We now report an assessment of the CSF proteome to determine the changes in protein composition that occur during this period.Understanding the early relationship between brain tumor cells and their environment could lead to more sensitive biomarkers and new therapeutic strategies. We have been using a rodent model of neurocarcinogenesis in which all animals develop brain tumors by six months of age to establish two early landmarks in glioma development: the appearance of a nestin+ cell clusters and microtumors were assessed in 63 ethylnitrosourea-exposed rats on 30, 60, and 90 days of age. CSF was obtained from the cisterna magna from 101 exposed and control rats at 30, 60, and 90 days and then analyzed using mass spectrometry. Differentially expressed peaks were isolated and identified.Nestin+ cells were noted in all ethylnitrosourea-exposed rats assessed pathologically. Small microtumors were noted in 0%, 18%, and 67% of 30-, 60-, and 90-day old rats, respectively . False Discovery Rate analysis of peak intensities showed that the number of true discoveries with p<0.05 increased markedly with increasing age. Isolation and identification of highly differentially detected proteins at 90 days of age revealed increases in albumin and a fragment of α1 macroglobulin and alterations in glutathionylated transthyretin.NestinThe presence of increased albumin, fragments of cerebrospinal fluid proteins, and glutathione breakdown in temporal association with the development of cellular hyperplasia, suggests that, similar to many other systemic cancers, inflammation and oxidative stress is playing an important early role in the host’s response to brain tumor development and may be involved in affecting the early growth of brain tumor. The influence of the local environment in the development of cancer has been clearly established for several systemic neoplasms including colon, breast and prostate cancers in situ environmental changes preceding the development of clinical abnormalities may lead to novel diagnostic and therapeutic strategies in primary brain tumors. Although it would be difficult to visualize tumors at very early stages in brain parenchyma, cerebrospinal fluid (CSF) represents a readily accessible source that could serve as a reporter of early stages of tumor development. Approximately 10–30% of all CSF is extrachoroidal in origin and is represented by bulk flow of the interstitial fluid from brain parenchyma into the ventricles and subarachnoid space We believe that a better understanding of the ongoing Surface-enhanced laser desorption/ionization TOF mass spectrometry (SELDI TOF MS) has been used successfully to identify biomarkers in blood from various malignancies using comparative proteomic strategies in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Numerous pathological studies including those from our laboratory have established that gliomas invariably develop in this model. While the gliomas are not generally detectable pathologically until approximately 90 days of age (P90), and even later using available magnetic resonance imaging (MRI) technology, clear landmarks of developing tumors can be noted as early as P30 In order to control for these variables, we assessed changes in CSF protein composition during the period in which brain tumors develop after a single In this controlled paradigm in which matched ENU- and saline-exposed rats were examined, we demonstrate proteomic changes in CSF as early as P60, which increase by P90 in ENU-exposed rats. Furthermore, the identification of changes in glutathionylated products of transthyretin as well as a fragment of α1-macroglublin as two of the most significant changes that correlated with the development of early cellular hyperplasia suggests that increased proteolysis is present within the brain environment during a time before tumors are detectable by imaging.+ precursor lesions (nests) as well as microtumors (areas of cellular hyperplasia measuring less than 200 µm) as previously described p<0.001, Chi-square, compared to other two groups) (ENU exposed rats (n = 63)  were examined histologically for the presence of nestin groups) Figure 2Methods. The relative intensities of peaks were different in the CSF of rats obtained at these three ages. For this reason we grouped the spectra by postpartum age for baseline correction, noise reduction and intensity normalization. The spectra for all three ages were then grouped together for the purpose of finding peaks, and then separated again by age for further analysis of the peaks at each age.CSF was collected from a total of 51 ENU and 50 saline exposed rats over three independent experiments. At P30 , P60  and P90 , mass spectra of CSF applied to CM10 ProteinChip arrays were collected for the three postpartum ages  as described in p<0.05, Mann Whitney U-test comparison) increased from 20 to 34 to 61 for P30, P60 and P90, respectively . We noted that the number of peaks that were significantly different  for each peak. Using this approach, we observed that despite there being 21 (of 247) peaks that appeared to be significantly different at P30, the lowest local FDR of 0.28 for any peak indicated that all these peaks with  p<0.002 Table 1.p<0.05 and a local FDR<0.12 are listed in The first 27 P90 peaks with MW U-test of Figure S2), we performed Western analysis and slot blot immunoassay using anti-PGD2S to verify decreased protein expression. These techniques did not verify the decreased amount of PGD2S in ENU relative to control CSF that were noted using peak intensities. We hypothesize that the increased albumin present in the CSF in ENU rats suppressed the ionization or detection of PGD2S by mass spectrometry, resulting in an artifactual decrease in the PGD2S peak intensity. There was a near-perfect inverse correlation between the albumin peak intensity and the 22,893 peak intensity   and therefore did not bind to the anion exchange media at pH 9, suggesting the peptide had a high pI. The peak intensities of the m/z 3493 peak in the pH 9 fraction were still higher in the ENU than the control rats . The pH 9 fraction did not contain any albumin, thus confirming its peak intensities were not affected by the presence of albumin. The m/z 3493 peptide was purified and identified as a novel fragment of α1-macroglobulin by both direct sequence analysis (MALDI MS/MS), and by sequence analysis of tryptic peptides . The sequence of the 3493 peptide is SFSYKPRAPSAEVEMTAYVLLAYLTSASSRPT, which corresponds to amino acids 1212–1243 of rat α-1-macroglobulin (a 1500 amino acid protein). Of note, a 45 kDa subunit of α-1-macroglobulin has been reported, from amino acids 1245–1500. Amino acid 1244 is the amino acid arginine. Neither in the discovery spectra, nor during the purification, did we find evidence of a peptide with an m/z 3650, which would be the theoretical m/z of the 3493 peptide with an additional C-terminal arginine.A third peak (m/z 3493) was also higher in the ENU rats than in the control rats. To decrease the complexity of the CSF, we also fractionated the CSF samples by strong anion-exchange chromatography prior to acquisition of SELDI spectra  or post-translational modifications of transthyretin. The peaks were purified, characterized and identified. The peaks 13601, 13670, 13725, 13788, and 13913  were purified as a group and extracted from a single band on non-reduced SDS-PAGE . Hypothesizing that these peaks were the unmodified and various modifications of transthyretin, we digested one part of the gel band with the enzyme Arg C without reduction and alkylation, and another part of the band with Arg C after proteins in the band had been reduced and alkylated. The 13601 peak corresponds to unmodified transthyretin, while the identities of the other peaks were different sulfhydryl modifications of the lone cysteine within the primary structure of rat transthyretin. The 13725 peak was found to be cysteinylated transthyretin; the 13788 peak, the –Cys-Gly modified transthyretin; and the 13913 peak, the glutathionylated transthyretin. Peptides consistent for the unmodified parent transthyretin and these post-translational sulfhydryl modifications were found in the mass spectrum of the Arg C-digested non-reduced band. Peptides for only the parent, unmodified transthyretin were found in the reduced/alkylated Arg C-digested band, a result confirmed by sequencing of the peptide , there was one difference among their intensities in ENU CSF compared to the CSF of age-matched control rats. For three of the four peaks in the figure  the peak intensities in the ENU rats were higher than control rats. In contrast, the intensity of the 13913 peak (glutathionylated transthyretin) was lower in the ENU rats compared to the controls. To assess the relative values of each of these proteins as potential biomarkers, we used a standard scalable algorithm, LASSO, that automatically balances classification performance against the number of markers in the classifier Using this approach, we repeated the statistical analysis 1000 times. We subsampled 36 rats from the control/ENU population of P90 rats and computed a sequence of classifiers using the Homotopy algorithm. We observed that on the 9 held out subjects, the error was minimized using five makers Figure 8This is the first time that the CSF protein profile has been examined prior to either the imaging or symptomatic detection of brain tumors. It is made possible by the availability of an experimental rodent model in which brain tumors invariably develop several weeks after a prenatal exposure to a neurocarcinogen. Investigations in several laboratories, including ours, have established predictable pathological landmarks including the appearance at P30 of abnormal cell and cell clusters expressing the neuroepithelial marker nestin without hyperplasia that become associated with cellular hyperplasia by P90 CSF is produced mainly by the choroid plexus, an intraventricular structure formed by the invagination of the pia mater. The choroid plexus regulates chemicals in the brain not only by selectively limiting the access of blood-borne substances to the CNS, but also by serving as a unique source of essential molecules to the cerebral compartment including VEGF, IGFII transferrin, PGD2S and transthyretin Supporting Information S1, Figure S1).A major difficulty with interpreting results of CSF protein analysis, however, is that protein composition is dependent on subject age, the site where CSF is accessed , while hyperplasias were detected in only 18% of brains examined at P60 and then 67% at P90. In association with this, we found that the global protein fingerprint was not significantly different between ENU-exposed and control subjects at P30 based on FDR analysis, after which increasing differences were noted at P60 and P90. No rat was found to have a macroscopic tumor at any of the times studied. Based on this, we conclude that CSF protein alterations noted in this study are more reflective of the cellular hyperplasia stage of this process.We chose the ages to be studied based on the staggered appearance of nestin-expressing cell clusters (which start at P30) and the association of these cells with surrounding cellular hyperplasia (which can be detected as early as P60) et al’s conclusions that this reflects a blood brain barrier disturbance that is present long before imaging changes are apparent.To develop further insights as to the types of proteins that were altered and whether they pointed to a particular biologic process, we next identified those peaks that were most significantly different between ENU-exposed rats and controls. Several of the peaks represented albumin, which was consistently elevated in ENU-exposed rats after P60, a finding confirmed by Western blotting. Albumin levels in CSF have been noted to be elevated in patients with glioblastoma Another peak with a mass of 22.9 kD was decreased in ENU exposed rats and identified as prostaglandin D2 synthase, also known at β-trace. PGD2S is exclusively brain derived and is one of the most abundant proteins in the CSF Our subsequent studies however indicate that changes noted in PGD2S peak intensities probably reflect an artifact caused by the effect of albumin concentration on the intensity of the PGD2S peak, as supported by the observations of almost a perfect inverse correlation between the PGD2S and albumin peak intensities Figure 5The identification of an increased concentration of a cleaved fragment of α1-macroglobulin is a unique finding that points to the advantages of using a top down MS approach, since it would have not been detected using a bottom up MS/MS approach that starts with proteolytic digestion of the sample. This macroglobulin is synthesized as a single polypeptide chain that is subsequently cleaved to form heavy and light chains. The novel fragment reported here is near the C-terminus of the heavy chain that is created during processing of the full-length protein. The m/z 3493 peptide is missing the C-terminal Arg residue of the heavy chain, and no evidence of a peak representing the 3493 peptide with the additional C-terminal Arg  was found in either unfractionated or anion exchange-fractionated CSF. This macroglobulin is a broad spectrum protease inhibitor that is constitutively active , present in serum and interstitial fluids and inhibits all four types of proteinases by physical entrapment Finally, many of the most significantly different peaks between ENU-exposed and control rats represented different post-translational modifications of transthyretin. Transthyretin functions in the transport of both thyroxine and retinol in plasma and CSF. The disproportionately high concentration of soluble monomeric tranthyretin (14 kD) in human ventricular CSF is related to the fact that it is synthesized in high quantities within the cells of the choroid plexus In an analysis of different brain tumors, transthyretin mRNA and protein were not expressed in 23 anaplastic astrocytomas and glioblastomas, being exclusively limited to choroid plexus papillomas Glutathione  is a tripeptide utilized in disulfide exchange reactions resulting in the formation of mixed protein-glutathione disulfides. It serves as an important cellular antioxidant in the brain, where it plays a critical role in suppressing oxidative stress and maintaining cellular redox stability. In addition, glutathione can function as a storage depot for both cysteine and glutamate, thus serving an important cytoprotective function by preventing the inherent cytotoxicity of free cysteine and glutamate-dependent neuronal excitotoxicity GSH is synthesized intracellularly and frequently bound to passenger proteins upon export. It can be hydrolyzed to its γ-glutamyl moiety and cysteinylglycine, the latter of which can be further broken down in times of cellular stress so that the released amino acids can be taken up by corresponding transporters and reused by cells In a prior study that also examined the evolution of gliomas in this experimental paradigm, we reported the consistent appearance of a unique cell that coexpressed glial fibrillary acidic protein (GFAP) and osteopontin (OPN) at the time these lesions evolved from nestin+cell clusters to hyperplastic nodules The challenge at this juncture is thus the identification of which components of this host reaction are synergistic and antagonistic relative to favoring tumor growth. Further analysis of these host-tumor environmental interactions should therefore hopefully lead to further insights into these relationships so as to suggest novel therapies that could either prevent tumor formation or treat established tumors.All experiments performed in this study were approved by the Stanford IUCAC (Protocol #11936) and are in accordance with guidelines for animal safety and welfare.Ethylnitrosourea (ENU) 50 mg/kg was administered to timed pregnant Sprague-Dawley rats (Taconic Farms) at gestational day 18 (E18). Subjects were placed in a restrainer and injected i.p. with 50 mg/kg ENU (Sigma) with a 26-guage needle over a several minute period, as described previously et al, To obtain CSF for analysis, rats aged between 30 and 90 days were anesthetized with ketamine and xylazine and placed in a stereotactic headholder. Using methodology similar to that described by Pegg callosum to the posterior midbrain and every twelfth section stained with H&E and immunostained with anti-nestin antibody (BD Biosciences Pharmingen) to assess for the presence of hypercellular microtumors and tumor precursors , respectively, as described previously After perfusion, brains were removed and prepared for histological analysis by immersion in gradually increasing concentrations of sucrose. Frozen sections were cut spanning an area from the anterior Samples of CSF (6 µl) were denatured with 9 µl 9 M urea/2% CHAPS/0.05 M Tris pH 9 and incubated on ice for 30 minutes. An aliquot of denatured CSF (10 µl) was then added to 90 µl 0.1 M acetate buffer, pH 4, and incubated on a negatively charged weak cation exchange ProteinChip array . After binding for 30 minutes, the CM10 arrays were washed three times with binding buffer, and then twice briefly with water. After air-drying, a small amount (1 µL) of the energy absorbing molecule sinapinic acid  was added to the CM10 arrays and air-dried. After a second application of sinapinic acid and air-drying, the CM10 arrays were placed in a Bio-Rad PCS4000 SELDI mass spectrometer. Spectra were acquired using low-, and medium-, and high-laser energies, which were chosen to optimize for analysis of small , medium  and large  proteins, respectively. The data from all spectra were stored in a MySQL database, for analysis by a variety of techniques (see below).Some analyses of the P90 rat CSF samples were performed on CSF that was fractionated by strong anion exchange chromatography prior to SELDI analysis. CSF (50 µL) was mixed with 75 µL 9 M urea/2% CHAPS/0.05 M Tris pH 9 and incubated on ice for 30 minutes. After addition of 125 µL 1 M urea/0.2% CHAPS/0.05 M Tris pH 9, the denatured CSF was applied to 50 µL packed HyperD F Q anion exchange beads in a 96 well Silent Screen filtration plate. After agitation for 60 minutes at room temperature, the pass-through fraction was obtained by vacuum filtration and combined with a subsequent wash with 100 µL 0.05 M Tris pH 9. The beads were then successively extracted with two 100 µL washes of buffers with decreasing pH  and a final organic extraction with 33% isopropyl alcohol/17% ACN/0.1% TFA.For SELDI analysis, aliquots (50 µL) of the pH 9 F×1 fraction were mixed with 50 µL 2 M acetic acid and incubated on CM10 ProteinChip Arrays. After binding of the samples for 30 minutes, the CM10 arrays were washed with 0.1 M acetate buffer pH 4.0 and prepared for mass spectrometry as above.The mass spectra were processed using the ProteinChip Data Manager software version 3.5 from the vendor (Bio-Rad). The spectra were de-noised, filtered and baseline subtracted. The processed spectra were then grouped by the age of the rat for normalization of signal intensities. The age-grouped spectra were then recombined for feature extraction (peak finding). The tables of peaks at each age are then used for statistical and biomarker discovery purposes (see below).U tests were used to rank differences between peak intensities in CSF from ENU-exposed and control brains. Given the large number of peaks being tested for differential expression, multiple hypotheses testing methods were utilized during the analysis of mass spectra peak intensities. Methods based on false discovery rates (FDR) were used in this analysis  p value. Local false discoveries and rates were determined by permuting the rat identities for each peak 100 times.Mann Whitney t-tests were used to evaluate differences in Western blot and immuno-slot blot assays.Chi-square analysis was used for evaluation of nests and early tumors. Student’s Finally, a LASSO analysis was performed to determine the peaks that best discriminated between the ENU group and the control group. This LASSO was computed in Matlab on a standard laptop computer.Supporting Information S1.See CSF samples (4 µL) were subjected to SDS-PAGE under reducing conditions on Bio-Rad TGX 4–20% gradient gels. Proteins were transferred to PVDF membranes in a Thermo semi-dry electro-transfer apparatus. Membranes were blocked with 6% nonfat mild powder in tris-buffered saline pH 7.4 (TBS) (BLOTTO), and incubated with anti-albumin antibody. After washing with TBS supplemented with 1% Tween 20 (TBS/Tween), membranes were incubated with horse radish peroxidase-conjugated goat anti-rabbit IgG . After washing with TBS/Tween, the blots were visualized after exposure to chemiluminescent substrate SuperSignal Pico (Thermo/Pierce). The band intensities on X-ray film were analyzed using Quantity One software (Bio-Rad).PVDF membranes were placed in a Hoefer 48-well slot blot vacuum manifold. The membranes were first treated with 0.5 ml water:methanol (1∶1), and then 0.5 ml TBS twice. Aliquots of CSF (3 µL) were then added to 0.25 ml TBS in each well, and the vacuum applied. The wells were then washed two times with 0.5 ml TBS, after which the membranes were removed and allowed to dry. The membranes were blocked with BLOTTO as above, and then incubated with anti-PGD2S (Santa Cruz Biotechnology), secondary antibody, and chemiluminescent substrate as above. The chemiluminescence intensities were quantified using ImageLab software on a Bio-Rad ChemiDoc XRS+imager.Figure S1The effect of increasing blood contamination of CSF on the intensity of the SELDI peaks. The intensity of the m/z 15216 globin is plotted on the left ordinate axis as a function of the ratio of blood added to CSF. The intensities of three additional peaks are plotted on the right ordinate axis for the same samples of blood-doped CSF samples: the m/z 66610 albumin peak, the m/z 13913 glutathionylated-transthyretin peak (TTY-glut), and the m/z 3493 α1-macroglobulin fragment.(PDF)Click here for additional data file.Figure S2Purification and Identification of PGD2S. A) Mean±SEM SELDI intensities of m/z 22893 peaks in Control  and ENU-exposed  rat CSF; B) mass spectrum of pH 4 fraction showing partial purification of the m/z 22893 peak; C) mass spectrum of proteins extracted from candidate band for the m/z 22893 peak. The change in peak m/z 22975 probably reflects an acrylamide modification of the protein during SDS-PAGE; D) mass spectrum of in-gel reduced/alkylated and trypsinized band from (C).(PDF)Click here for additional data file.Figure S3Purification and Identification of α1-macroglobulin fragment. A) Mean±SEM SELDI intensitites of m/z 3493 biomarker in Control  and ENU-exposed  rat CSF; B) mass spectrum of CSF showing the m/z 3493 peak; C) Purification scheme; D) mass spectrum of purified m/z 3493 peak on NP20 ProteinChip arrays; E, F) MS/MS sequence identification following reduction/alkylation and trypsinization of sample from (D), showing the sequence of the 2628.3 and 884.4 ions, respectively.(PDF)Click here for additional data file.Figure S4Transthyretin and post-translationally modified transthyretin biomarkers. A) Maldi mass spectrum following in-gel Arg C-digestion of candidate SDS gel band, without reduction and alkylation; B) MALDI mass spectrum of same band from (A), except that the proteins in the gel band were first reduced and alkylated before Arg C digestion.(PDF)Click here for additional data file.Supporting Information S1Identification of differentially expressed proteins.(DOCX)Click here for additional data file.


Pinus sylvestris L.), but has not been applied because of vegetative propgation of the species is difficult. With a common interest in Scots pine breeding, forest research institutions from Sweden, Finland, and Latvia  joined in a collaborative project to develop pine cutting propagation for breeding purposes. The main objective of this effort was to find protocols for sufficient shoot production, and at the same time, maintain a high rooting response. Secondly, the aim was to increase the knowledge on the influence of different rooting agents, watering regimes and substrates on the rooting of cuttings.Testing of candidates as clones would greatly benefit breeding of Scots pine (From Finland and Latvia, respectively, 15 full-sib families from parents with high breeding values were included. From Sweden, 15 families from each of two research stations, one northern and one southern, were included. These sets of the families were used locally. Another collection of five families was shared among the participants, which means that 5 families extra were included at each location.All donor plants were pruned according to the same principles. However, the timing of the pruning varied in the five propagation models tested:A. One-year-old donor plants pruned to yield 2x winter cuttings / 4-year methodB. One-year-old donor plants pruned to yield winter cuttings and late summer cuttings/ 3-year methodC. “Turbo line” i.e. one-year-old donor plants pruned 2 x cuttings in one year/ 2-year methodD.One-year-old donor plants pruned to yield 2x late summer cuttings/ 3-year methodE. Two-year-old donor plants pruned to yield winter cuttings/ 4-year methodAccording to the results, the average production of cuttings with propagation models including two harvests in consecutive years on the same donor plant can be predicted to be 10-15 cuttings per a donor plant, with a substantial variation among families and donor plants. Even though rooting responses above 50 % can be achieved, this could not be repeated for a large number of propagations. The generally low and erratic rooting response leads to conclusion that the project was unsuccessful in developing improved and reliable protocols for Scots pine cutting propagation. However, a propagation model enabling two harvests on a 1-year-old donor plant within the same year is a promising option but needs further studies.http://www.metla.fi/julkaisut/workingpapers/2011/mwp198.htmMore detailed information on the project and its results is available at:


Anthropometric measurements, biochemical variables and systemic RBP4 levels were determined. RBP4 levels were significantly higher in patients with metabolic syndrome and type 2 diabetes than in euglycemic subjects  and correlated with triglycerides but not with those of HOMA-IR in the whole population. The multivariate regression model revealed that triglycerides were the strongest predictor of systemic RBP4 levels. Analysis of lipoprotein subfractions in a subpopulation of 80 subjects showed an altered profile of insulin resistant states characterized by higher VLDL, sdLDL and small HDL percentages and lower large HDL percentage. Although RBP4 levels correlated significantly with LDL particle size and small HDL percentage, the latter parameter was independently associated only with RBP4. Our study reveals that systemic RBP4 levels could play an important role in lipid metabolism in morbid obesity, increasing triglyceride levels and contributing to the formation of small HDL.Retinol binding protein 4 (RBP4) is an adipokine that may contribute to the development of insulin resistance. However, how this adipokine is affected and its possible involvement in lipid metabolism in obese patients with varying degrees of insulin resistance is yet to be determined. A total of 299 middle-aged morbid obese patients (BMI>40 kg/m Obesity is a prevalent condition characterized by the excessive accumulation and storage of fat in adipose tissue. It is a major cause of insulin resistance, which is implicated in the development of metabolic syndrome (MetS) and type 2 diabetes (T2D). These pathologies are related with the development and progression of atherosclerosis, one of the leading causes of morbidity and mortality in industrialized countries Dyslipidemia is a known risk factor for atherosclerosis and is commonly linked to insulin resistance and, hence, to obesity, MetS and T2D Retinol binding protein 4 (RBP4), secreted primarily by the liver and adipocytes, is a recently identified adipokine In addition, growing evidence suggests that RBP4 plays a role in lipid metabolism to an even greater extent than insulin resistance. In fact, many human studies have found a strong relationship between RBP4 and triglycerides, some finding an association with insulin resistance Thus, in order to gain further insight into the relation between RBP4, insulin resistance and lipid metabolism, we studied a large cohort of obese subjects with different degrees of insulin resistance. The following aspects were explored: 1) the potential association of serum RBP4 levels with insulin resistance; 2) metabolic parameters as independent predictors of RBP4; and 3) the involvement of RBP4 in contributing to the atherogenicity of both LDL and HDL particles.2 who were referred to the Outpatient's Department of the Endocrinology and Nutrition Service at the University Hospital Dr. Peset in Valencia to be treated for obesity. The study was conducted according to the guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee of the University Hospital Dr Peset. Written informed consent was obtained from all the participants.Our study population was composed of patients with a BMI >40 kg/mThe MetS was based on the updated National Cholesterol Education Program Adult Treatment Panel III criteria Exclusion criteria were pregnancy or lactation, recent change of oral contraceptive formulation, severe disease, history of cardiovascular disease or chronic inflammatory disease, pharmacological treatment for diabetes-antidiabetic agents or insulin-, secondary obesity , and pharmacological treatment for dyslipidemia or hypertension. According to these criteria, subjects were divided into three groups: euglycemic without MetS (Euglycemic group); metabolic syndrome patients without T2D (MetS group) and metabolic syndrome patients with newly diagnosed type 2 diabetes (Type 2 diabetic group).th rib and the iliac crest in the former case and at the height of the major trochanter using a metric tape in the latter, with approximations of 0.5 cm in both cases.Anthropometrical parameters were evaluated as follows: weight was determined using electronic scales with an approximation of 0.1 kg and a capacity of up to 200 kg; height was measured with a stadiometer with an approximation of 0.5 cm; BMI was calculated by dividing the weight in kilograms by the square of the height in meters; blood pressure was measured twice consecutively using a mercury sphygmomanometer; and waist and hip circumference was measured at the natural indentation between the 10Venous blood samples were collected from patients after 12-h overnight fasting and placed in a BD Vacutainer® Rapid Serum Tube . To separate serum from blood cells, samples were centrifuged at 2,000g for 15 min at 4°C following 5-min clotting. Freshly separated serum was employed for determination of lipid profile and hydrocarbonated parameters, and the remaining aliquots of serum were stored at −80°C until they were assessed for circulating levels of RBP4 and lipoprotein subfractions.Glucose, total cholesterol and triglycerides were measured by means of enzymatic assays and HDLc using a direct method and a Beckman LX-20 autoanalyzer . The intraseries coefficient of variation was <3.5% for all these determinations. LDLc was calculated using Friedewald's formula for values of triglycerides below 300 mg/dl. Apolipoproteins (Apo) AI and B were determined by immunonephelometry  (intra-assay coefficient of variation <5.5%) and insulin was determined by immunochemiluminescence with an Immulite analyzer  (intra-assay coefficient of variation <4%). Insulin resistance was estimated by the homeostasis model assessment of insulin resistance (HOMA-IR) index (fasting insulin (µIU/ml) x fasting glucose (mg/dl) /405). Serum RBP4 concentrations were measured by nephelometry . This method has a sensitivity of 0.1 µg/ml. The intra- and interassay coefficients of variation were 3.1% and 2.2%, respectively.LDL and HDL subfractions were separated using the Quantimetrix Lipoprint system  th and 75th percentiles for parametric and non-parametric data, respectively. Qualitative data were expressed as percentages. Data with normal distribution were compared with a one-way analysis of variance (ANOVA) and those without normal distribution were compared with a Kruskal-Wallis, followed in each case by a post hoc test  or an independent T-test or Mann-Whitney U when the dependent variable was not normally distributed. The Chi-Square test was used to compare proportions. Spearman's correlation coefficient was employed to measure the strength of the association between RBP4 and anthropometric and metabolic parameters. In the multivariable regression models, the relationship between two or more explanatory variables (independent variables) and a response variable (dependent variable) was evaluated by fitting a linear equation to the data obtained. After identification of independent variables, these variables were introduced as covariates in a univariate general linear model. All the tests had a confidence interval of 95% and differences were considered significant when p<0.05.The statistics program SPSS version 17.0 was employed for statistical analysis. Continuous variables were expressed as mean and standard deviation (SD) or as median and 25This study analysed a total of 299 patients (102 men and 197 women). When the distribution of RBP4 was assessed according to gender, we found it to be significantly higher in men than in women .Anthropometric and biochemical parameters for obese patients with MetS, T2D and euglycemia are shown in In contrast, no differences were observed among the patient groups in relation to BMI (p = 0.444), total cholesterol (p = 0.152) or LDLc (p = 0.303). Serum levels of RBP4 were higher in MetS and T2D patients than in euglycemic subjects and were similar for the two insulin-resistant states.Spearman correlation analysis demonstrated the strongest correlation between RBP4 and triglycerides , which was observed in the whole population. This positive correlation was detected for each of the populations studied –euglycemic, MetS and T2D- as shown in In multivariable regression models, the association of RBP4 with sex, age, BMI, waist circumference, total cholesterol, HDLc, LDLc, triglycerides, glucose, insulin and HOMA-IR was evaluated in each case as a potential independent predictor using the stepwise method. Our results showed that total cholesterol, triglycerides and gender were independently associated with RBP4 . To furtLDL and HDL subfractions were also analysed in a subpopulation of 80 subjects to determine the link between plasma RBP4 and lipoprotein metabolism. An altered lipoprotein profile -higher VLDL and sdLDL percentages and lower HDL percentage with a more proatherogenic profile- was associated with insulin-resistant state . This waThe current study provides evidence that serum RBP4 levels are higher in middle-aged morbid obese subjects with MetS and T2D than in euglycemic ones. We also show that triglycerides are the main independent predictor for determining systemic RBP4 levels, independently of the degree of insulin resistance. In addition, we demonstrate an independent association among small HDL percentage and LDLc, HDLc, triglycerides and RBP4, but not between LDL particle size and RBP4. Our data suggest a central role of this adipokine in the hypertriglyceridemia associated with insulin-resistant state and point to it as an independent predictor of the formation of small HDL.In accordance with previous reports In the present study we have observed a positive correlation between RBP4 and waist circumference –a subrogate marker of central obesity- which is in line with previous findings It has previously been shown that obesity, presence of MetS and T2D -all pathogenic conditions accompanying insulin resistance- are associated with increased serum RBP4 levels In this context, growing evidence suggests that RBP4 plays a more important role in lipid metabolism than in insulin resistance. Several studies in humans have shown that RBP4 levels are positively correlated with those of triglycerides but not with insulin resistance The possible effect of this adipokine on lipoprotein subfractions has been the focus of little research until now, but our findings suggest a strong association between RBP4 and triglycerides and indicate that RBP4 is not directly involved in determining LDL particle size, despite the negative correlation between the two. In a multiple linear regression, the independent predictors of LDL particle size included LDLc, HDLc and triglycerides, but not RBP4. This finding contrasts with the results of two previous studies in which RBP4 was independently associated with sdLDL Patients with insulin resistance exhibit an increased flux of free fatty acids to the liver that increases the cytosolic triglyceride storage pool and promotes VLDL production. In the presence of increased plasma levels of VLDL and normal activity of cholesterol ester transfer protein (CETP), VLDL triglycerides can be exchanged for LDL- and HDL cholesterol. This exchange produces LDL particles enriched in triglycerides, which are rapidly lipolyzed by hepatic lipase, resulting in smaller, denser particles. Numerous studies have demonstrated that the predominance of sdLDL particles  correlates with the progression of atherosclerosis and earlier and more severe cardiovascular disease Therefore, our results provide further evidence that RBP4 is involved in different stages of the atherosclerotic process; on the one hand, it may be partially responsible for the enhancement of triglyceride levels, which, in turn, promotes the production of VLDL. On the other hand, it seems to be implicated in the formation of small HDL.We should point out that our study has several limitations. First, causality could not be assessed due to the cross-sectional nature. Therefore, a longitudinal study needs to be performed in the future in order to explore further the independent association of triglycerides, formation of small HDL and RBP4 levels. Second, our results cannot be applied to the general human population, since our population was exclusively Caucasian.To summarise, circulating RBP4 may play a major role in lipid metabolism in morbid obesity in which it is associated with an increase in triglyceride levels and contributes to the formation of small HDL, independently of insulin-resistant state. These findings are in accordance with the premise that systemic RBP4 levels promotes the development of atherosclerosis.


Rhinella marina) in Australia contain several types of parasites, raising concern that the toads may increase rates of parasitism in local anuran species. We sampled cane toads and sympatric native frogs  at the southern invasion front of cane toads in north-eastern New South Wales (NSW). We dissected and swabbed these anurans to score the presence and abundance of nematodes , myxozoans, and chytrid fungus. To determine if cane toad invasion influences rates of parasitism in native frogs, we compared the prevalence and intensity of parasites in frogs from areas with toads, to frogs from areas without toads. Contrary to the situation on the (rapidly-expanding) tropical invasion front, cane toads on the slowly-expanding southern front were heavily infected with rhabditoid lungworms. Toads also contained gastric-encysting nematodes, and one toad was infected by chytrid fungus, but we did not find myxozoans in any toads. All parasite groups were recorded in native frogs, but were less common in areas invaded by toads than in nearby yet to be invaded areas. Contrary to our predictions, toad invasion was associated with a reduced parasite burden in native frogs. Thus, cane toads do not appear to transfer novel parasites to native frog populations, or act as a reservoir for native parasites to ‘spill-back’ into native frogs. Instead, cane toads may reduce frog-parasite numbers by taking up native parasites that are then killed by the toad’s immune defences.One of the most devastating impacts of an invasive species is the introduction of novel parasites or diseases to native fauna. Invasive cane toads ( This process might reduce invader viability, and hence slow the advance of the invasion front . AlthougRhinella marina) through Australia provides a robust model system within which to explore the impacts of an alien species not only in terms of direct effects . First, we determined the prevalence and intensity of parasites in cane toads, to test the prediction that toads would exhibit heavy parasite loads even at the invasion front . Second, we compared the prevalence and intensity of parasites in native frogs from areas where toads had recently invaded, to those of frogs in adjacent areas yet to be invaded by toads. If toads affect parasitism rates of native frogs (either by direct transmission or spillback), we expect to see higher rates of parasitism in frogs from areas that also contain toads, than from areas where toads have yet to arrive. Overall, our study was designed to clarify the consequences of cane toad invasion for host-parasite biology (of both toads and native frogs) in southern Australia.22.12.1.1Frogs and toads were obtained from roads adjacent to waterbodies  between the towns Kingscliff and Broom’s Head, along the northern coast of NSW . This reLimnodynastes peronii and Litoria latopalmata) for which we were able to obtain robust sample sizes  occurs in cane toads in Australia, whereas native frogs are infected by at least three species of the native lungworm, all currently lumped under the name R. cf. hylae  occurs in the gall bladders of cane toads (R. marina) and Australian frogs belonging to at least six families , or examining the lungs (n = 5), stomach (n = 3), or gall bladder (n = 21).Each road-killed anuran was placed into a separate clean 17 × 18 cm plastic, zip-lock bag. Prior to dissection, each specimen was measured , weighed (g), and the sex determined .To detect 2.2.2L. peronii.Parasitic nematodes have been reported within many organs of anurans . We focu2.2.3B. dendrobatidis infection by firmly running a sterile cotton swab 10 times across the animal’s ventral surface, sides, groin and underside of foot , internal positive controls were included in one replicate of each sample. Where inhibition was detected, a 1/100 dilution of the originally extracted DNA was prepared to dilute inhibitory agents and the reaction repeated. Where amplification occurred in any of the replicates, the mean number of genomic equivalents detected at a standardised cycle threshold was calculated, providing a relative measure of individual infection load.Anurans were swabbed for  of foot . Each spocedures , using a2.2.4Cystodiscus axonis and Cystodiscus australis) have been discovered in the gall bladders of anurans on the east coast of Australia; they are morphologically indistinguishable from some other myxozoan species . Anurans were tested for presence or absence of myxospores; individual myxospores were not counted. Recently, two new myxosporean species  and frog phenotype , with presence or absence of the parasite as the dependent variable. Infection intensities were compared among species using one-factor ANOVA. In addition, to compare the mean intensity of parasites in native frogs between “toad-present” sites and “toad-absent” sites, we conducted ANOVAs with the same independent variables as above, plus the site was included as a random factor; the dependent variable was the number of parasites per host . All statistical tests were conducted using JMP Pro .3R. marina) than in native frogs  that were sympatric with cane toads had fewer, not more, parasites than did conspecific frogs that we collected from nearby sites where toads were absent. This comparison is based upon only two anuran species, because we were unable to obtain enough Litoria nasuta from “toad-present” sites.Prevalence and intensity of parasites were higher in cane toads ; the two native species were infected at similar intensities . The mean intensity of lungworm infection was generally low in both native frogs (<5) and in cane toads (<20), although some cane toads contained more than 50 lungworms  but not its species . Frogs from toad-invaded areas had a lower prevalence of lungworm infection . Lungworm intensity showed the same pattern as prevalence; that is, frogs from “cane toad-absent sites” contained more lungworms per infected host , frog species  or frog mass . The overall parasite burden due to Rhabdias infection was thus higher in frogs collected from “toad-present” versus “toad-absent” sites .Overall, the prevalence of adult ungworms . Both L.t” sites A. ANOVA ted host B, as wel3.2χ22 = 0.94, p = 0.62; L. peronii overall , but L. peronii and L. latopalmata were infected at similar intensities . The numbers of parasitic larval nematodes detected per host showed similar patterns to Rhabdias lungworm infections. That is, cane toads contained more larval nematodes than did native frogs, and frogs collected from “toad-absent” sites tended to be more heavily infected than were frogs of the same species from “toad-present” sites  or body mass . The intensity of infection with larval nematodes in frogs from “toad-present” sites did not differ significantly from those in “toad-absent” sites , and nor did overall parasite burden due to these taxa . We detected no larval nematodes in L. latopalmata from “toad present” sites , and cane toad-absent sites (n = 1). These were detected under the throat (n = 1), under the left armpit (n = 1), and near the groin (n = 2). Physaloptera nematodes have not been previously reported from L. peronii  from a “toad-absent” site . That difference is consistent with mathematical models of transmission probability that attribute the lack of lungworms in tropical invasion-front toads to the rapid rate of spread of the invader in that system , and compared parasite prevalence and intensity in frogs sympatric with cane toads, to frogs from sites where cane toads were absent. Laboratory experiments and field surveys on tropical Australian anurans have concluded that cane toads are unlikely to transmit rhabditoid parasites to native frogs . Likewist system . The appt system . The hist system . Nonethe4.1Rhabdias lungworms were found in all of the anuran species that we examined, with cane toads containing a significantly higher intensity overall  were able to retain even more lungworms than did cane toads. These results suggest that, under the right conditions, native frogs may be susceptible to R. pseudosphaerocephala infection  associated with cane toad presence. overall ; Table 3in frogs . In fact methods . AlthougR. hylae , a parasocephala . Most nanfection . Howevernfection . HoweverCounter to our initial predictions, these two species of native frogs not only are not at risk from invading cane toads introducing parasites; the toads may actually reduce rather than increase the parasite burdens of native frogs. The mechanisms underlying this unanticipated effect warrant further research. Plausibly, cane toads might selectively consume insects that otherwise would transmit parasites to native frogs; or cane toad parasites might trigger changes to frog immune systems that make the frog more capable of dealing with infective larvae of native parasite taxa. More likely, many native parasites are taken up by cane toads but fail to survive because of the host’s ability to mount an effective immune defence. Experimental studies show that native frogs can deal effectively with the toad’s lungworm species in this way , suggestR. pseudosphaerocephala was as high (70%) in cane toads at the southern invasion front as in well-established cane toad populations in Queensland  in response to low host-densities . However, this may not be the case with the southern cane toad invasion. Nematodes are particularly vulnerable to desiccation  identified eight species of nematodes across all anuran taxa. All species were present in cane toads, and six of the eight were present in native frogs; however, native frogs were never detected with more than two different nematode species concurrently, whereas cane toads could bear up to three. Cane toads host more than 80 species of helminths across their entire geographic range, of which at least 30 are nematodes . Thirtee4.3B. dendrobatidis, and hence spread the fungus to native frogs  in 1985  were Myxidium-free, and that the parasites of Brazilian cane toads are genetically distinct from the Australian species ; cane toads then acquired Myxidium from native frogs and assisted its spread. In more recent work, Myxidium in native Australian frogs than was first realised; two new species of myxozoa  have now been described (Surveys in eastern Australia first detected myxosporean spores within the gall bladders of Australian cane toads in 1983, and native frogs ( in 1985 . M. immeon front . Hartiga species . This faescribed .L. peronii and L. nasuta, both species known to carry this parasite (Cystodiscus infection rates in native frogs.parasite . Howeverparasite . Our mic4.5Cane toads are continuing to spread down the east coast of Australia  and will


Microglia are the primary immune cells of the brain whose phenotype largely depends on their surrounding micro-environment. Microglia respond to a multitude of soluble molecules produced by a variety of brain cells. Macrophage colony-stimulating factor (M-CSF) is a cytokine found in the brain whose receptor is expressed by microglia. Previous studies suggest a critical role for M-CSF in brain development and normal functioning as well as in several disease processes involving neuroinflammation.Using biopsy tissue from patients with intractable temporal epilepsy and autopsy tissue, we cultured primary adult human microglia to investigate their response to M-CSF. Mixed glial cultures were treated with 25 ng/ml M-CSF for 96 hours. Proliferation and phagocytosis assays, and high through-put immunocytochemistry, microscopy and image analysis were performed to investigate microglial phenotype and function.1-42 peptide.We found that the phenotype of primary adult human microglia was markedly changed following exposure to M-CSF. A greater number of microglia were present in the M-CSF- treated cultures as the percentage of proliferating (BrdU and Ki67-positive) microglia was greatly increased. A number of changes in protein expression occurred following M-CSF treatment, including increased transcription factors PU.1 and C/EBPβ, increased DAP12 adaptor protein, increased M-CSF receptor (CSF-1R) and IGF-1 receptor, and reduced HLA-DP, DQ, DR antigen presentation protein. Furthermore, a distinct morphological change was observed with elongation of microglial processes. These changes in phenotype were accompanied by a functional increase in phagocytosis of AβWe show here that the cytokine M-CSF dramatically influences the phenotype of adult human microglia. These results pave the way for future investigation of M-CSF-related targets for human therapeutic benefit. Microglial cells are brain-resident immune cells that have many protective roles but can also contribute to neurological disease processes. The functional phenotype of microglia depends on the cell types and specific activating factors in their surroundings, and for this reason microglia are said to have an adaptive or acquired phenotype, reflecting their response to a collection of external signals . Secreteet al. (1997) have also demonstrated M-CSF expression in neurons of the adult human brain [One molecule that can influence microglial phenotype is the cytokine macrophage colony-stimulating factor (M-CSF). M-CSF is found in the brain and its receptor is expressed by microglia ,12. M-CSet al. (1996) found that sense PU.1 expression constructs increased M-CSF-dependent proliferation in mouse bone-marrow macrophages, and antisense PU.1 constructs reduced proliferation in response to M-CSF [c-fms promoter [Signaling through the M-CSF receptor (CSF-1R) is required for the development and differentiation of microglia  and M-CSto M-CSF . They alpromoter .et al. (2009) reporting beneficial effects of M-CSF on cognitive impairment and Aβ1-42 deposition in a mouse model of Alzheimer’s disease, it has been suggested that increased M-CSF expression could contribute to Alzheimer’s disease pathogenesis [et al. (2001) cultured glia from adult human brains and found that M-CSF was elevated in Alzheimer’s disease compared to non-demented microglia [While M-CSF is important for normal brain development and function , severalogenesis ,30. Lue icroglia . M-CSF ricroglia . On the icroglia . M-CSF iDespite this comprehensive body of literature, there are several caveats which may prohibit the linking of functional studies to observations in human brain tissue. Although most of the research on M-CSF and microglia has been carried out using rodent cells and models, it is becoming increasingly clear that there are important differences between rodent microglia and their human counterpart ,34. Age Autopsy human adult brain tissue was obtained from the Neurological Foundation of New Zealand Human Brain Bank under the University of Auckland Human Subjects Ethics Committee. Biopsy adult human brain tissue was obtained from patients undergoing surgery for intractable temporal lobe epilepsy and was approved by the Northern Regional Ethics Committee with informed consent from all tissue donors. The specimens used in this paper were from confirmed temporal lobe epilepsy cases with varying degrees of mild and moderate to marked mesial temporal sclerosis . Patients had taken a range of medications alone or in combination, including phenytoin, tegretol, topiramate, lamotrigine, and sodium valproate. There were no obvious associations between the results of any of the reported experiments and medication use or degree of sclerosis.2+ and Mg2+ free, Gibco BRL, Carlsbad, CA, USA). The meninges and visible blood vessels were removed. The tissue was then diced into small (approximately 1 mm3) cubes and placed in 10 ml/g tissue warm enzyme dissociation mix containing 2.5 U/ml papain  and 10 U/ml DNase  in HBSS and incubated for 30 minutes at 37°C with agitation. An equal volume of DMEM/F12 supplemented with 10% FBS, 1% penicillin-streptomycin-glutamine  , streptomycin (100 μg/ml) and L-glutamine (0.29 mg/ml)) was added to the tissue which was then gently triturated. The cell suspension was passed through a 100 μm nylon cell strainer , centrifuged for 10 minutes at 160 g, the pellet resuspended in 20 ml medium and plated into 75 cm2 tissue culture flasks  which were incubated at 37°C in 95% air/5% CO2. After 24 hours, the debris and unattached cells were removed, centrifuged for 10 minutes at 160 g and replated onto the adhered cells for a further 24 hours. Finally, the debris was removed and the cells carefully washed with medium. Cells were cultured for one to two weeks (in the same medium as for isolation) prior to plating for experiments at 50,000 cells/ml on 96-well plates.Glial cells were isolated from adult human brain tissue as previously described ,39. ApprMixed primary human glial cell cultures were given two concentrations of 25 ng/ml M-CSF at 0 and 48 hours. Total time of M-CSF treatment was 96 hours.Following 72 hours exposure to 25 ng/ml M-CSF, 10 μM BrdU was added to the cells for 24 hours. Cells were washed twice with PBS to remove excess BrdU and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature (RT). For immunocytochemistry, cells were first incubated with 2 M HCl at 37°C for 30 minutes. Cells were then washed twice in 0.1 M borate buffer (pH 8.5) and three times in PBS before applying anti-BrdU antibody.Cells were fixed in 4% PFA for 15 minutes at RT then washed for 10 minutes with phosphate-buffered saline containing 0.2% Triton X-100 (PBS-T). Antibodies were diluted in immuno-buffer (PBS-T containing 1% goat serum and 0.04% merthiolate). Cells were incubated with primary antibody , phagocytosis assays were performed as previously described [1-42 was added to the cells for 24 hours. Cells were washed twice with PBS to remove excess Aβ1-42 and fixed with 4% PFA for 15 minutes at RT. Thioflavin S was used to visualize phagocytosed Aβ1-42[To evaluate the effect of M-CSF on primary adult human microglial phagocytosis of amyloid-βescribed ,40. FollImmunocytochemical, phagocytic and morphological observations have been quantified using a Discovery-1 automated fluorescence microscope  and Metamorph  image analysis system as previously described ,41. Resu2, P = cell perimeter, A = cell area) to determine cell shape.For quantification of microglial morphology, the Journal ‘Microglial Shape’ was written in Metamorph. The journal automatically thresholded each image to isolate CD45-positive microglia, then applied the Integrated Morphometry Analysis tools Elliptical Form Factor (length/breadth) and Shape Factor . Experiments were replicated with cells from at least six different individuals. Number of replicates (n) for each experiment is indicated in figure legends. Statistical analysis was carried out using Primary mixed glial cultures containing microglia were prepared from biopsy and autopsy adult human brain tissue as previously described ,39.In mixed cultures of adult human glial cells, the M-CSF receptor CSF-1R) was expressed by microglia. We found that protein expression of CSF-1R coincided with microglial cell surface protein CD45 (leukocyte common antigen) and microglial transcription factor PU.1 ). However, treatment with M-CSF markedly increased microglial division ). M-CSF treatment resulted in a greater number of microglia expressing the cell division protein Ki67 Figure A and B a1-42 peptide, M-CSF-treated microglia were more phagocytic than vehicle-treated microglia . In the majority (six out of seven) of cases which had basal microglial HLA-DP, DQ, DR expression, M-CSF treatment was found to decrease the level of expression Figure A and B. On the other hand, no change in CD45 expression was observed with M-CSF treatment Figure . When thTo investigate the molecular events precipitated by M-CSF treatment, we looked for changes in factors known to be associated with PU.1. From the CCAAT enhancer-binding protein (C/EBP) transcription factor family, we detected an increase in C/EBPβ expression within microglia in mixed glial cultures with M-CSF treatment Figure . A quantDAP12 is a myeloid adapter protein found in microglia in the human brain . Signaliin vitro. Furthermore, on exposure to M-CSF (25 ng/ml) for 96 hours, microglial expression of CSF-1R was increased , which has previously been reported to be linked to M-CSF signaling . M-CSF tM-CSF has numerous interesting effects on adult human microglia, many of which may have significance for a range of neurological diseases.in vitro. The proportion of microglia that undergo division is specific to each individual patient. However, the result of increased microglial division with M-CSF treatment is consistent between cases.The numbers of PU.1 and CD45 immunopositive cells were increased by M-CSF treatment and we have shown that this is at least partially through an increase in microglial division. This division effect was immediately apparent as adult human microglia do not frequently divide when cultured in basic medium (DMEM/F12 + FBS + PSG). This observation is in line with previous studies noting a proliferation effect with M-CSF for human fetal microglia , and up-It has previously been shown that microglia in the rat brain, as well as the BV2 rodent microglial cell line, constitutively express a high level of PU.1 in both the ‘normal’ and post-injury state . We have1-42 peptide by microglia from different cases, regardless of the starting level of phagocytosis. In the context of Alzheimer’s disease it is thought that microglia may be helpful in clearing the brain of extracellular deposits of Aβ1-42 protein [1-42[Phagocytosis is an important innate function of microglia as part of their role to respond to cell injury and regulate the extracellular environment. We found that M-CSF increased adult human microglial phagocytosis of Aβ protein ,47. M-CSein [1-42. In anotein [1-42.We have previously found that PU.1 is necessary for basal phagocytosis in human adult microglia . DAP12 a1-42 which induces changes to their cytoskeletons necessary for phagocytosis. Even though the M-CSF-treated microglia are more rod-shaped and ‘ramified’ than vehicle-treated microglia, they have an increased propensity to phagocytose compared to control microglia of heterogeneous morphology.Another prominent effect of M-CSF on adult human microglia is their change in morphology to bipolar, elongated (‘ramified’) cells. Microglial morphology is presumed to relate to their function, although exactly how is currently unclear. Round ‘amoeboid’ microglia are traditionally viewed as activated, inflammatory microglia. The M-CSF-induced morphology change could be a sign of microglia being ‘primed’ towards a particular activation state. However, microglial phenotype is multifaceted and M-CSF-induced elongation doesn’t prohibit microglia from changing morphology when exposed to other molecules, for example Aβet al. (2012) used the same concentration of M-CSF as part of an ‘alternative’ macrophage/microglia activation polarizing protocol. Whereas macrophages under these conditions had more extended processes, no change was observed for microglia. It may be that the CD45 marker of microglia used here is better for observing morphological differences, or that their addition of IL-4 and IL-13 reduced the morphology effect seen in microglia [Durafourt icroglia . The M-Cicroglia .in situ in rodent models of neurological injury including ischemia [et al. (2002) also noticed proliferating cell nuclear antigen co-labeling with rod microglia and reduced expression of HLA compared to more rounded microglia [Elongated rod-shaped microglia have also been reported ischemia ,52. Graeischemia . Rod-shaischemia  and in sischemia . In concicroglia .in vitro correlates of the ‘rod’ microglia reported in diseased adult human brain tissue, this will provide an invaluable tool for investigation into this particular microglial phenotype. Furthermore, our method for quantifying microglial morphology is a quick, high throughput and unbiased way of assessing these changes, compared to laborious and subjective quantification by eye.Rod-shaped microglia are relatively under investigated  and our To determine the functional relevance of the morphology change and to look further at the activation state of these M-CSF-exposed microglia, we investigated whether their expression of HLA was affected. HLA-DP, DQ, DR is an inducible protein and we find that its expression by microglia varies widely between cases. We asked whether this inducible expression of HLA-DP, DQ, DR was modulated by M-CSF and found that, despite an increased number of microglia, fewer microglia express high levels of HLA-DP, DQ, DR with M-CSF. HLA-DP, DQ, DR expression by microglia is often taken to represent an ‘activated’ or inflammatory microglial phenotype. Our results suggest that M-CSF-treated microglia may be alternatively activated and have reduced antigen presentation capacity.et al., (2002) found that M-CSF-induced maturation increased MHC class II expression with IFNy [et al. (2012) have recently shown that the ‘alternative’ activating cytokine IL-4 increases HLA-DR mRNA expression whilst also inducing an elongated morphology in adult human microglia [This M-CSF effect of reduced HLA-DR has also been noted for human fetal microglia . Furtherith IFNy . This fiicroglia . OverallAn HLA-DQ-derived peptide has been found to have anti-proliferative effects , suggestet al. showing that increased PU.1 was associated with an activated microglial phenotype in rodents [Of particular interest in our study is that an up-regulation of PU.1 with M-CSF correlates, in general, with a less activated microglial phenotype , although phagocytosis was stimulated. This contrasts with a recent report by Ponomarev  rodents . The mosTo decipher the mechanisms by which the M-CSF-induced phenotypic changes occur in microglia, we looked for changes in factors known to be associated with the transcription factor PU.1.CCAAT enhancer-binding protein (C/EBP) transcription factors are expressed throughout the body including the brain ,57. The C/EBPβ plays numerous roles in activation and differentiation of macrophages . It has C/EBPβ forms heterodimers with members of its own family and interacts with several other transcription factors  includinDAP12 is a myeloid adapter protein found in microglia in the brain. We found that M-CSF treatment of adult human microglia increased their DAP12 expression. It has been shown to be involved in M-CSF-induced proliferation and survival of mouse bone marrow-derived macrophages . The conet al. (2002) demonstrated an upregulation of DAP12 in PU.1-rescued monocytic precursor cells and Weigelt et al. (2007) have shown that DAP12 expression is dependent on PU.1 via a binding site in the DAP12 proximal promoter [Henkel promoter ,49. Thuspromoter .c-fms transcript expression [et al. (2010) have also found that M-CSF increases microglial CSF-1R expression in the context of the rat axotomized facial nucleus [CSF-1R expression is shown here to be restricted to microglia, and not detected on other cell types in our cultures, both basally and with M-CSF treatment. In addition, we found that M-CSF increased the expression of its receptor on microglia. The increase in PU.1 found with M-CSF treatment is likely to directly increase CSF-1R levels as it has been reported to regulate pression . Yamamot nucleus .et al. (2004) found C/EBPβ to have a critical role in cell survival, in part by regulating expression of IGF-I. Furthermore, M-CSF was found to compensate for IGF-1 and could rescue IGF-1-deficient cells [Coincidentally, M-CSF treatment also increased microglial expression of the IGF-1 receptor. There have been a number of associations previously reported between the growth factors M-CSF and IGF-1 . Both fant cells . The ovein vivo role of IL-34 may differ between rodents and humans [Another CSF-1R ligand, IL-34, has recently been discovered and is expressed throughout the body, including the brain . Like M-d humans , and resin vivo implications of these results are yet to be determined. Therapeutically, it would be desirable to have the ability to modulate microglial phenotype towards a protective role. To this end, M-CSF-induced human microglia are worthy of further investigation.M-CSF dramatically influences the phenotype of adult human microglia. We have found that M-CSF has many fascinating effects on these human brain cells and has an important role in determining microglial phenotype and function in the context of the adult human brain. However, the 1-42 protein; BrdU: 5-bromo-2′-deoxyuridine; C/EBP: CCAAT enhancer-binding protein; CSF -1R: Colony-stimulating factor-1 receptor/M-CSF receptor; DAB: 3,3′-diaminobenzidine tetrahydrochloride; FBS: Fetal bovine serum; GFAP: Glial fibrillary acidic protein; HLA: Human leukocyte antigen; IGF-1: Insulin-like growth factor-1 receptor; IL: Interleukin; M-CSF: Macrophage colony-stimulating factor; PFA: Paraformaldehyde; SEM: Standard error of the mean.Aβ1-42: Amyloid- βThe authors have no competing interests.AMS, HMG and MD conceived and designed the experiments. AMS performed cell isolation and cell culture experiments, immunocytochemistry, image acquisition and analysis. AMS and MD interpreted data and wrote the manuscript. RLO, PSB, EWM, MAC and RLMF contributed materials and revised the manuscript. All authors read and approved the final manuscript.


In this study, according to some recently reported drawbacks with PS CpG, the adjuvant potential of liposomal PO CpG as a substitute for PS CpG was evaluated. CpG oligodeoxynucleotides (CpG ODNs) have been shown to have potent immunostimulatory adjuvant activity for a wide range of antigens. Due to susceptibility of phosphodiester CpG ODNs (PO CpG) to nuclease degradation, nuclease-resistant phosphorothioate CpG ODNs (PS CpG) were currently utilized in an Leishmania antigens (SLA) as a model antigen and distearoylphosphatidylcoline (DSPC) as a neutral lipid were employed to prepare liposomes. Susceptible BALB/c mice received buffer, SLA, Lip-SLA, Lip-SLA-PS CpG, Lip-SLA-PO CpG, SLA+PS CpG, or SLA+PO CpG subcutaneously 3 times with 3 weeks intervals and then were challenged with Leishmania major’s live promastigotes. Blood and spleen samples were analyzed to determine the level and type of antibodies and cytokines. The number of live parasites in the spleen of immunized mice was determined. Moreover, the lesion size progress was assessed weekly by footpad swelling measurement.Soluble The results showed that mice immunized with Lip-SLA-PS CpG or Lip-SLA-PO CpG developed a significantly smaller footpad swelling, higher level of anti SLA IgG antibodies before and after challenge, and lower spleen parasite burden compared with the control groups. However, there was no significant difference between mice received Lip-SLA-PS CpG and those received Lip-SLA-PO CpG. The results demonstrated that liposomal PO CpG ODN could be used instead of PS CpG ODN to overcome the possible drawbacks. Accelerating antigen-specific immune responses by 5-500-fold was shown when they are in close physical contact with immunogen (Cytosine phosphate guanine oligodeoxynucleotides (CpG ODNs) are DNA motifs with high frequency in bacteria, so mimic the immunostimulatory activity of bacterial DNA , 2. Theymmunogen -3. Whilemmunogen , 5, clinmmunogen . Despitemmunogen . Moreovemmunogen -11. Admimmunogen . In addimmunogen , transiemmunogen , lymphoimmunogen , and PS mmunogen  dependinmmunogen , 16-18. One strategy to protect and extend the activity of PO CpG is encapsulation of CpG ODNs into the liposomes , 20. MorInduction of cell-mediated immune (CMI) response to poorly immunogenic Ags is possible through encapsulation of Ags into the liposomes . LiposomLeishmania, a parasite transmitted by the bite of the sand fly, causes a group of diseases ranging from a self-healing cutaneous lesion (CL) to potentially fatal visceral form of disease, known as leishmaniasis. It was well established that people were infected with cutaneous leishmaniasis, naturally, or with leishmanization, won’t be infected with the disease again. Protection against leishmaniasis is associated with development of a Th1 type of immune response and activation of CD8+ T cell population . The mice were maintained in the animal house of Pharmaceutical Research Centre and fed with tap water and laboratory pellet chow . Animals were housed in a colony room 12/12 hr light/dark cycle at 21 °C with free access to water and food. Experiments were carried out according to the Ethical Committee Acts  of the Mashhad University of Medical Sciences, Mashhad, Iran.Leishmania major strain (MRHO/IR/75/ER) used in this experiment is the one which was used to prepare experimental Leishmania vaccine, leishmanin and leishmanization , and then the preparation was incubated in ice-water bath for 10 min and the parasites were lysed using freeze-thaw method followed by probe sonication  in an ice bath at 4 °C with 20 pulses of 15 sec each at medium amplitude. The supernatant of centrifuged lysate parasites was collected, dialyzed against buffer solution, sterilized using a 0.22 µm membrane, and stored at -70 °C until use. The protein concentration of the preparation was determined using BCA protein assay method .SLA preparation was carried out using the protocol developed by Scott et al  with minTwo type of the CpG ODNs  were used in this study which contains two CpG motifs, 20-mer termed 1826 (5´- ACG ACG TT-3´) with a nuclease-resistant phosphorothioate backbone (PS CpG ODN) and natural phosphodiester backbone (PO CpG ODN), with known Th1 type immunostimulatory effects on murine model , 41.Liposomes preparation and characterization Liposomes containing SLA and CpG ODNs were prepared using lipid film preparation method. Briefly, the lipid phase consisting of 20 µmol/ml DSPC  and 10 µmol/ml cholesterol  was dissolved in chloroform in a sterile tube. The solvent was then removed using rotary evaporator  resulting in deposition of a thin lipid ﬁlm on the tube’s wall. The lipid ﬁlm was then freeze – dried  overnight to ensure complete removal of the solvent. The lipid ﬁlm was then hydrated and dispersed in sterile buffer containing SLA (1 mg/ml) and CpG ODNs (200 µg/ml) at 65 °C. The resulting multilamellar vesicles (MLVs) were converted to large unilamellar vesicles (LUVs) using a bath sonicator . Then, vesicles size was reduced to 200 nm by a mini-extruder . To prepare liposomes containing only SLA (i.e. Lip-SLA), the same procedure was followed except the CpG ODNs was omitted. Particle size analyzer  was used to estimate the mean diameter and zeta potential of the liposomes. The concentration of SLA encapsulated in liposomes was determined using BCA protein assay kit . Spectrophotometry was used to determine the concentration of CpG ODNs encapsulated in liposomes by using their absorption at 260 nm. Dialysis method (cut-off 300 kD) has been used for separation of entrapped SLA and CpG OND from their free forms. Immunization of BALB/c miceDifferent groups of mice (ten per group) were subcutaneously (SC) immunized in their left hind footpad 3 times in 3-week intervals with one of the following formulations: HEPES buffer , SLA (50 µg SLA/50 µl buffer/mouse), Lip-SLA (50 µg SLA/50 µl liposome/mouse), Lip-SLA-PS CpG (50 µg SLA-10 µg CpG ODNs/50 µl liposome/mouse), Lip-SLA-PO CpG (50 µg SLA-10 µg CpG ODNs/50 µl liposome/mouse), SLA (50 µg SLA/50 µl buffer/mouse) plus PS CpG ODN in buffer (10 µg CpG ODNs/50 µl buffer/mouse), SLA (50 µg SLA/50 µl buffer/mouse) plus PO CpG ODN in buffer (10 µg CpG ODNs/50 µl buffer/mouse). Challenge with L. major promastigotes6L. major promastigotes harvested at stationary phase in 50 µl volume, at week 2 after the last immunization booster. Lesion development was recorded in each mouse by measurement of footpad swelling using a metric caliper . Grading of lesion size was done by subtracting the thickness of the uninfected contralateral footpad from that of the infected one.The immunized mice (seven per group) were challenged SC in the left footpad with 1×10Quantitative parasite burden in spleenL. major parasites in the spleen of mice was estimated by a limiting dilution assay method as described previously . The cells were stimulated in vitro with either SLA (10 µg/ml), Con A (2.5 µg/ml), or medium alone and incubated at 37 °C in 5% CO2 for 72 hr. The culture supernatants were collected and the levels of IL-4 and IFN-γ were checked using ELISA method according to the manufacturer’s instructions .Three mice from each group were sacriﬁced at week 2 after the last booster injection  and the spleens were aseptically removed. Mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation method from spleen cell suspension. The cells were washed and resuspended in RPMI 1640- and seeded at 2×10Statistical analysisP< 0.05 were considered to be statistically signiﬁcant.One-way ANOVA statistical test was used to assess the significance of the differences among various groups. In the case of signiﬁcant F value, Tukey–Kramer multiple comparisons test was carried out as a post-test to compare the means in different groups of mice. Results with Liposome characterizationAs shown in Table 1, liposomes used in this study were 230-310 nm in size as calculated by particle size analyzer with a mean diameter of 272.83±29.35 and 296.60±24.17 nm (n= 3) and the zeta potential of -23.20±1.49 and-33.57±1.94 mV for Lip-SLA-PS CpG and Lip-SLA-PO CpG, respectively. The amount of SLA entrapment measured by BCA protein assay was 52±5% (n= 3). The encapsulated PO CpG and PS CpG percent in liposomes were 9.8±1.2% and 9.2±0.8% (n= 3), respectively.Challenge resultsP< 0.001). There was no significant differences between the groups which received Lip-SLA-PS CpG and those immunized with Lip-SLA-PO CpG. The lesion size in mice immunized with Lip-SLA-PO CpG was significantly (P< 0.05) smaller than those received SLA+PO CpG at week 4 after challenge and even more at week 6 (P< 0.01). The lesion size in mice immunized with buffer, SLA alone or SLA+PO CpG was progressed continuously and no protection was observed in these groups and the footpad swelling reached a plateau after 6 weeks but interestingly, the groups that received Lip-SLA-PS CpG or Lip-SLA-PO CpG showed significant reduction in the swelling size from week 5 to 6 post-challenge.Lesion development was monitored by weekly measurement of footpad thickness . The lesSplenic parasite burdenL. major was determined in the spleen of different groups of mice at week 7 after challenge (P< 0.001) received Lip-SLA-PS CpG or Lip-SLA-PO CpG. Interestingly, no difference was observed between these two groups. However, there was no difference between the mice immunized with Lip-SLA-PS CpG or Lip-SLA-PO CpG and those received SLA+PS CpG or SLA+PO CpG.The number of viable hallenge . The estAntibody responseIn order to determine the type of generated immune response, the anti-SLA IgG antibodies and IgG1 and IgG2a subclasses were titrated before  and afteP< 0.001) higher levels of IgG1, IgG2a, and IgG total antibodies compared with the control groups (buffer or SLA) with just an exception in IgG1 titer so that the level of IgG1 antibody in sera of mice immunized with Lip-SLA-PO CpG was lower than SLA in soluble form. The level of IgG1, IgG2a, and IgG in the group of mice immunized with Lip-SLA-PS CpG were higher than groups immunized with Lip-SLA-PO CpG (P< 0.001) or SLA+PS CpG (P< 0.001). Comparison between groups received Lip-SLA-PO CpG or SLA+PO CpG showed significant differences (P< 0.001) in all of evaluated antibody isotypes. The level of IgG2a antibody in mice immunized with Lip-SLA-PO CpG was significantly (P< 0.001) higher than those received SLA+PO CpG, although there was no significant difference in IgG titer. Interestingly, the level of IgG1 antibody in groups received SLA+PO CpG was higher than those received Lip-SLA-PO CpG. The sera of mice immunized with Lip-SLA-PS CpG or Lip-SLA-PO CpG before challenge showed a signiﬁcantly  higher levels of IgG2a and IgG total antibodies and lower levels (P< 0.001) of IgG1 antibody compared with the control groups. The levels of IgG1, IgG2a, and IgG total in the group received Lip-SLA-PS CpG was significantly higher than group received Lip-SLA-PO CpG (P< 0.001). The mice immunized with Lip-SLA-PO CpG showed higher levels of IgG2a and IgG total and lower level of IgG1 than those immunized with SLA+PO CpG (P< 0.001). The ratio of IgG2a/IgG1 in sera of mice immunized with Lip-SLA-CpG was higher than all the other groups and even higher than this ratio before challenge .Challenge with hallenge . The serIn vitro cytokine production by splenocytesin vitro with SLA and was analyzed to determine the level of IFN-γ and IL-4 and cytokines indicative of Th1 and Th2 response, respectively. The results showed that the group of mice immunized with Lip-SLA produced the highest amount of IFN-γ compared with the control groups but the lowest amount compared with all the groups which received CpG ODNs (P< 0.001) either in free or liposomal form. Moreover, the level of IFN-γ in mice immunized with Lip-SLA-PS CpG or Lip-SLA-PO CpG showed significantly higher level than SLA+PS CpG or SLA+PO CpG, respectively (P< 0.001). Surprisingly, there was no significant difference between the groups received Lip-SLA-PS CpG or Lip-SLA-PO CpG The supernatant of cultured splenocytes given from mice sacrificed 2 weeks after the last booster restimulated in vivo was assessed and compared with PS CpG ODN. Moreover, their adjuvanticity effect was studied against leishmaniasis in murine model.In the current study, the role of liposomes in the protection of PO CpG ODN when administered in vivo  higher than those received SLA+PO CpG, but it was opposite regarding IgG1 level. The mice immunized with Lip-SLA-PO CpG showed the same response as those received Lip-SLA-PO CpG and stimulated Th1 type of immune response but it was not true for mice which were immunized with SLA+PO CpG. On the other hand, PO CpG in free form cannot induce Th1 type of immune response because of its instability.The results of antibody assay in groups received Lip-SLA-PO CpG or SLA+PO CpG showed a significant difference in IgG2a and gG1 subclasses. The level of IgG2a antibody in mice immunized with Lip-SLA-PO CpG was significantly  in groups immunized with Lip-SLA or SLA. At APCs level, CpG ODNs augment both activation and maturation of DC as well as the induction of proinflammatory cytokines . Thus, tLeishmania antigens such as gp63 or LAg, SLA has already shown better immune response when used in liposomal form (c= 54 ºC). The role of phospholipids in extent of induced immune response against leishmaniasis was assessed previously and results showed that DSPC is more efficient than DPPC, DMPC, or EPC regarding stimulation of Th1 type of immune response (Tc have greater membrane rigidity in vivo and therefore entrap solutes (e.g. antigens) and antigens remain with the carrier for a longer period of time (The idea of using SLA was based on a theory that says a cocktail Ag vaccine, which contains most of parasite antigens, provides a wider range of potentially protective epitopes than any of parasite’s antigens alone . In compmal form . Howeverresponse , 61. LipL. major infection was developed using SLA and PS CpG ODNs. The prepared formulation was appropriate to induce Th1 type of immune response against leishmaniasis (Leishmania (The effect of bilayer charge on the cellular uptake of liposomes is a matter of considerable discussion -64. Somemaniasis . Howevermaniasis . Lipophomaniasis . Sialic ishmania .The current data pointed out that there is no significant difference between PS CpG and PO CpG in terms of their ability to induce Th1 response when encapsulated into the liposomes. Therefore, our results suggested that liposomal form of PO CpG might be used instead of PS CpG in future vaccine formulations as an efficient adjuvant. In addition, the promising role of CpG ODNs and neutral DSPC liposomes as adjuvants to enhance stronger immune response against SLA was confirmed.


Primary closure of a large anterolateral thigh (ALT) flap donor site defect with the assistance of an external tissue expansion system is presented. The dimensions of this donor site (12 cm × 40 cm) and its percentage of leg circumference (34%) would make this site likely to require skin grafting or further flap coverage based on the results of previously published literature. The anterolateral thigh (ALT) flap has become a popular choice of reconstructive surgeons when soft tissues are required for a given defect. Although the donor site morbidity is generally thought to be minimal, a significant cosmetic deformity can result, especially when a skin graft is required for closure of larger donor sites –3. AdditWith regard to potential primary closure of the ALT donor site, several studies have been published to elucidate which defects are likely to necessitate a skin graft for closure. In their experience with 672 ALT flaps, Wei et al. stated that 40% of their donor sites, often those with width greater than 6 to 9 cm, would require a skin graft .In order to facilitate the closure of the donor site without the need for a skin graft, several techniques have been described. These techniques include additional flaps and internal tissue expansion, the placement of which has been described before, during, and after the time of flap harvest , 10. To The patient was a 55-year-old diabetic male who developed necrotizing fasciitis of the left foot and leg. After the infection was eradicated, a large soft tissue deficit was present. This deficit was reconstructed with an ALT flap  was then applied in the shoelace technique with six anchors as described in the product insert at the cranial aspect of the remaining defect. Mechanical creep could be appreciated within minutes, which then allowed for primary closure in this portion of the wound. The expansion system was then removed and reapplied in a caudal direction and the process was repeated. Simultaneously, an additional external tissue expansion system was utilized in a caudal to cranial fashion until the wound was closed with the expanders meeting in the middle of the incision Figures  and 3. TThe external tissue expanders were left in place for one week postoperatively to reduce tension on the closure and to provide further expansion. During this time, the patient experienced a sensation of tightness in the thigh but had no other complaints in regard to the donor site. The expanders were then removed at the bedside with only one dose of intravenous morphine required for the comfort of the patient. The appearance of the donor site at one month postoperatively is shown in We have described the utilization of an external tissue expansion system to aid in the primary closure of a large ALT donor site. According to the aforementioned published literature, the dimensions of this donor site (12 cm × 40 cm) would make it likely to require a skin graft or an additional flap procedure in order to achieve primary closure. Further, the width of this flap was 34% of the circumference of the thigh, which by the work of Boca et al. would also make the site unlikely to be amendable to primary closure.Skin grafting of the ALT donor site is a possible means of wound coverage that has several potential drawbacks, including but not limited to poor durability, contour irregularity, and the necessity of graft harvesting with resultant scarring.When compared to skin grafting, additional flap coverage of the donor site provides more durable tissue and lessens contour irregularity, yet the requisite of an additional donor site and any associated complications are not eliminated. To obviate the creation of an additional donor site, internal tissue expanders have been utilized at all stages of reconstruction; however, this involves at the very least a second procedure. Applying an external tissue expansion system that provided intraoperative expansion and continuous postoperative tension relief at the suture line, we were able to close this large donor site primarily, without sacrificing any additional tissue. Removal of the devices was performed at the bedside one week postoperatively, and coverage has been stable at a follow-up time of six months.


Exosomes are released from multiple cell types, contain protein and RNA species, and have been exploited as a novel reservoir for disease biomarker discovery. They can transfer information between cells and may cause pathology, for example, a role for exosomes has been proposed in the pathophysiology of Alzheimer's disease. Although studied in several biofluids, exosomes have not been extensively studied in the cerebrospinal fluid (CSF) from humans. The objective of this study was to determine: 1) whether human CSF contains exosomes and 2) the variability in exosomal protein content across individuals.CSF was collected from 5 study participants undergoing thoraco-abdominal aortic aneurysm repair (around 200 - 500 ml per participant) and low-density membrane vesicles were concentrated by ultracentrifugation. The presence of exosomes was determined by western blot for marker proteins, isopycnic centrifugation on a sucrose step gradient and transmission electron microscopy with immuno-labelling. Whole protein profiling was performed using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR).Flotillin 1 and tumor susceptibility gene 101 (TSG101), two exosomal marker proteins, were identified in the ultracentrifugation pellet using western blot. These markers localized to a density consistent with exosomes following isopycnic centrifugation. Transmission electron microscopy visualized structures consistent with exosomes in size and appearance that labelled positive for flotillin 1. Therefore, the pellet that resulted from ultracentrifugation of human CSF contained exosomes. FT-ICR profiling of this pellet was performed and 84-161 ions were detected per study participant. Around one third of these ions were only present in a single study participant and one third were detected in all five. With regard to ion quantity, the median coefficient of variation was 81% for ions detected in two or more samples.Exosomes were identified in human CSF and their proteome is a potential new reservoir for biomarker discovery in neurological disorders such as Alzheimer's disease. However, techniques used to concentrate exosomes from CSF need refinement to reduce variability. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller 'real life' clinical samples; a key challenge in the development of exosomes as translational tools. Exosomes are lipid and protein rich vesicles that are formed as part of the intra-cellular endosomal pathway . During in vitro, for example, primary cultures of rat cortical neurons [Exosomes represent a novel reservoir for biomarker discovery because they contain protein, messenger RNA and microRNA that has been demonstrated to change with the disease state of the affected organ ,9. To da neurons , differe neurons  and glia neurons ,13 have  neurons ,15. The  neurons ,16.in vitro evidence that exosomes may represent the mechanism of prion protein transfer between cells [In addition to being a source of biomarkers there is evidence that exosomes may mediate neurological disease. In Alzheimer's disease (AD), cleavage of amyloid precursor protein by the enzyme β-secretase occurs in a subset of endosomes and a fraction of the Aβ protein is released into the extracellular milieu in association with exosomes ,18. Furten cells ,25. In sen cells . Given tThe protein composition of a complex biofluid can be investigated using mass spectrometry -30. ThisCSF was collected from 5 patients undergoing thoraco-abdominal aortic aneurysm repair. None of the patients had a history of neurological illness. This study was approved by NHS Lothian research ethics committee and prospective informed consent was obtained from all patients. This patient group was chosen because they have a CSF drain inserted peri-operatively as part of routine clinical management and large volumes of CSF are available for study (around 200 - 500 ml). Men were recruited to this study because female sex hormones may alter exosome release. The CSF was carefully inspected for evidence of blood contamination before collection. Protease inhibitors and preservative  were added to the collected CSF. The collected CSF was stored in aliquots at -80°C before use .g for 10 minutes to pellet any shed cells, large membrane fragments and other debris. The supernatant was then centrifuged at 200,000 × g for 60 minutes. The pellet was resuspended in phosphate buffered saline (PBS) and then re-centrifuged at 200,000 × g for 60 minutes before final resuspension in PBS. The protein content of the exosomal fraction was determined using the BCA protein assay kit .After thawing, the CSF was vigorously vortexed then centrifuged at 15,000 × Samples were solubilised with Laemmli sample buffer and separated on a 1D SDS-PAGE gel before transfer to polyvinylidene fluoride  membrane. The membrane was probed with the following primary antibodies: mouse anti-human flotillin-1  and mouse anti-human TSG101 .g. Six fractions were collected from the gradient and stored for density determination on a DMA 35 N density meter  and for western blot analysis.Sample was diluted into the top fraction of a step gradient comprising layers of 2, 1.3, 1.16, 0.8, 0.5 and 0.25 M sucrose. The gradients were centrifuged for 2.5 hours at 100,000 × The exosomal fraction was mixed 1:1 with 4% paraformaldehyde. A drop of this solution was then placed on a Petri dish and a formvar-coated 200-mesh gold grid floated on top for 20 minutes. The grid was washed in PBS before low and high molecular weight blocking using 0.05 M glycine/PBS and 1% BSA/PBS, respectively. The grid was re-washed and transferred to a 1:200 dilution of the anti-flotillin-1 antibody or the isotype control in 0.02% Triton X-100 PBS for 45 minutes at room temperature. The grid was then incubated with a 10 nm gold conjugated anti-mouse IgG antibody  for 60 minutes at room temperature. The grid was washed again and incubated with 1% glutaraldehyde for 5 minutes. The grid was washed and then contrasted and embedded with 0.5% uranyl acetate/2% methyl cellulose. Excess fluid was removed, the grid allowed to air dry and then the grid was examined on a Philips CM120 BioTwin transmission electron microscope.Proteomic studies were performed on the ultra-centrifugation pellet from each study participant, with each pellet being analysed in duplicate as per previous studies . Initialm/z 200 and 3000, yielding 1 Mword time-domain transients.The proteins present in the ultra-centrifugation pellet were separated from the lipid components using a chloroform/methanol precipitation . Followihttp://www.scipy.org, and Matplotlib [The mass spectrometry data was processed using the DataAnalysis  software package version 4 SP4. The Progenesis LC-MS v2.6 software package  was used for the quantitative mass spectrometry. This package matched the peaks from the ion chromatograms of our 5 study participants by the elution time and m/z ratio. The quality of the matching was checked manually. This matching process allowed determination of the number of samples which contained a given protein or peptide. The ion current was integrated over the elution window, then normalised against the total ion current of the chromatogram, and comparative quantitation of the abundance of an ion detected in 2 or more samples was calculated. Data analysis was performed in the python language with the Scipy tplotlib  packages3 .CSF was collected from 5 patients whose demographics are presented in Table 3 Figure . IsopycnTEM revealed a number of structures of exosomal size and appearance Figure  but therth percentile (25% of ions had a CV < 68%) and the median was 81%  and isobaric tag for relative and absolute quantification (iTRAQ). Both thWe have identified exosomes in the CSF from humans. These cell-derived particles represent a new reservoir for neurological disease protein and RNA biomarker discovery. However, the techniques used to concentrate exosomes from CSF need refinement to improve the quality of exosome isolation. In this study we used relatively large starting volumes of human CSF, future studies will focus on exosome isolation from smaller 'real life' clinical samples; a key challenge in the development of exosomes as translational tools.AD: Alzheimer's disease; CNS: Central Nervous System; CSF: Cerebrospinal fluid; ESCRT-I: Endosomal Sorting Complex Required for Transport; FT-ICR: Fourier transform ion cyclotron resonance mass spectrometry; TEM: Transmission electron microscopy; TSG101: Tumor susceptibility gene 101.The authors declare that they have no competing interests.JMS isolated exosomes and performed density studies, TEM and mass spectrometry with supervision of JWD  DJW (for exosome isolation), PB (mass spectrometry), CLM (mass spectrometry) and SW (mass spectrometry). CB performed immunoblotting on exosomes. TSW and RTAC identified and recruited patients and organised sample collection. All authors read and approved the final manuscript.Mass Spectrometry data. Whole dataset from FT-ICR MS on human CSF exosomes.Click here for file