Primary or secondary abnormalities of glycosylation have been reported in various brain diseases. Decreased asialotransferrin to sialotransferrin ratio in cerebrospinal fluid (CSF) is a diagnostic marker of leukodystrophies related to mutations of genes encoding translation initiation factor, EIF2B. We investigated the CSF glycome of eIF2B-mutated patients and age-matched normal individuals in order to further characterize the glycosylation defect for possible use as a biomarker.We conducted a differential N-glycan analysis using MALDI-TOF/MS of permethylated N-glycans in CSF and plasma of controls and eIF2B-mutated patients. We found in control CSF that tri-antennary/bisecting and high mannose structures were highly represented in samples obtained between 1 to 5 years of age, whereas fucosylated, sialylated structures were predominant at later age. In CSF, but not in plasma, of eIF2B-mutated patient samples, we found increased relative intensity of bi-antennary structures and decreased tri-antennary/bisecting structures in N-glycan profiles. Four of these structures appeared to be biomarker candidates of glycomic profiles of eIF2B-related disorders.Our results suggest a dynamic development of normal CSF N-glycan profiles from high mannose type structures to complex sialylated structures that could be correlated with postnatal brain maturation. CSF N-glycome analysis shows relevant quantitative changes associated with eIF2B related disorders. This approach could be applied to other neurological disorders involving developmental gliogenesis/synaptogenesis abnormalities. Genetic defects of N-glycan metabolisms due to abnormal hydrolysis (oligo saccharidosis), transport or storage  and synthesis as in congenital disorders of glycosylation (CDG) are responsible for severe alterations of the CNS including myelination impairment Studies have been performed with the ethical agreement of the “centre de protection des personnes Sud-Est VI, France” and the Institutional Review Board of the National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA. A signed written informed consent was obtained from the patients or their legal guardians. Our ethics committee specifically approved this study. Only excess CSF and plasma drawn for other clinical or research purposes was used for these analyses. Samples from eIF2B-mutated patients came from the French Leukobank, approved by the ethical committee “centre de protection des personnes Sud-Est VI, France”. We obtained written informed consents for this original human work. In total, CSF and EDTA plasma from respectively 12 and 9 eIF2B-mutated patients and from 17 and 15 control individuals without neurologic disorders were collected. The biological fluids were centrifuged at 500×g during 10 min and supernatants were transferred to clean Eppendorf polypropylene tubes and stored at −80°C until analysis.www.rochediagnostics.fr), SPE Carbo from GRACE Alltech , Sep-Pack Plus C18 were from Waters . Methanol, acetonitrile are LCMS grade. Nonidet P40; sodium dodecyl sulphate; 2 mercaptoethanol; DMSO; iodomethane; Sodium hydroxide; TFA, sodium phosphate salts were purchased from SIGMA Aldrich . 2, 5-Dihydroxybenzoïc acid and peptide calibrant C104 were purchased from LaserBio Labs .Endoglycosidase, peptide-N-Glycosidase F  was from Roche Diagnostics  on MALDI TOF DE PRO  with DHB as matrix . External calibration of spectra allowed obtaining a mass accuracy of 20 ppm. MALDI-PSD fragmentation was performed on selected ions to confirm structures.http://ca.expasy.org/tools/glycomod/) and Glycoworkbench. Glycans and glycan types  were compared between groups using ANOVA. Selected ions were fragmented by MALDI-PSD to further elucidate the corresponding glycan structures. Since a clear distinction of relative intensities of the isobaric complex bisecting and triantennary structures was not possible, these structures were not differentiated in the quantitative test. A difference between two groups of data was considered statistically significant when p-values were less than 0.005. Receiver Operating Characteristics (ROC) curves were used to assess the sensitivity and specificity of potential diagnostic variables with ANOVA p-values<0.001. Analyses were performed using SAS ver. 9.1.3. A principal component analysis was also performed according to TANAGRA MALDI–MS data were processed using DataExplorer 4.0 to generate files listing m/z values and to compare intensities of 34 ions corresponding to the main N-glycan structures from plasma and CSF samples. Interpretation of N-glycan structures corresponding to monoisotopic masses was performed using Expasy GlycoMod tool  appeared statistically significant (p = 0.0016). The most abundant structure in profile I CSF was high mannose type structure (Man)5 (GlcNAc)2 (at m/z 1580). Hybrid glycan structures decreased threefold. Profile II CSF was characterised by an increase in complex tri-antennary/bisecting (29.6±2.7% versus 48.6±1.7%) (p = 0.0016) and fucosylated type structures (43.0±2.3% versus 57.0±1.4%) (p = 0.0022). A 6-fold increase in sialylated N-glycan structures (6.8±2.1% versus 43.0±3.7%) (p = 0.0016) also distinguishes the two profiles. In contrast, complex bi-antennary type structures showed no significant difference between the two profiles.In order to further characterize these differences, relative intensities of a total of 34 individual N-glycans falling into 6 separate N-glycan families were determined for each MALDI spectra. The relative intensities of glycans within these N-glycan families were calculated for both profiles and statistically significant differences could be detected . Princip3 (GlcNAc)2 core containing N-glycan could not be assigned to this ion. MALDI-PSD fragmentation 1 (GlcNAc)1, corresponding probably to a degradation fragment of polysialic acid (PSA)-containing N-glycans, found on glycoproteins, such as NCAM, within the CNS.MALDI-TOF spectra revealed the presence of an intense ion at m/z 1602 for profile I control CSF , which ientation  allowed After having established CSF reference profiles, glycoprofiling of samples from eIF2B-mutated patients was performed and spectra were compared to their respective reference.We established MALDI-TOF MS profiles of permethylated N-glycans from 12 CSF samples of patients with eIF2B-related disorders (age at sampling from 3.5 to 39 years) .versus 45.0±2.5%) (p<0.0001) and the concomitant decrease of complex tri-antennary/bisecting structures (34.2±2.4% versus 48.6±1.7%) (p<0.0001) which are generally considered as brain or CNS type glycan structures were consistent among all samples (3 (Fuc)1 (Man)3 GlcNAc)2 reveals a fragment ion at Y1372 specific for a tri-antennary glycan (1 (GlcNAc)3 (Fuc)2 (Man)3 GlcNAc)2 reveals the presence of fragment ions at m/z B486 and Y1996 specific for a presence of bisecting structures but also a minor fragment ion at Y1751 suggesting the presence of tri-antennary glycans (1 (GlcNAc)1 (Fuc)1- 1- . No variRelative intensity of individual N-glycans showed statistically relevant differences for 11 structures  between patient and control CSF . Three gMeasuring relative intensity variation of these four N-glycan structures in CSF glycan profiles as potential biomarkers for identifying eIF2B-related disorders is strongly suggested. As lumbar puncture is more invasive than a simple blood sampling, we decided to investigate whether the observed differences in CSF N-glycan profiles were also found in eIF2B-mutated patients' plasma.MALDI-TOF MS profiles of permethylated N-glycans from 8 plasma samples from patients with eIF2B-related disorders (age at sampling from 8 to 38 years) were compared to 15 healthy age and sex matched controls (age at sampling from 3 to 58 years) .Twenty µl of plasma were sufficient to generate permethylated N-glycan profile MS spectra with a good signal to noise ratio over an m/z range of 1500–5000. N-glycan profiling of plasma from control and eIF2B mutated individuals resulted in highly reproducible spectra showing the specific profiles represented in We thus show that the differences in N-glycan profiles found in CSF from eIF2B-mutated patients as compared to control CSF are specific for this biological fluid and cannot be detected in corresponding plasma samples. We also did not find any other differences when comparing N-glycan profiles from mutated and control plasma.Using MALDI TOF MS-based analysis of permethylated glycans, we discovered an evolution of the control CSF glycome from N-glycan profiles rich in high mannose type structures to profiles with complex sialylated structures that could be correlated with postnatal brain maturation. We identified significant disease correlated differences in CSF but not in plasma N-glycan profiles of eIF2B-mutated patients suggesting for the first time a global change in N-glycosylation of CSF proteins in this pathology.The MALDI-TOF-based glycoprofiling method has been used previously to identify and quantify N-glycan structures in glycomic studies 3 1 (GlcNAc)1 in CSF, which might correspond to a degradation product of polysialic acid (PSA)-containing N-glycans. This type of glycans is notably found on glycoproteins, such as NCAM, that are highly implicated in the development and the formation of plasticity of the CNS The analysis of CSF control samples led to the discovery of a dynamic progression of CSF N-glycan profiles. Profiles I were found in control CSF from young patients, with age at sampling between 1.5 and 5 years, and may correspond in the brain to the achievement of the intense primitive myelinating phase associated with active synaptogenesis. Profiles II were found in control CSF with age at sampling between 2 and 50 years. An overlap between the two CSF glycan profiles was observed between 2 and 5 years, suggesting that inter-individual variations might occur during brain development and might lead to different delays in N-glycosylation dynamics. Increased sampling with CSF collected between 2 and 6 years and before 1 year of age would be useful to delineate this interphase more precisely. For this study it was not necessary to pool CSF samples from several donors as it was previously described for HPLC based glycoprofiling of CSF N-glycan profiles obtained from 12 eIF2B-mutated CSF samples corresponded exclusively to profile II and were therefore compared to profile II references. Patient samples showed a significant decrease of tri-antennary/bisecting structures and an increase in bi-antennary glycans. Within these global CSF N-glycan profiles the glycan structure at m/z 2081 decreases while the sialylated structure at m/z 2792 increases. Knowing that these structures are the main glycans found on transferrin from CSF, we can conclude that their quantitative variations between eIF2B-mutated and control CSF samples are in accordance with the findings of Vanderver and coll  concerning a decrease of the asialotransferrin/total transferrin ratio in eIF2B-mutated CSF The permethylated A complex monogalactosyl biantennary fucosylated glycan (at m/z 2040) increased with the pathology and appeared as potentially promising marker candidate. Knowing that this structure is a dominant N-glycan on IgG which are quantitatively important in biological fluids, we could suspect an increase in IgG in CSF of patients. Clinical presentation and CSF protein analyses do not support a supposed inflammation. A contamination of eIF2B-mutated CSF by plasma during lumbar puncture was also excluded as the ion at m/z 2431, second major N-glycan present in plasma, was stable (3.3±0.2%) in N-glycan profiles of patient and control CSF. The three other potentially promising N-glycan marker candidates are GlcNAc3 containing triantennary/bisecting type structures at m/z 2111, 2459 and 2489 . One eIFThe observed quantitative variations within CSF glycan profiles might be correlated to a down-regulation on the translational level of the expression of specific enzymes involved in glycan biosynthesis influenced by the mutation of translation initiation factor eIF2B. A decrease in the activity of beta-1.4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase III (GlcNAc-T III) which is responsible for the synthesis of bisecting glycans, might thus explain the observed reduced levels of triantennary/bisecting structures. Stanta et coll. (2010) described a comparable correlation between down-regulation of the MGAT3 gene encoding GlcNAc-T III and other genes on the transcriptional level and variations of the CSF glycome related to Schizophrenia. Based on our results, an association of this glycomic approach and the determination of GEF activity in patients' lymphoblasts can be used to screen patients with 100% specificity and 100% sensitivity before sequencing or not the five EIF2B genes. In a patient diagnostic setting, we propose to first measure the eIF2B GEF activity in lymphoblastoid cell lines from patients selected based on MRI and clinical symptoms to carry potentially eIF2B-related disorder. If the GEF activity is >77.5% in patients with a classic or moderate clinical form of eIF2B-related disorders, analyses of CSF glycome will be useful to validate or not the eIF2B-related disorders. In order to enhance clinical value of such glycan analyses, perspective of this work will be to analyse CSF glycan profiles from patients with other white matter disorders not linked to eIF2B mutations.Eif2b5 knock in (KI) mice model of the pathology reported a delay in brain WM development in vitro models or in patients' brains showed a defective glial maturation with a significant increase in premyelinating oligodendrocyte progenitor proliferation and a defect in the maturation of astrocytes Analysis of the recent Table S1Relative intensities for the 34 m/z corresponding to N-glycans for 24 individuals, the average for 12 control samples and 12 eIF2B-mutated samples with the ANOVA p values. In grey, the 4 structures with p values<0.001.(DOCX)Click here for additional data file.Figure S1MALDI-PSD fragmentation of selected glycan molecules. MS peaks corresponding to specific fragment ions are indicated within each of the three spectra and characteristic fragmentation patterns are annotated for each of the glycan structures. For figures B and C both bisecting and triantennary structures are annotated with fragment ions in the spectra potentially indicating the presence of both types of glycan structures within the corresponding mass peak. A) PSD fragmentation of the ion at m/z 1602 (NeuAc)3 1 (GlcNAc)1 indicates the presence of a linear glycan fragment probably originating from PSA glycans. B) Fragmentation of the ion at m/z 2081 (GlcNAc)3 (Fuc)1 (Man)3 (GlcNAc)2 and annotation of the corresponding triantennary and bisecting glycan structures. C) Fragmentation of the ion at m/z 2459 1 (GlcNAc)3 (Fuc)1 (Man)3 (GlcNAc)2 and annotation of the corresponding triantennary and bisecting structures showing the presence of the Lewis type antennary structure 1 (GlcNAc)1 (Fuc)1 represented by specific fragmentation ions B 660 and Y 1822.(DOCX)Click here for additional data file.


The mechanism by which nanoparticles cross the placental barrier was studied by using isotopic tracers. The abortion rates and other related data were counted and analysed in pregnant mice with different pregnancy times. Results showed that oxidised multi-walled carbon nanotubes (oMWCNTs) crossed the placental barrier and entered the foetus body. The abortion rates in the pregnant mice depended on pregnancy times. The abortion rates in the first-time, second-time and fourth-time pregnant mice were 70%, 40% and 50%, respectively. The maternal body weight gain was inhibited until gestational ages of 13, 10 and 11 d. oMWCNTs decreased the serum progesterone level and increased the serum oestradiol level in a dose- and time-dependent manner. However, this effect decreased with gestational age. The histology and vascular endothelial growth factor/reactive oxygen species content in the placenta showed that oMWCNTs narrowed the blood vessel and decreased the number of blood vessels in the placenta. They found that polystyrene particles with diameters of up to 240 nm can cross the placental barrier without affecting the viability of the placental explant. However, the toxic and damaging effects of nanoparticles on the mother and offspring remain unclear, respectively. Lim et al.et al.et al.et al. indicated that various types of malformation were observed in all MWCNT-treated groups given intraperitoneally to pregnant ICR mice, but not observed such malformation until the MWCNT dose high to 4 or 5 mg/kg body weight by given intratracheally521et al.A pregnant body or foetus is more sensitive to environmental toxins than adults. Many scholars have investigated the reproductive toxicity of nanomaterials181920In this work, we studied the mechanism by which CNTs cross the placental barrier by using radiotracers and determined the abortion rates caused by oxidised MWCNTs (oMWCNTs) in mice with different pregnancy times. We also investigated the maternal and foetal toxicity of oMWCNTs. The results of this study can help doctors alleviate the negative effects of nanomaterials and warn women who work or live in environments with heavy nanoparticles pollution to take safety measures during pregnancy.99mTc-oMWCNTs were determined by paper chromatography, with normal saline as the solvent  and Raman spectroscopy to further prove the reliability of the radiolabelling compounds. The oMWCNT structure is shown in The radiolabelling yields and stability of  solvent . The rad99mTc-oMWCNTs in the maternal and foetal bodies is shown in Figs. 299mTc-oMWCNTs were principally distributed in the lungs (up to 90% ID/g), followed by the liver, spleen and kidney  by determining abortion and fertility rates. The experimental results are shown in The effect of oMWCNTs on pregnancy was studied by exposing pregnant mice to oMWCNTs  at the gestational age of 14 d. p < 0.05 vs. control groups). This result indicated that oMWCNTs inhibited the secretion of progesterone after entering the placental system . Multiple exposure to a low dose  further increased the serum progesterone level than single heavy exposure . As shown in p < 0.05). The serum oestrogen level in the exposure groups injected once with a heavy dose was higher than that in the exposure groups injected multiple times with a low dose (*p < 0.05).As shown in The damaging effects of oMWCNTs on the placenta and foetus were also examined, and the results are shown in Figs. 11As shown in 99mTc-labelled oMWCNTs with diameters of 20 nm to 30 nm , they loaded the venous blood from the right atrium into the left ventricle, the middle part of which included the pulmonary capillary bed. Most oMWCNTs were captured by the pulmonary capillary bed and highly accumulated in the lungs at 1 h99mTc-oMWCNTs in the lungs were eliminated and entered the circulatory system with time, whereas some were absorbed by macrophage organs . As a result, high accumulation of 99mTc-oMWCNTs was observed in the spleen and liver  in the foetus was significantly lower at the gestational age of 14 d (99mTc-oMWCNTs 20 mg/kg.bw exposure) than at the gestational age of 18 d. This result suggests that the capability of 99mTc-oMWCNTs to cross the placental barrier improves with time. Enders and BlankenshipAfter the intravenous injection of nd liver . These rmembrane . After enjection . These nnjection . The livnjection . At 6 h njection . The foenjection . Possiblnjection . PlacentAs shown in 38et al.In the exposure groups, the development of dead foetus after abortion (20 mg/kg.bw oMWCNTs) was better in the second-time or fourth-time pregnant mice  than in in vivo. The average weight change before and after production is mainly due to the negative effect of oMWCNTs on fecundity. Small weight change before and after production suggests that more embryos or foetuses have been absorbed in the uterus before production or abortion. Specifically, oMWCNTs cause severe damage on pregnancy. As shown in in vivo. In addition, the high abortion rate of 70% occurred correspondingly in the early part of pregnancy of the first-time pregnant mice from the exposure groups  in environments with heavy pollutionmust be subjected to regular pregnancy tests and must be injected with appropriate amounts of progesterone or other hormones to protect their pregnancy.After exposure to pregnant mice through intravenous injection, oMWCNTs could cross the placental barrier into the foetus and mainly accumulate in the foetal liver, lungs and heart. oMWCNTs decreased progesterone levels and increased oestradiol levels in the serum. The effects of oMWCNTs on pregnancy depended on the exposure dose but weakened with gestational age. The damaging effects of oMWCNTs were more serious on first-time pregnant mice than on multiple pregnancy mice. oMWCNTs damaged placental dysfunctions, thereby delaying foetal growth and further harming the foetal heart and brain. This condition could lead to abortion. These results suggest that CNTs elicit severe toxicity to pregnant women. Proper safety precautions must be taken to prevent damages caused by carbon nanoparticle pollution during pregnancy.3 to remove the hemispherical caps of the nanotubes. The mixture of 3 g MWCNTs and 400 mL 3 mol/L HNO3 was ultrasonically stirred for 24 h. The suspension was filtered, rinsed with deionised water until the pH of the suspension reached approximately 6, and then dried at 80°C. The oMWCNTs were then characterised through TEM, Raman spectroscopy (LabRaM-HR-800) and Fourier-transform infrared spectroscopy (170SX). The results are shown in MWCNTs prepared by chemical vaporisation deposition were commercially obtained from Shenzhen Nanotech Port Co. Ltd., Guangdong, China. According to the product specification, as-received MWCNTs were determined by TEM to be 1 μm to 2 μm in length and 10 nm to 30 nm in diameter. Purity was > 96% (wt.%), containing < 3% amorphous carbon and < 0.2% ash. The as-grown MWCNTs  were added into the solution of 3 mol/L HNO99mTc labelling of oMWCNT and determination of labelling yields were performed  according to the method of Li et al.99mTc-oMWCNTs was formed. oMWCNTs were dissolved in deionised water with an ultrasonic device for 5 min. Ascorbic acid, stannous chloride and 99mTcO4− were then added into the suspension. This mixture was stirred at 90°C for 20 min. After centrifugation, the supernatant was decanted, and the remaining solid was identified to be 99mTc-oMWCNT. The radiolabelling yields and stability of 99mTc-oMWCNTs were measured by paper chromatography using Whatman 1 paper strips  (99mTc (6.02 h), we measured the radio counts in 1 d for accurate determination of the tag. Otherwise, the tag (99mTc) would decay in 10 half times.The ent, UK) . Considead libitum. All animal protocols were in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC) and approved by Institutional Animal Care and Use Committees of Gansu Province Medical Animal Center and Lanzhou University Animal Committees Guideline (China). When a mouse was up to sexual maturity (approximately 30 g), the cage  was closed at approximately 10 p.m. The pessary was then then detected to determine pregnancy at approximately 7 a.m. the next day. The pregnant mice were organised into five groups (five mice/group) on the gestational age of approximately 17 d. The body weights of the mice were determined before they were injected intravenously with 20 mg/kg.bw of 99mTc-oMWCNTs solution . The mice were killed at 1, 2, 6, 16 and 24 h after injection. Tissues from the heart, lungs, liver, spleen, kidney, stomach and foetus were immediately dissected. The maternal blood and the heart, lung and liver of the foetus were also collected. Each tissue or organ was wrapped in foil, weighed and counted for 99mTc. Data were corrected for physical decay of radioactivity. The distribution in the tissues was presented in percent injected dose per gram of wet tissue (%ID/g), which could be calculated by the percent injected dose  per gram of the wet tissue5899mTc labelling of oMWCNTs.Kunming mice  initially weighing 15 g to 18 g were provided by the Laboratory Centre for Medical Science, Lanzhou University, Gansu, China. All animals were housed in individual cages in a temperature-controlled (21°C to 22°C) and light-controlled (turned on from 08:00 h to 20:00 h) environment and were fed food and tap water CNaCl = 0.9%, 0.2 mL) was administered to the control group (10 mice). The daily maternal body changes were recorded, and the status of the pregnant mice was observed. Genital tract bleeding during pregnancy marked the occurrence of abortion. A 1 mL blood sample was collected by anaesthetising the mice after abortion or parturition. The blood samples were kept in the refrigerator at 4°C for 24 h and then centrifuged at 3000 rpm/min to obtain the serum. The serum contents of progesterone and oestradiol were measured. It provided a similar dispose to the second-time pregnant and fourth-time pregnant mice as that of the first-time pregnant mice groups. The effects of oMWCNTs on the pregnant mice with different pregnancy times were then studied.The first-time pregnant mice were injected with 0.2 mL to 0.3 mL of oMWCNTs (20 mg/kg.bw) at the gestational age of 7 d until abortion or parturition to serve as the experimental group (10 mice). Saline solution  was intravenously injected to the experimental pregnant mice at the gestational ages of 4, 11 and 15 d every morning. Approximately 0.2 mL of saline solution was injected to the mice from the control groups (five to six mice/group). All mice were then killed at the gestational ages of 7, 14 and 18 d. Approximately 1 mL of maternal blood was collected. The blood samples were kept in the refrigerator at 4°C for 24 h and then centrifuged at 3000 rpm to obtain the serum. Afterward, the author studied the effect of oMWCNTs on the different gestational ages of pregnant mice by measuring the progesterone and oestradiol contents in maternal serum. The experimental groups (five to six mice/group) were exposed to 4, 20 and 30 mg/kg.bw of oMWCNTs (injection volume of 0.2 mL to 0.3 mL) at the gestational age of 11 d. Approximately 0.2 mL of saline solution was administered to the control groups, and approximately 1 mL of blood samples was obtained from all mice. The blood samples were processed as previously mentioned to investigate the dose of oMWCNTs that affected the pregnant mice. One group of pregnant mice (five mice) was exposed to 20 mg/kg.bw equivalent oMWCNTs at the gestational age of 13 d and to saline solution on the following days. Another group was injected with approximately 0.2 mL of oMWCNTs (4 mg/kg.bw) for five consecutive days . The same volume of saline solution was administered to the mice from the control groups. Approximately 1 mL of blood sample was collected at the gestational age of 18 d and processes as previously described to determine the effects of the exposure time of oMWCNTs on pregnant mice.VEGF and ROS ELISA kits were purchased from Shanghai Heng Hitter Trade Co., Ltd. and then kept in the refrigerator at 4°C. Approximately 0.2 mL of oMWCNTs (20 mg/kg.bw) was injected intravenously to the experimental pregnant mice at the gestational ages of 9 d to 11 d every morning for 3 d (five to six mice). Approximately 0.2 mL of saline solution was administered to the mice from the control groups. All mice were then killed. Approximately 1 mL of maternal blood with anti-caking agent was collected to obtain the plasma. The plasma contents of ROS and VEGF were then measured. At the same time, the placenta, liver, lung, spleen and foetus were harvested immediately. These tissues were fixed in 10% buffered formalin and processed for routine histology with haematoxylin and eosin staining by the Centre for Medical Science, Lanzhou University . Microscopic observation of tissues was performed with an Olympus Microphot-CX41 microscope coupled with a digital camera. The placental homogenate was made from part of the placenta tissues to investigate the ROS and VEGF contents.SD of multiple determinations. Statistical significance in the difference was evaluated by analysis of variance (ANOVA) or Kruskal-Wails-test method after One-sample Kplmogorov-Smirnov Npar-test and Homogeneity of Variances test, and all statistical calculations were carried out using SPSS software.Data are reported as mean values +Q.W. and L.Z. wrote the main paper; Q.W., B.J., W.J. and L.P. carried out the experiments; Q.W. prepared all the figures and tables; Z.X., W.J. and W.W. revised the language of the paper; and all authors reviewed the paper.Supplementary Information


Amyloid accumulation in the brain parenchyma is a hallmark of Alzheimer's disease (AD) and is seen in normal aging. Alterations in cerebrospinal fluid (CSF) dynamics are also associated with normal aging and AD. This study analyzed CSF volume, production and turnover rate in relation to amyloid-beta peptide (Aβ) accumulation in the aging rat brain.Aging Fischer 344/Brown-Norway hybrid rats at 3, 12, 20, and 30 months were studied. CSF production was measured by ventriculo-cisternal perfusion with blue dextran in artificial CSF; CSF volume by MRI; and CSF turnover rate by dividing the CSF production rate by the volume of the CSF space. Aβ40 and Aβ42 concentrations in the cortex and hippocampus were measured by ELISA.p < 0.01); 3-30 months (p < 0.001). CSF production rate increased from 3-12 months (p < 0.01) and decreased from 12-30 months (p < 0.05). CSF turnover showed an initial increase from 3 months (9.40 day-1) to 12 months (11.30 day-1) and then a decrease to 20 months (10.23 day-1) and 30 months (6.62 day-1). Aβ40 and Aβ42 concentrations in brain increased from 3-30 months (p < 0.001). Both Aβ42 and Aβ40 concentrations approached a steady state level by 30 months.There was a significant linear increase in total cranial CSF volume with age: 3-20 months (In young rats there is no correlation between CSF turnover and Aβ brain concentrations. After 12 months, CSF turnover decreases as brain Aβ continues to accumulate. This decrease in CSF turnover rate may be one of several clearance pathway alterations that influence age-related accumulation of brain amyloid. The cerebrospinal fluid (CSF) circulation is critical to maintaining a healthy environment for the brain, and its functional decline with normal aging and the age-related dementias remains of particular interest and concern. For example, there is decreased CSF production and turnover, diminished clearance of proteins, peptides and other potentially toxic metabolites, altered ion and solute transport, and decreased resistance to oxidative stress -3. CSF iReduced CSF turnover may have consequences for clearance of many toxic peptides, proteins, and other metabolites. Recently, it was found that albumin (a soluble CSF protein) levels are elevated in elderly sheep associated with decreased CSF production and turnover . AlbuminEnlarged cerebral ventricles and decreased CSF turnover are also associated with decreased CSF Aβ concentrations in non-AD patients . In sevea separate group from those used to measure both CSF volume (n = 16) and Aβ concentrations (n = 31). The rats were housed in the Central Animal Facility at Rhode Island Hospital and had food and water ad libitum. The Institutional Animal Care and Use Committee (IACUC) at Rhode Island Hospital approved all experiments. Rats were euthanized by intra-peritoneal (i.p.) pentobarbital (125 mg/kg) after the acute experiments were completed. Rats used for ELISA measurements were perfused with phosphate-buffered saline (PBS) via left ventricular cannulation prior to removing the brain.Male F344/BN hybrid rats aged 3, 12, 20, and 30 months were examined. F344/BN rats were chosen because they are long-lived and available at most ages up to 30 months from the National Institute of Aging colony. Rats used to measure CSF production (n = 24) were 3; TR = 2700 ms; TE = 3.89 ms; inversion time = 900 ms; flip angle = 9 degrees; acquisition time = 12 min 38 s) for a total of 8 acquisitions. All acquisitions were aligned to the original image and averaged using the Analysis of Functional Neuroimages (AFNI) software package [Total cranial and ventricular CSF volumes were measured using a Siemens 3T TIM Trio MR scanner. The scanner was equipped with an AC88 high performance gradient insert and a 4.5 cm custom volume transmit-receive coil. Rats were imaged under continuous 1.8-2.2% isoflurane anesthesia. T1-weighted MPRAGE data were acquired /Co], where V represents the rate of CSF flow, C the concentration of dye, and the subscripts f, i, and o represent formation, inflow, and outflow [-1.CSF production was calculated as a function of dye-dilution using ventriculo-cisternal (V-C) perfusion with Blue Dextran 2000 dye . Complete methodological details have been previously described . Briefly outflow . CSF turThe fronto-parietal cortex plus the complete hippocampal formation was excised from the rat brains post-perfusion with PBS. These samples were then thoroughly ground into a powder over a bed of liquid nitrogen, and 250 mg of that powder was taken for the homogenization and protein extraction. The concentration of Aβ40 and Aβ42 in cortical and hippocampal rat brain samples was measured using a sandwich ELISA method. Details can be found in our previous publication . High-sep < 0.05.All statistics were conducted using GraphPad Prism v4.01 . Data values for CSF volume and CSF production were first subject to analysis of variance (ANOVA) and then to Bonferroni's multiple comparison test. CSF turnover was calculated for each age group from the means of the CSF production values and the CSF volume measurements. Significance was set at p < 0.001, r2 = 0.72 , 3 and 30 months (p < 0.001), and 12 and 30 months (p < 0.05). Bonferroni's method was used to adjust the level of significance. Figure Ventricular CSF volume  increased linearly with age, 2 Figure . The meap < 0.001, r2 = 0.99 , 3 and 30 months (p < 0.001), 12 and 30 months (p < 0.0001), and 20 and 30 months (p < 0.05). Bonferroni's method was used to adjust the level of significance. Figure Total cranial CSF volume also increased linearly with age, 9 Figure . The meap < 0.0008  and decreased between 12 and 30 months (p < 0.05). There was no significant difference between 3 and 30 month, 3 and 20 month, and 12 and 20 month production rates (p > 0.05), although a continuous decreasing trend is apparent from 12 to 30 months. CSF turnover increased initially between 3 and 12 months (9.41 day-1 to 11.3 day-1) and then decreased to 20 months (10.23 day-1) and further to 30 months (6.62 day-1)  Figure .p < 0.001). The mean ± SEM value for Aβ42 in rats at 3 months was 1.65 ± 0.19 pg/mg total protein (tp) (n = 8), at 12 months 4.94 ± 1.11 pg/mg tp (n = 8), at 20 months 7.79 ± 0.57 pg/mg tp (n = 8), and at 30 months 8.67 ± 0.13 pg/mg tp (n = 7). ANOVA with Bonferroni's multiple comparison adjustment revealed a significant difference between 3 and 12 months (p < 0.01), 3 and 20 months (p < 0.001), 3 and 30 months (p < 0.001), 12 and 20 months (p < 0.05), and 12 and 30 months (p < 0.01). The mean ± SEM value for Aβ40 in 3 months old rats was 3.25 ± 0.23 pg/mg tp (n = 8), at 12 months 4.79 ± 0.39 pg/mg tp (n = 8), at 20 months 5.58 ± 0.23 pg/mg tp (n = 8), and at 30 months 5.66 ± 0.32 pg/mg tp (n = 7). There was a significant difference between 3 and 12 months (p < 0.01), 3 and 20 months (p < 0.001), and 3 and 30 months (p < 0.001). Aβ42 and Aβ40 concentration increased rapidly between 3 and 12 months, followed by a decreased rate of rise in Aβ40 between 12 and 30 months, approaching a steady state between 20 and 30 months. Aβ42 continued to rise to 20 months and then the rate of increase slowed to 30 months. There was a reversal of the Aβ40/Aβ42 ratio in brain parenchyma with advancing age. Figure The concentrations of parenchymal Aβ42 and Aβ40 in specimens of cerebral cortex plus hippocampus increased significantly with age  CSF bulk flow and turnover.Determining the causes of Aβ accumulation in the brain with aging may be crucial to the development of effective AD therapies. Aβ accumulates as a result of an imbalance between production and clearance . StudiesPrevious reports have shown Aβ clearance pathway alterations with age. There is a significant decrease in Aβ efflux transporter expression with age and an increase in influx transporter expression at the BBB ,36. InteCSF turnover is a modulator of CSF and brain protein concentrations ,10. AlthThe procedure for measuring CSF production, V-C perfusion, is extremely invasive, and the rats experienced high mortality rates, particularly in the elderly. However, the number of rats at 3 and 12 months was sufficient for showing a significant rise in CSF production, and the decrease from 12 to 30 months fits with existing studies ,10. In aThere is an increase in CSF production and turnover from 3 to 12 months in this rat aging model at a time when Aβ concentrations increase, showing that before 12 months of age there is no relationship between amyloid deposition in the brain and CSF production and turnover alterations. CSF turnover is inversely related to brain Aβ accumulation in elderly rats. The decreasing CSF turnover rate in older rats suggests a possible therapeutic target to improve CSF solute clearance by shunt insertion in the aging F344/BN rat.Aβ: amyloid-beta peptide; Aβ40: 40 amino acid Aβ; Aβ42: 42 amino acid Aβ; AD: Alzheimer's disease; ANOVA: analysis of variance; BBB: blood-brain barrier; BCSFB: blood-CSF barrier; CP: choroid plexus; CSF: cerebrospinal fluid; ELISA: enzyme-linked immunosorbent assay; F344/BN: Fischer 344/Brown-Norway hybrid rat; IACUC: Institutional animal care and use committee; i.p: intra-peritoneal; MRI: magnetic resonance imaging; AFNI: Analysis of Functional Neuroimages; NIfTI: Neuroimaging Informatics Technology Initiative; 3D: 3-dimensional; PBS: phosphate-buffered saline; SEM: standard error of the mean; tp: total protein; V-C: ventriculo-cisternal.There are no actual or potential competing interests (conflicts of interest) to disclose for any of the authors or their families.CC wrote the first draft of the manuscript, did the MRI volume measurements, and did the CSF turnover calculations. MCM did the ELISA measurements for Aβ40 and Aβ42. INC measured the CSF production rates by V-C perfusion. MW did the MRI scanning and contributed the CSF space software. TB analyzed the data statistically and edited the manuscript. ZNG helped develop the V-C perfusion technique in the lab. CEJ co-directed the research and critically read the manuscript. GDS conceived the research plan, directed the research and edited the manuscript. All authors have read and approved the final version of the manuscript.


Decisions regarding choice and dose of anti-epileptic drug (AED) are driven by considering the potential benefits of reducing seizure frequency against the potential harms of alternative AEDs. Such decisions should be made using the best available evidence, which often requires a quantitative synthesis of data from multiple randomised controlled trials (RCT). However, the systematic review and meta-analysis of harms data is hindered by problems such as inadequate reporting, heterogeneity of harms definitions, and selective reporting bias. Here we will evaluate the quality of reporting of harms data in epilepsy trials, and assess the potential added value of incorporating harms data beyond the clinical indication of epilepsy.To evaluate the quality of reporting of harms data in RCTs of AEDs in patients with epilepsy we have undertaken a systematic review . We searFor the reporting quality review we identified 152 RCTs that met the eligibility criteria. None of the trials satisfied all criteria. The mean number of criteria per trial was 11.3 . No improvement could be detected following publication of the CONSORT statement for harms (difference in means: 0.6 with 95% CI (-0.9 to 1.8) p=0.53). Items that were not frequently reported were; definition of adverse events , use of a validated dictionary , use of a validated instrument , reporting of both number of patients and number of adverse events  and methods for handling of recurrent events . In the summary of harms data, borrowing strength from other indications resulted in a more precise effect estimate, and indicate that there is evidence for some adverse events across the range of indications.Reporting of harms in RCTs of AEDs is poor and has not improved since the publication of the CONSORT guidelines on the reporting of harms. To allow reliable meta-analyses of harms data, improvements to reporting quality are essential. Preliminary results suggest that harms data from AEDs prescribed for headache and neuropathy may be useful to inform the harms profile of AEDs prescribed for epilepsy.


Instead, the study revealed an inverse correlation between inappK in left ventral striatum and alcohol craving scores. To resolve the influx and efflux phases of radiolabeled molecules, we independently estimated the unidirectional blood-brain FDOPA clearance rate (K) and the washout rate of [18F]fluorodopamine and its deaminated metabolites (kloss), and we also calculated the total distribution volume of decarboxylated metabolites and unmetabolized FDOPA as a steady-state index of the dopamine storage capacity (dV) in brain. The craving scores in the 12 alcoholics correlated positively with the rate of loss (lossk) in the left ventral striatum. We conclude that craving is most pronounced in the individuals with relatively rapid dopamine turnover in the left ventral striatum. The blood-brain clearance rate (K), corrected for subsequent loss of radiolabeled molecules from brain, was completely normal throughout the brain of the alcoholics, in whom the volume of distribution (dV) was found to be significantly lower in the left caudate nucleus. The magnitude of dV in the left caudate head was reduced by 43% relative to the 16 controls, consistent with a 58% increase of lossk. We interpret the findings as indicating that a trait for rapid dopamine turnover in the ventral striatum subserves craving and reward-dependence, leading to an acquired state of increased dopamine turnover in the dorsal striatum of detoxified alcoholic patients.A previous study of the DOPA decarboxylase substrate 6-[ The ratio between the enzymatic clearance of FDOPA (K) and the rate of loss (lossk) constitutes a steady-state volume of distribution for the decarboxylated and deaminated metabolites, which reflects the total distribution volume in brain , an index of dopamine storage capacity, as detailed in the earlier publications The measure of the net blood-brain transfer rate of FDOPA obtained in these studies of alcoholic patients (Kinapp) and alcohol craving scores 18F]fluoro-dopamine formed in ventral striatum and subsequent washout of the breakdown products 18F]fluorodopamine washout (lossk) in the left ventral striatum would correlate with the severity of craving in the group of alcoholics, as previously noted for the net blood-brain transfer (Kinapp), and second, that the analysis would reveal increased lossk throughout striatum of detoxified alcoholic patients, relative to an age-matched control group.We predicted that the previous finding of an inverse correlation between the net blood-brain transfer . The control group consisted of the 12 volunteers from the earlier study The mean (± SD) alcohol lifetime alcohol consumption of the patients was 1038±828 kg, and they smoked a mean (± SD) of 17±13 cigarettes per day, yielding a weak trend towards correlation between individual alcohol and cigarette consumption . The lifetime alcohol intake was measured with the Lifetime Drinking History questionnaire 18F]fluoro-L-DOPA (OMFD) were determined in selected samples by reverse-phase high performance liquid chromatography Subjects fasted overnight, and received carbidopa  one hour prior to the PET scan in order to minimize the decarboxylation of FDOPA in peripheral tissues In the present study, dynamic PET sequences were realigned and corrected frame-wise for head motion, and then summed and registered to the Montreal Neurological Institute (MNI) stereotaxic brain, as described previously 1-k2K neurokinetic model. The OMFD curve was regenerated mathematically for subtraction of OMFD contamination. This OMFD subtraction method permits the two hour tomography, which is sufficient for subsequent parameter estimation of the present study. Following this mathematical subtraction of brain OMFD, FDOPA kinetics in brain simplifies to an “inlet-outlet” model K, ml g−1 min−1), and the fractional rate constant for the process of elimination of [18F]fluorodopamine together with its deaminated metabolites from brain . The ratio (K/lossk) is analogous to an effective distribution volume (EDV ml g−1), as defined in dV, ml g−1) equals the sum of the FDOPA plasma volume , the distribution volume of unmetabolized FDOPA in brain , and the decarboxylated metabolite pool, consisting of [18F]fluorodopamine and its deaminated metabolites . In the common MNI stereotaxic space after anatomical standardization, mean dV maps were calculated for the two groups, and difference maps were calculated by subtraction. Voxel-wise statistical analysis was performed using the general linear model (GLM), in order to search for cluster volumes and partial out the effects of smoking rates. Group differences and voxel-wise regression of dV with ACQ scores for the 12 patients were calculated using the GLM implemented in Glim Image, a program developed at the MNI. Then, cluster VOIs were defined directly from the raw t-maps, where the t-value threshold was set arbitrary to form a cluster (t>2.5–2.8), but as high as possible to avoid false positive voxels. The cluster VOIs identified in the dV voxelwise contrast and regression analyses were applied to the OMFD-subtracted dynamic 4D recordings for TAC extraction, and the individual magnitudes of K, lossk and dV were calculated separately from the TACs. The kinetic estimates obtained from the cluster VOIs were tested using the GLM analysis for the contrast between alcoholic and control groups as well as the regression with ACQ scores, treating individual smoking rates as a nuisance variable.To search for brain regions with perturbed [18F]fluorodopamine storage capacity, as revealed by both the mean parametric dV map  and the mean magnitude of dV was reduced by 43% (P = 0.002)  ; there wdV as a function of the ACQ scores revealed a cluster (t >2.5) with inverse correlation in the left ventral striatum  revealed no difference from an age-matched control group, but a highly significant negative correlation between estimates of the net transfer rate (inappK) in the left ventral striatum and individual ACQ scores. In the present study, the regression analysis with the ACQ scores of the alcoholic patients, employing the concurrent and separate estimation of the unidirectional blood-brain clearance (K) and the metabolite washout rate (lossk), revealed a positive correlation with lossk and a negative correlation with dV, but no correlation with the magnitude of the clearance (K) in left ventral striatum, in which no significant group differences of the three kinetic estimates were detected. We conclude that the earlier finding of a negative correlation between inappK and craving reflects the underestimation of the unidirectional clearance of FDOPA (K) in relation to the magnitude of washout of [18F]fluorodopamine and its deaminated metabolites (lossk), which we determined separately from the 120 min PET recordings in the present analysis.Two previous PET studies of dopamine synthesis capacity with FDOPA in alcoholic patients yielded partially disparate results dV and high estimates of lossk in left ventral striatum, predicting a relatively small dopamine pool, characterized by rapid turnover. In functional anatomic terms, the convergence within the ventral striatum of glutamatergic inputs from limbic structures and the dopamine innervation arising in the ventral tegmental region of the mesencephalon is thought to subserve gating of affective signaling from limbic afferents. Given the role of dopamine in incentive salience 18F]fluorodopamine pool and its increased washout in ventral striatum of alcoholic patients may serve to link craving with disinhibition of dopamine turnover.Those patients who experienced the highest craving had relatively low estimates of 11C]raclopride studies of post-synaptic D2/3 receptors revealed reduced binding in striatum of acutely withdrawn alcoholic patients, as compared to healthy control subjects 18F]fallypride 18F]desmethoxyfallypride ([18F]DMFP) 123I]IBZM 123I]-epidepride 11C]dihydrotetrabenazine to the neuronal vesicular monoamine transporter (VMAT2) was reduced in caudate and putamen of seven males with severe chronic alcohol abuse 11C]-methylphenidate found no such reduction 11C]-raclopride binding to psychostimulant challenge in alcoholic patients Chronic alcohol abuse is likely to alter the integrity of dopaminergic pathways in human brain. Indeed, a number of fluorodopamine together with its deaminated metabolites. These results in alcoholic patients stand in clear distinction to our earlier findings of increased K and decreased dV estimates in patients with schizophrenia, a circumstance which we termed “poverty in the midst of plenty” dV in left caudate nucleus of the present alcoholic group exceeds that seen in patients with early Parkinson’s disease The present ion maps  and the ic group , in the dV in the caudate nucleus of abstinent alcoholics, since it may reflect focal degeneration, increased dopamine signaling, or conversely “futile” [18F]fluorodopamine synthesis in a cellular compartment containing monoamine oxidase. However, given the preserved estimates of K, the present findings are more consistent with altered functional state of dopamine innervations, rather than an actual degenerative process. The caudate nucleus is implicated in cognitive performance pertaining to selection of actions and evaluation of outcomes 18F]fluoro-meta-tyrosine As noted in the introduction, there is no simple physiological interpretation of decreased 11C]raclopride studies of healthy pigs, we noted greater nicotine-evoked binding reductions on the left side 11C]raclopride binding changes on the left side 11C]raclopride binding changes on the right ventral striatum of healthy males Significant findings of the present study were asymmetric, as noted in earlier PET studies. For example, in [18F]-fluorodopamine turnover in the alcoholic group. However, the putative transition from a vulnerability trait mediated by greater dopamine turnover in the ventral striatum to an acquired state of elevated dopamine turnover in the dorsal striatum might best be investigated in prospective molecular imaging studies of individuals at risk for alcoholism.There is a significant correlation between sensation-seeking propensity and differences of extracellular dopamine and dopamine receptors in the striatum inappK in the left caudate of two patients In general, comparisons between molecular imaging studies of alcoholism are difficult because of factors such as age, smoking, and other co-morbidities, cumulative alcohol intake, and duration of abstinence. This may explain the neurochemical heterogeneity of type I alcoholics noted in one of the previous PET studies with FDOPA, which reported reduced


Macrophages are terminally differentiated cells of the mononuclear phagocytic lineage and develop under the stimulus of their primary growth and differentiation factor, CSF-1. Although they differentiate into heterogeneous populations, depending upon their tissue of residence, motility is an important aspect of their function. To facilitate their migration through tissues, macrophages express a unique range of adhesion and cytoskeletal proteins. Notably, macrophages do not form large, stable adhesions or actin stress fibers but rely on small, short lived point contacts, focal complexes and podosomes for traction. Thus, macrophages are built to respond rapidly to migratory stimuli. As well as triggering growth and differentiation, CSF-1 is also a chemokine that regulates macrophage migration via activation the CSF-1 receptor tyrosine kinase. CSF-1R autophosphorylation of several intracellular tyrosine residues leads to association and activation of many downstream signaling molecules. However, phosphorylation of just one residue, Y721, mediates association of PI3K with the receptor to activate the major motility signaling pathways in macrophages. Dissection of these pathways will identify drug targets for the inhibition of diseases in which macrophages contribute to adverse outcomes. Despite their heterogeneity, macrophages all originate from the pluripotent hematopoietic stem cell and, under the influence of hematopoietic growth factors, differentiate through several multipotent progenitor stages to lineage committed mononuclear phagocytic precursors in the bone marrow–3. The m defects –9. CSF-1 defects , to trig (IL-34) –13.op/Csf1opCsf1 mouse and their reconstitution upon restoration of CSF-1 expression indicates that CSF-1 regulates the differentiation and migration of trophic and/or scavenger macrophages that are physiologically important for normal development and tissue homeostasis rather than in immune function [Macrophages are professionally motile cells that carry out a variety of roles in immune surveillance and normal tissue development by secreting cytokines and growth factors and phagocytosing foreign material and apoptotic cells. Transendothelial and interstitial motility is an essential aspect of their function as they must be able to move to specific sites upon demand. From studies in primary macrophages and CSF-1 dependent macrophage cell lines, it is evident that CSF-1 is not only a mononuclear phagocyte lineage growth factor but is an important regulator of macrophage motility –16. Deplfunction , 11, 17.function . Thus, Cfunction , 18, 19.function . To partfunction . Yet litfunction , 18, 19,μm/min [μm/min in vitro, in vivo they respond rapidly to wounding or inflammatory signals and can migrate over considerable distances. Indeed, their migration speed has been measured at over 10 μm/min when attracted into a wound in a fish model [μm/min) [Almost all cell types are capable of migration but, in the adult organism, motility is particularly important for cells participating in immune cell function and wound healing. Leukocytes move rapidly compared to other cells, with neutrophils and lymphocytes measured at speeds of up to 25–30 μm/min , 22. Whish model . Compare[μm/min) , macrophDictyostelium discoides [The fundamental locomotory mechanisms are broadly similar in most cell types , 25. Motiscoides . The difiscoides , 22. Indiscoides , 31. Coniscoides , 33. Moriscoides .in vitro of the actin cytoskeleton and adhesion structures in macrophages and fibroblasts indicates important mechanistic differences in the motility machinery between the two cell types (μM) that anchor thick actin bundles or stress fibers  family. Upon binding of homodimeric CSF-1, the CSF-1R dimerizes, becomes activated and autophosphorylates at least 7 of its 20 intracellular tyrosine residues . To ident CSF-1R , 77. Thet CSF-1R  and thatt CSF-1R . Individγ2 [in vitro and, perhaps more importantly, these macrophages moved less well in vivo and there was a significant reduction in their capacity to enhance tumour cell invasion in vitro [γ2, was responsible for initiating pY721-based motility signaling. While PLCγ2 associated with the activated CSF-1R in a pY721-independent manner, CSF-1 rapidly stimulated a prolonged Y721-dependent association of PI3K with the receptor, which resulted in PIP3 production [Macrophages elongate when they polarize and begin to move , 79 so aγ2 –83. A dein vitro . Underlyin vitro . This syoduction . Thus th721VEM in the CSF-1R [3) from PI 4, 5-bisphosphate (PIP2) at the cell membrane [3 at the leading edge stimulates migration by inducing plasma membrane translocation of pleckstrin homology (PH) domain-containing molecules, PDK1, Akt, and Rho family GTPase regulators, or molecules with other PIP3 binding motifs such as WASP and its homologues [3 levels are rapidly returned to baseline levels by the phospholipid phosphatase, PTEN [α and β are ubiquitously expressed, p110δ expression is highly enriched in hematopoietic cells, including macrophages. The three PI3K p110 isoforms have nonredundant biological roles and their function differs between primary and immortalised macrophages such that while p110δ is the main isoform recruited to the CSF-1R in bone marrow-derived macrophages, all three are recruited to the receptor in BAC1.2F5 macrophages [δ appears to be the main regulator of migration in both primary macrophages and BAC1.2F5 cells, in which it triggers actin polymerization, cytoskeletal remodeling, and cell adhesion [3 mediates these disparate effects of CSF-1 stimulation have yet to be identified, but regulation of individual elements further downstream in macrophage migration are becoming clearer and appear to converge on the Rho family GTPases.Class IA PI3Ks consist of a p110 catalytic subunit bound to a p85 regulatory subunit that translocates to activated RTKs upon interaction of its SH2 domains with pYXXM motifs, including Ye CSF-1R , 84. Upomologues , 87. RTK−/− macrophages did not form ruffles, normal lamellipodia, or podosomes [in vivo [−/− and RhoA−/− macrophages as well as FRET studies should prove illuminating. Nevertheless, further mechanistic insights into Rho family GTPase regulation of macrophage motility have been gained through examination of their main downstream effectors for actin polymerization, the WASP and WAVE complexes [Rho family GTPases are well-known regulators of actin polymerization and cell adhesion downstream of RTKs in many different cell types . The maiodosomes . Moreove[in vivo , 97. In [in vivo , 94. Int[in vivo . Thus, tomplexes , 98.−/− dendritic cells also fail to form podosomes, instead forming longer lived focal contacts [−/− macrophages were more motile towards CSF-1 in vitro and responded to an inflammatory stimulus more readily in vivo [−/− macrophages demonstrated increased adhesion on fibronectin and increased CSF-1-stimulated F-actin levels in association with increased PIP3 levels. ROCK1 was shown to negatively regulate CSF-1-induced migration through regulation of PTEN activity [In macrophages, Cdc42 and Rac stimulate actin polymerization by activating the Arp2/3 complex through their GTP-dependent association with the Arp2/3 activating scaffold proteins, WASP and WAVE, respectively , 99. WAScontacts . The WAVcontacts , 98. WAVcontacts . Thus, b in vivo . Underlyactivity . Rho alsactivity .in vitro and in vivo, apparently via the same pathway, as loss of Pyk2 does not further reduce migration and invasion in FAK−/− macrophages [−/− macrophages demonstrated high levels of Rac activity in association with hyperprotrusiveness [−/− macrophages showed reduced integrin-mediated Rho activation [The specific roles of individual Rho family proteins in the regulation of macrophage adhesion is less well understood than their roles in actin polymerization and actomyosin contractility and is made more complex by the fact that there is crosstalk between actin polymerization, actomyosin contractility, and adhesion formation and turnover , 104. Nerophages , 72. Thesiveness  while Pytivation . Moreovetivation , 47. Thutivation , 104.γ receptor-mediated phagocytosis in RAW264.7 macrophages reveal distinct spatiotemporally regulated patterns of Rac1, Rac2, and Cdc42 activation underlying actin polymerization in the phagocytic cup [γ receptor-mediated phagocytosis in macrophages [An area where the role of individual Rho GTPases has been more clearly defined in macrophages is phagocytosis, which is a highly ordered process of membrane protrusion and actin polymerization that uses many of the same elements of cellular machinery as locomotion . FRET stytic cup . In addiytic cup , is recrytic cup . Rho GTProphages . Further−/−), when transduced with individual wild-type or tyrosine mutant CSF-1Rs, allows examination of specific signaling pathways triggered by individual tyrosine residues in mature macrophages [It is clear that the interplay between the different elements of the adhesion and motility apparatus, coordinated in large part by Rho family GTPases, is complex and will require the use of many different approaches to unpack these complexities. Mature macrophages have proven difficult to adapt to some approaches as, not only do they selectively express a number of important adhesion and motility proteins but they are difficult to transfect . Neverthrophages . Using trophages . Furtherrophages , and wor


An association between testosterone therapy (TT) and cardiovascular disease has been reported and TT use is increasing rapidly.We conducted a cohort study of the risk of acute non-fatal myocardial infarction (MI) following an initial TT prescription  in a large health-care database. We compared the incidence rate of MI in the 90 days following the initial prescription  with the rate in the one year prior to the initial prescription  (post/pre). We also compared post/pre rates in a cohort of men prescribed phosphodiesterase type 5 inhibitors , and compared TT prescription post/pre rates with the PDE5I post/pre rates, adjusting for potential confounders using doubly robust estimation.trend = 0.03), while no trend was seen for PDE5I (ptrend = 0.18). In men under age 65 years, excess risk was confined to those with a prior history of heart disease, with RRs of 2.90  for TT prescription and 1.40  for PDE5I, and a RRR of 2.07 .In all subjects, the post/pre-prescription rate ratio (RR) for TT prescription was 1.36 . In men aged 65 years and older, the RR was 2.19  for TT prescription and 1.15  for PDE5I, and the ratio of the rate ratios (RRR) for TT prescription relative to PDE5I was 1.90 . The RR for TT prescription increased with age from 0.95  for men under age 55 years to 3.43  for those aged ≥75 years (pIn older men, and in younger men with pre-existing diagnosed heart disease, the risk of MI following initiation of TT prescription is substantially increased. Testosterone therapy (TT) has been used in healthy older men to treat diminished extremity strength and physical function associated an age-related decline in serum testosterone. The Truven Health MarketScan® Commercial Claims and Encounters Database includes employees, dependents and retirees with commercial or Medicare insurance whose employers license healthcare data to Truven Health Analytics (Truven). The MarketScan data contributors include Fortune 500 employers (60 percent) and health plans covering numerous other companies and unions (40 percent). The data include diagnoses, procedures, and prescriptions for all enrollees. Enrollees in 2011 are distributed regionally within the U.S. as Northeast (24 percent), North Central (37 percent), South (20 percent), and West (19 percent). We used the data from 2006 to 2010, including patient-specific enrollment history, year of birth, gender, inpatient and outpatient diagnoses (ICD-9 codes) and treatments, and outpatient prescriptions. The data for the study were hosted in secure facilities of Consolidated Research, Inc. (CRI), as required by the Agreement between CRI  and Truven Health Analytics . We received IRB exemption for this study from the NIH Office of Human Subjects Research Protections (OHSRP) since the study was conducted in claims data that were anonymized or de-identified by Truven prior to release. The database is available from Truven under licensing agreements similar to ours.We formed cohorts from the database from men with a minimum of 22 months of continuous enrollment for analyses with post-prescription follow-up intervals of 90 days, and 25 months for analyses with post prescription follow-up intervals of 91 to 180 days. From this cohort, we selected men who filled a first prescription for any of several TT prescriptions not containing estrogen  and, for a comparison population, men who filled a first prescription for phosphodiesterase type 5 inhibitors , between January 1, 2008 and September 30, 2010. We did not have data on how much of the prescribed medication was consumed. The most common TT prescriptions were testosterone gel, testosterone micronized, testosterone cypionate, and testosterone transdermal system. We selected men receiving PDE5I prescriptions as a comparison group because some indications for prescription are similar to those for TT prescription. In addition, PDE5I is commonly prescribed to older men, does not have androgenic effects, and is not metabolized to other sex steroid hormones, such as dihydrotestosterone or estrogens. Also, while PDE5I is recommended for men healthy enough to engage in sexual activity, the drugs themselves, after extensive scrutiny, have not been associated with adverse cardiovascular events The covariates were those recorded in the 18 to 12 month interval prior to the initial prescription for TT prescription or PDE5I. The pre-prescription interval was the one year prior to the initial prescription, the post-prescription interval was 90 days following the initial prescription, thus the prescription cohorts were restricted to those men with a minimum of 18 months enrollment prior to and three months after their initial prescription. In the post-prescription interval, patients were followed until a diagnosis of acute non-fatal myocardial infarction, refilled first prescription, or 90 days following initial prescription, whichever occurred first.For those who did not refill their initial prescription, we analyzed an additional 90-day interval (91–180 days post-prescription), a time when this group likely had minimal use of these drugs. A refill and a subsequent prescription were treated equally in the analysis. Patients with first prescriptions for both TT prescription and PDE5I during follow-up were excluded from this analysis.The study outcome was a diagnosis of acute MI (ICD-9: 410). Men with a history of MI prior to the first prescription for TT or PDE5I were excluded from the post-prescription analyses.Age at the time of initial prescription was included as a covariate. Diagnostic covariates were identified by the ICD 9 codes recorded for inpatient or outpatient diagnoses, reported to be associated with MI, We examined risk of MI with TT prescription in all subjects. Since previous studies indicated that cardiovascular events appeared early in treatment, we focused on the 90 day interval following the filling of a first prescription. For those who did not refill their prescription, we also assessed the interval of 91–180 days. To examine potential effect modification by pre-existing disease, we estimated the effect of TT prescription by prior history of a heart disease diagnosis . We also examined the RR for PDE5I and compared the RRs in the TT prescription and PDE5I cohorts.We examined the effect of the medications by estimating the ratio of the MI incidence rate in the post-prescription interval to the MI incidence rate in the pre-prescription interval (post/pre RR). To estimate the effect of TT prescription relative to PDE5I, we weighted the PDE5I patients with weights derived from propensity scores, specifically their estimated odds of being prescribed TT prescription rather than PDE5I. We also estimated the ratio of rate ratios (RRR). trend = 0.03). Men aged 65 years and older, whose follow-up was not right-censored because they did not refill their prescription, were followed for an additional 90 day period (91–180 days post-prescription) during which the RR was 0.98 . In men under age 65 the corresponding RR was 1.15 .For all TT prescription subjects combined, the post/pre prescription rate ratio for MI (RR)was 1.36  . In men For the comparison group of PDE5I users, the baseline distributions of prior cardiovascular diagnoses, risk factors, and medication use were less common than in the TT prescription cohort, but after weighting, the distributions of covariates in each prescription cohort were nearly identical . This prThe data for both prescription groups were also divided into men with a previously recorded diagnosis of any heart disease, and those without . For TT As odds-of-treatment weighting is less familiar than unweighted regression methods, we also examined the effect of controlling for the covariates using an unweighted Poisson regression. In subjects aged 65 years and older, the RRR was 2.27 , about 20 percent higher than the weighted estimate of 1.90, and in those under age 65 years with a history of heart disease, the RRR was 2.33 , about 13 percent higher than the weighted estimate of 2.07. The lower estimates suggest that weighting in the primary analysis helped reduce possible upward confounding.Among men aged 65 years and older, we observed a two-fold increase in the risk of MI in the 90 days after filling an initial TT prescription, the risk declined to baseline in the 91 to 180 days after initial TT prescription, among those who did not refill their prescription. Since we censored follow-up at the first refill, and the supply for most prescriptions was 30 to 90 days, it is likely that there was little use of the medication in the 91 to 180 day post-prescription interval when the risk declined. Thus, the pattern of change in risk by supply of testosterone is consistent with an effect of the drug, and underscores the concerns raised by three recent studies in predominantly older men P = 0.41) Among younger men with a history of heart disease, we observed a two to three-fold increased risk of MI in the 90 days following an initial TT prescription and no excess risk in younger men without such a history. Among older men, the two-fold increased risk was associated with TT prescription regardless of cardiovascular disease history, although this analysis was based on relatively small numbers of MI cases in each subgroup. More relevant perhaps is the rapid increase with age in the prevalence of diagnosed and undiagnosed coronary artery disease reported from autopsy studies, both overall and among accident victims, serious adverse cardiovascular-related events  which included myocardial infarction along with other conditions. Our findings are consistent with a recent meta-analysis of placebo-controlled randomized trials of testosterone therapy lasting 12 or more weeks among mainly older men, which reported that testosterone therapy increased the risk of adverse cardiovascular-related events , as well as endogenous testosterone levels may also be positively associated with cardiovascular events Taken together, the evidence supports an association between testosterone therapy and risk of serious, adverse cardiovascular-related events–including non-fatal myocardial infarction–in men. However, there is some evidence that low Despite plausible biologic mechanisms linking TT prescription to an elevated risk of MI, our study has limitations related to use of a health-care database that did not include information on the serologic or diagnostic indications for treatment. It also identified only subjects with non-fatal MIs, typically representing about 75% of the total incidence, and was based on the diagnosis of an attending physician, rather than a structured evaluation as might occur in a randomized trial. However, the accuracy of an MI diagnosis is considered to be reliable in such databases, We addressed potential confounding from measured and unmeasured risk factors by using each treated group as its own control, comparing risk before versus after the start of medication use during the short time-frame of our study, and by controlling for differences in the prevalence of risk factors between the two treatment groups. In the prescription-odds weighted regressions, we found no association between PDE5I prescriptions and the risk of MI, suggesting that the TT prescription-related risk of MI is more likely a drug effect, rather than a result of behavioral or other factors associated with prescription.Furthermore, there is no reason to suspect that physicians excluded high-risk individuals from TT prescription or monitored them more closely, since the hypothesis relating TT prescription to adverse cardiovascular events was not widely known during the study period. Indeed, the initial prescription for over 90 percent of the patients in our study occurred prior to publication of this potential concern, Further study is needed to examine the risk of a variety of specific serious adverse cardiovascular events in relation to TT dose and duration, and to assess if the risks of TT vary by level of serum testosterone and presence or absence of hypogonadal disease. The observed excess MI risk in younger men with a history of heart disease is a particular public health concern, as about 10 percent of the men in our study under age 65 years with a TT prescription had a history of heart disease.Given the rapidly increasing use of TT, the current results, along with other recent findings emphasize the urgency of the previous call for clinical trials adequately powered to assess the range of benefits and risks suggested for such therapy. Until that time clinicians might be well advised to include serious cardiovascular events in their discussions with patients of potential risks, particularly for men with existing cardiovascular disease.Table S1Distribution of baseline covariates in men 65 and older in the TTand PDE5 inhibitor cohorts before and after weighting. The TT patients were unweighted and the PDE5 inhibitor patients were weighted to match the TT cohort based on odds of TT prescription.(DOC)Click here for additional data file.Table S2Distribution of baseline covariates in men under 65 in the TT and PDE5 inhibitor cohorts before and after weighting. The TT patients were unweighted and the PDE5 inhibitor patients were weighted to match the TT cohort based on inverse prescription probability.(DOC)Click here for additional data file.


To study the relevance of high-sensitive troponin measurements in the acute workup in patients admitted to the emergency department of a large university hospital due to syncope.In this retrospective study all patients admitted to the emergency department because of syncope of the Inselspital, University Hospital Bern between 01 August 2010 and 31 October 2012, with serial determination of high-sensitive troponin (baseline and three hours control) were included. Of all identified patients we obtained data on demographics, laboratory data, ECG as well as on outcome. A change in high-sensitive troponin in the three hours control of +/−30% compared to baseline was considered significant.A total of 121 patients with a mean age of 67 years (SD 16) were included in the study. 79 patients (65%) were male and 42 (35%) were female. There was no significant difference in the median high sensitive-troponin level at baseline and in the three hours control . Median percent change in high-sensitive troponin level between baseline and control was 0% (−9.1 to 5). 51 patients (42%) had elevated high-sensitive troponin levels at baseline with 7 patients (6%) showing a dynamic of +/−30% change from the baseline measurement in the 3 hours control. 3 of these patients received coronary angiography due to the dynamic in high-sensitive troponin, none of whom needed intervention for coronary revascularization.On basis of the current study, where no single patient took benefit from determination of high-sensitive troponin, measurement of cardiac troponins should be reserved for patients with syncope presenting with symptoms suggestive for the presence of an acute cardiac syndrome. In the Guidelines of the European Society of Cardiology, syncope is defined as a transient loss of consciousness caused by a self-limiting global cerebral hypoperfusion characterized by rapid onset, short duration, and spontaneous complete recovery th percentile of the reference population. However, the study did not answer the question in which way a determination of troponin is of any benefit for the patient in the workup of syncope.So far only one clinical study has determined the role of high-sensitive troponin in the evaluation of syncope In the present study we aimed to determine the relevance of serial high-sensitive troponin determination in patients presenting to a large emergency department of a university hospital. Additionally, we wanted to identify the number of patients with acute myocardial infarction detected by serial high-sensitive troponin measurements in patients admitted to an ED for syncope.www.kek-bern.ch). The need for written consent was waived by our institutional review board due to the retrospective design of the study.The study was approved by our local institutional review board  or median and first and third quartile, as appropriate. Wilcoxon matched pairs test was used to compare serial high troponin measurements. A p-value <0.05 was considered to be significant.During the study period a total of 55.422 patients were admitted to the ED. Of these, 1.218 were admitted for syncope. 121 patients had serial measurements of high-sensitive troponin and were included in the study. Mean age of patients was 67 years (SD 16). 79 patients (65%) were male and 42 (35%) were female. 4 patients (3%) presented with minor head trauma along with the syncope.8 patients (6%) complained of chest pain on admission to the ED. 41 patients (34%) had abnormalities in the ECG: 19 patients (46%) showed ST segment depression, 8 (20%) right bundle branch block, 5 (12%) a negative T wave, 3 (7%) a left bundle branch block, 2 (5%) presence of Q-waves and bifascicular block, respectively. Tachycardic atrial fibrillation and atrioventricular block grade I was present in 1 patient (2%), respectively. All patients received a baseline measurement of high-sensitive troponin in the ED and a control measurement three hours after ED admission. Median high sensitive-troponin level at baseline was 0.01 mcg/L (0.003 to 0.022) and three hours control was 0.011 mcg/L (0.003 to 0.022). 70 patients (58%) had a high-sensitive troponin measurement of <0.014 mcg/L at baseline, while 4 of them had increases to a level equaling or exceeding the cutoff for the normal range of 0.014 mcg/L at the control measurement. However, only in one of these patients a significant troponin rise exceeding 30% of the baseline value was found (790% rise or from 0.01 to 0.89 mcg/L). The patient was found to suffer from intracranial bleeding, finally ending lethal.51 patients (42%) had elevated high-sensitive troponin levels at baseline with 7 patients  showing a dynamic of +/−30% change from the baseline measurement in the 3 hours control. In 3 of the patients with a high-sensitive troponin dynamic exceeding 30% from baseline to control no none-cardiac cause could be attributed to the rise in troponin and they finally received coronary angiography. However, none of the patients was found to have a significant stenosis in the coronary angiography leaving the cause for the elevated troponin unclear. None of the 7 patients with a high-sensitive troponin change of +/−30% from baseline measurement died.No single patient included in our study finally received the diagnosis of acute coronary syndrome. One patient (1%) died during hospitalization from intracerebral hemorrhage.In the present study, we found that 121 patients received measurement of high-sensitive troponin during their initial evaluation at the ED for syncope. Although 42% of patients showed initial high-sensitive troponin levels of ≥0.014 mcg/L, only 6% had a significant high-sensitive troponin dynamic of +/−30% at the 3 hours control measurement. 3 of the patients received coronary angiography, all of which ended without need for intervention.Although troponin measurements are not recommended in the current guidelines for the evaluation of syncope of the large societies, it is often performed by physicians in daily clinical practice It is well described today that various patient populations with an elevation of high-sensitive troponin are at risk for adverse outcome But there is also a financial aspect: Currently one determination of high-sensitive troponin in our institution costs the amount of 23 Swiss Francs (approximately 24 United States Dollar). The routine determination of high-sensitive troponin in a common (and often benign) condition such as syncope would result in serious amounts of unnecessary health care spending.Our study is limited by its retrospective and single site design. The detailed thoughts behind ordering high-sensitive troponin determination of the physician in charge are unknown. Additionally, we do not have information on long-term outcome of patients. On the other hand, long term outcome is not necessary in order to answer our study question.In conclusion, based on our study in which no single patient took benefit from the determination of high-sensitive troponin, routine measurements of cardiac troponins cannot be recommended for the evaluation of syncope in the ED. In the workup for syncope, the measurement should be reserved for patients who present with symptoms suggestive for the presence of acute coronary syndrome.


P < 0.001) in the CSF of patients with ICH than in controls. TFa levels were significantly (P < 0.05) higher in ICH than in patients with meningitides or NDD. Higher levels (P < 0.05) of TFa were observed in patients with ICH who died than in survivors. TFa measurement in the CSF of patients with ICH could constitute a new prognostic marker.Brain contains large amounts of tissue factor, the major initiator of the coagulation cascade. Neuronal apoptosis after intracerebral haemorrhage (ICH) leads to the shedding of procoagulant phospholipids (PPLs). The aim of this study was to investigate the generation of PPL, tissue factor activity (TFa), and D-Dimer (D-Di) in the cerebrospinal fluid (CSF) at the acute phase of ICH in comparison with other brain diseases and to examine the relationship between these factors and the outcome of ICH. CSF was collected from 112 patients within 48 hours of hospital admission. Thirty-one patients with no neurological or biochemical abnormalities were used to establish reference range in the CSF (“controls”). Thirty had suffered an ICH, and 51 other neurological diagnoses . PPL was measured using a factor Xa-based coagulation assay and TFa by one home test. PPL, D-Di, and TFa were significantly higher ( Intracerebral haemorrhage (ICH) is a type of stroke caused by bleeding within the brain tissue itself. Primary ICH accounts for ~80% of all incidences of ICH and is more likely to result in death or major disability than ischemic stroke or subarachnoid haemorrhage. ICH has a higher incidence among populations with a higher frequency of hypertension, including African Americans and Asian populations, possibly due to environmental factors  and/or genetic factors . In contComputed tomography (CT) is now widely available in the developed world and has become the diagnostic test of choice in ICH to determine the site of the haemorrhage and estimate the volume of the haematoma . The abiOver the past decade, numerous studies have reported a positive correlation between S100B levels in blood or CSF and impaired neurological function. S100B is a calcium-binding protein concentrated in glial cells  and serum S100B is considered as a relevant diagnostic and prognostic tool in acute spontaneous ICH. However, a recent study in patients with ICH suggested that its sensitivity and specificity were not as high as previously described at least in patients with traumatic brain injury  and consDuring stroke, the blood-brain barrier (BBB) is compromised by endothelial cell death. Cytosolic contents released from injured brain tissues have the potential to cross the barrier. We hypothesise that the measurement of molecules which are expressed in high concentration in the brain but in trace amount in normal CSF could be used to monitor ICH severity and prognosis. As the brain is a major source of tissue factor (TF), the main coagulation cascade activator  and richFresh CSF was collected from 112 hospitalised patients by lumbar puncture in the first 2 days after admission . Among these, 31 patients that had been admitted to the emergency ward with suspected meningitis or subarachnoid haemorrhage but were found to had no CSF biochemical abnormalities and normal neurological explorations were considered as controls. Of the remaining patients, 30 had suffered a cerebral haemorrhage, and 51 had other neurological disorders: 13 with confirmed viral meningitis, 15 with bacterial meningitis, and 11 with a neurodegenerative disease (NDD) Alzheimer's type; 12 were treated with ventricular drainage (VD) following brain surgery. ICH patients, defined as an acute and persisting focal neurological deficit, were to be admitted to the neurological department within six hours after symptom onset. No patients received intrathecal fibrinolytic therapy, antiplatelet agents, or prophylactic calcium channel blockers before sample collection. Informed consent was obtained from all appropriate family members.The CSF was immediately placed on ice to prevent enzyme activation and transported to the laboratory where each was centrifuged at 1,000 ×g for 15 min to remove cells and debris. A small number of CSF samples were bloody and were additionally centrifuged for 15 minutes. Medical treatment was started soon after the lumber puncture when cranial CT had proved the existence of ICH.9/L in normal CSF did not change the time of PPL determination, while CSF specimens included in this study contain less than 45 × 109 RBC/L.PPLs in the CSF were measured on a STA-R analyser using a factor Xa-based coagulation assay . The test consists of the measurement of clotting time in the presence of CSF, in a system in which a phospholipid-depleted substrate plasma makes the test dependent on the PPL of the test sample. Bovine factor Xa triggers the coagulation cascade downstream from factor Xa, thus eliminating the influence of coagulation factors acting upstream . A shortTFa was measured by a one-stage kinetic chromogenic method . This asFree tissue factor pathway inhibitor (f-TFPI) was determined by ELISA . D-Dimer (D-Di) was measured on a STA-R analyser by latex immunoassay  according to the manufacturer's instructions.CSF proteins, glucose, chloride, and lactate measurements were done using the routine laboratory techniques .r) were calculated using Spearman's rank test. A two-sided P value <0.05 was considered significant. For survivors to nonsurvivors comparison, a Mann-Whitney test was used. Receiver operating characteristic (ROC) analysis was performed to calculate cut-off values discriminating survivors and nonsurvivors.All statistical calculations were done using NCSS, version 9  and R statistical Package, version 3.0.2 . Data are presented as median (1st and 3rd quartiles). Since all tests showed significant departure from normal distribution, between—group comparisons of continuous variables between different subgroups were performed using Kruskal—Wallis non parametric analysis of variance. When this led to global significance, pairwise comparisons were done between all groups and control and all groups and ICH, using Dunn's test. The Bonferronni-Simes correction was used to preserve the nominal significance level. Coefficients of correlation (P < 0.001). PPL was also significantly increased in comparison with controls in the different groups of patients analyzed, even in patients with NDD. PPL in patients with ICH was also significantly higher when compared to the different pathological groups, except in patients with VD and NDD.Clotting time in the PPL assay and levels of TFa and D-Di, in the CSF of the controls and patients are shown in When compared to controls, TFa was significantly increased only in patients with ICH. D-Di was significantly increased in patients with ICH and in patients with VD.Compared to controls, CSF proteins, glucose, and lactates were significantly increased in patients with ICH. Significantly higher levels of CSF glucose were found in ICH patients when compared to patients with both types of meningitis and NDD and of CSF lactates when compared to NDD.μg/L, data not shown).The levels of f-TFPI in the CSF of control and in patient CSF of the different groups were below the detection level  was found only between TFa and D-Di. In the CSF of patients with ICH, clotting time in the PPL assay was significantly inversely correlated  with CSF lactate levels, which means that the quantity of PPL increases with the lactate level. In the CSF of patients with bacterial meningitis, a significant positive correlation  was found between TFa and D-Di and an inverse correlation between TFa and chloride . In CSF of patients with VD, a positive correlation was found between glucose and lactate . In the CSF of controls, a significant positive correlation (P < 0.05). There was no significant difference in CSF D-Di levels (P = 0.48) and PPL (P = 0.07). ROC curve analysis indicated PPL to be more sensitive but less specific than TFa  mainly of platelet and endothelial origin . In thisIt was beyond the scope of this study, but it would, in future studies, be of interest to identify the origin of these cells in the different groups of patients tested in this study, since CSF PPL levels were significantly increased in each group, where different physiopathological mechanisms are suspected.The brain is rich in TF. The cellular sources of TF are the astrocytes in the parenchyma, glia limitans, and arachnoid cells, with the latter two being in contact with the CSF , 16. HowThe third parameter studied was D-Di. In agreement with previous studies , 23, we In conclusion, PPL and TFa assays presented in this study are rapid tests which can be performed in an emergency context of ICH and can provide useful diagnostic information. Further studies are required to better assess the relation between these markers and the occurrence of cerebral vasospasm or oedema formation and to evaluate whether the time-course of these factors may be helpful in predicting patient complications. The limitations of our study are the small sample size and relatively small number of patients included in each group. This makes it difficult to draw firm conclusions. These assays should be compared to new parameters such as copeptin or resistin which were recently described as having a prognostic value in ICH , 26.


High-sensitivity (hs) cardiac troponin is a valuable biomarker of myocardial injury and frequently elevated in patients with hypertrophic cardiomyopathy (HCM). Using cardiovascular magnetic resonance (CMR) the HCM phenotype can be characterized in great detail with fibrosis as a key finding with prognostic impact. Therefore, our aim was to investigate whether elevated troponin levels in patients with clinical HCM were associated with LV hypertrophy and fibrosis as assessed with CMR.th percentile reference limit (≥ 0.014 pg/l). CMR with late gadolinium enhancement (LGE) was performed (Philips Achieva 1.5T) to assess LV mass indexed to body surface area, maximal wall thickness (MWT) and fibrosis. LV mass, MWT and the presence and extent of LGE were assessed  and compared between HCM patients with and without an elevated hs-troponin using Mann Whitney U or Fisher exact testing.In 62 clinical HCM patients  hs-troponin T was determined with a high-sensitivity assay (Roche Diagnostics). The lower detection limit is 0.003 pg/l and an elevated hs-troponin is defined as a concentration ≥ the 99Hs-troponin was detectable in 46 HCM patients (74%), of whom 16 patients had an elevated hs-troponin level. The median LV mass and MWT were higher in HCM patients with an elevated hs-troponin (p<0.001). The difference in the presence of fibrosis was almost statistically significant between groups. Notably, if present, the extent of fibrosis was higher in patients with an elevated hs-troponin (p= 0.04) (Table Elevated troponin levels assessed with a high-sensitivity assay are common in HCM patients and associated with specific features of HCM such as hypertrophy and maximal wall thickness. Our findings are indicative of troponin as a potential surrogate marker of myocardial injury in the form of fibrosis. Future studies will have to address this issue more elaborately especially with regard to the potential prognostic value of troponin in these patients.None.