of human cerebrospinal fluid (CSF) provides an accessible window into the molecular changes associated with the disease progression, we characterised soluble aggregates present in CSF samples from individuals with AD, mild cognitive impairment (MCI) and healthy controls using a range of sensitive biophysical methods. We used super-resolution imaging and atomic force microscopy to characterise the size and structure of the aggregates present in CSF and correlate this with their ability to permeabilise lipid membranes and induce an inflammatory response. We found that these aggregates are extremely heterogeneous and exist in a range of sizes, varying both structurally and in their mechanisms of toxicity during the disease progression. A higher proportion of small aggregates of Aβ that can cause membrane permeabilization are found in MCI CSF; in established AD, a higher proportion of the aggregates were larger and more prone to elicit a pro-inflammatory response in glial cells, while there was no detectable change in aggregate concentration. These results show that large aggregates, some longer than 100 nm, are present in the CSF of AD patients and suggest that different neurotoxic mechanisms are prevalent at different stages of AD.The online version of this article (10.1186/s40478-019-0777-4) contains supplementary material, which is available to authorized users. TheAs previously published, every 70-year-old in Gothenburg, Sweden, born during 1944 on prespecified birthdates was invited to the examination in 2014–2016, and 1203 participated (response rate 72.2%). Of these, 430 (35.8%) consented to a lumbar puncture . ParticiFor each participant, the clinical MCI diagnosis was determined in a clinical consensus conference, comprised of a neurologist and psychiatrist, taking into account the participants history and information from informants. CSF biomarker levels of Aβ 42, total-tau and Phopho-tau were considered in choosing participants with MCI and underlying preclinical AD pathology. All MCI participants had a clinical dementia rating (CDR) score of 0.5. Healthy controls had to be free of MCI and Dementia and had a CDR of 0. Dementia diagnoses were an exclusion criterion in this study.All samples were collected in the morning (before lunch) at the highly coordinated memory clinics in south-western Sweden, using clinically implemented CSF sampling protocol that has been detailed in: Blennow et al. . All sam2+ containing buffer, which we denote as blank (Fblank). Then a 30 μL CSF aliquot was added to the glass coverslip and incubated for 15 min before images of the exact same fields of view were recorded (Fsample). Then, 10 μL of ionomycin was added to the same coverslip and images of the vesicles were acquired in the same fields of view (Fionomycin). The recorded images were analysed to determine the fluorescence intensity of each vesicle under the three different conditions and the average Ca2+ influx for each vesicle was calculated using the formula (Fsample - Fblank) / (Fionomycin - Fblank) X 100%. The stage movements were performed using a bean shell-based program which allows to select fields of view without any user bias. For antibody experiment, CSF and 300 nM antibody were incubated for 15 min and added to the vesicle containing glass coverslip for membrane permeabilisation study.A detailed protocol and further description of the membrane permeabilisation can be found elsewhere . BrieflyImaging of individual vesicles were performed using a home-built total internal reflection fluorescence (TIRF) microscope. For excitation, a 488 nm laser (Toptica) beam was focussed in the back focal plane of the 60x, 1.49 NA oil-immersion objective lens . Fluorescence emission from the dyes were collected by the same objective and imaged onto an air-cooled EmCCD camera .6 cell/mL. 200 μL of CSF was diluted in 1 mL of DMEM and added to BV2 microglia cells. Every 24 h the supernatant was removed for analysis and replaced by fresh diluted CSF. The TNF-α concentration in the supernatant was quantified using the Duoset® enzyme-linked immunosorbent assay (ELISA) development system . Three wells for each CSF were used to estimate variation in the experiments. For antibody experiment, CSF and antibody were diluted in DMEM buffer and added to the vesicle containing glass coverslip.The BV2 cell line was derived from immortalised murine neonatal microglia and grown in 10% foetal bovine serum and 1% L-Glutamine supplemented Dulbecco’s Modified Eagle’s (DMEM) medium. The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, until the cell density reached approximately 1.6 × 1022. Briefly, glass-slides were cleaned with argon plasma, rinsed with 1% tween-20 and washed with PBS. All buffers were first passed through a 0.02 μm filter . The CSF was diluted ten-fold in PBS and added to wells on the coverslip formed by a multiwell chamber coverslip (CultureWell CWCS-50R-1.0). After leaving any aggregates to adhere to the surface for 5 min the CSF was removed, the wells were washed with PBS and then filled with imaging mix (1 nM imaging strand (sequence CCAGATGTAT-CY3B) and 100 nM aptamer-docking strand (sequence GCCTGTGGTGTTGGGGCGGGTGCGTTATACATCTA) in PBS). The wells were then sealed using another clean coverslip. The samples were imaged on a home-built TIRF microscope using a 1.49 N.A., 60x TIRF objective  mounted on a Ti-E Eclipse microscope (Nikon) fitted with a perfect focus system. The Cy3B was excited at 561 nm  passed through a FF01–561/14–25 excitation filter (Semrock). Fluorescence was separated from the excitation light using a dichroic mirror , passed through a filter  and focussed on the EMCCD camera described above operating in frame transfer mode (electron-multiplying Gain of 11.5 e-1/ADU and 250 ADU/photon). To eliminate user bias an automated script (Micro-Manager . T. T43]. TTo examine the aggregate morphologies, we plotted the relative differences between normalised Fig. d,e and cThe increase in the relative proportion of large aggregates means that there is also a small increase in the total mass of Aβ present in AD CSF, as also previously detected by ELISAs. However, ELISA cannot measure the mass and aggregate number independently. This may explain why it is difficult to detect a clear difference between control and AD CSF since both factors contribute to the total aggregate mass detected in a given sample. This also suggests that the observed reduction in Aβ monomer concentration in AD CSF is partially due to this increased mass of aggregates, although this cannot account for the entire reduction and presumably there is also significant deposition of aggregates in the brain.p < 0.001). The AD CSF samples showed the coexistence of highly heterogeneous elongated aggregates. These species belonged roughly to two populations of height, the first with cross sectional height ranging between 0.3–1 nm which is similar to the species present in MCI CSF aggregates, often referred to as protofilaments, and the second with cross-sectional height between 1 and 3 nm, often referred to as protofibrils. A larger cross-sectional aggregate height has been associated in vitro to an increased content of intermolecular β-sheet and maturity of amyloid structure [To measure the heterogeneity and three-dimensional morphological properties of the protein aggregates present in the CSF samples, we utilised a phase controlled AFM technique to resolve the structures of individual protein aggregates at Angstrom resolution , 36 Fig. This altructure . Interestructure . TLR3 sitructure . Since tIn conclusion, we have reported that there is a higher proportion of small aggregates that can cause increased membrane permeabilisation present in MCI CSF than in AD and control CSF, while in AD CSF there is an increased number of larger and longer aggregates, corresponding with enhanced neuroinflammation. These larger aggregates appear to be protofibrillar in nature, with distinct structural features from those of the smaller aggregates and appear to be the species that initiates cytokine production via TLR4 signalling.It has to be noted that our study has been performed on a small number of samples and in future work it would be of interest to see if we observe the same changes in aggregate size distributions in bigger cohorts. If replicated then following the changes in aggregate size distribution provides a method to follow disease progression that does not require the measurement of the absolute number of aggregate species, with the proportion of larger aggregates increasing. Our results indicate that as the disease progresses there are changes in the aggregate size distribution in CSF but not in the total number of species. It has been reported that there is a three-fold increase in Aβ aggregates deposited in the brain during the development of AD . Taken tAdditional file 1:Table S1. Characterisation of the CSF samples used in this study. Figure S1 Toll like receptor 4 (TLR4) antagonists block AD CSF-induced aggregate induced inflammation. Figure S2 a portion of the toxic aggregates present in MCI and AD CSF are composed of Aβ To understand the composition of toxic aggregate present in human CSF. we employed a series of Aβ-specific antibody which are known to counteract the toxicity induced by soluble aggregates of Aβ. Figure S3 Detection of aggregates present in control, MCI and AD CSF using pFTAA. (DOCX 197 kb)