Central nervous system infection is a daily concern in neurointensive care; however, diagnosis remains difficult because classical criteria based on cerebrospinal fluid (CSF) analysis are difficult to interpret in post-trauma or neurosurgery patients after recent bleeding. A rapid, specific, sensitive test to diagnose CSF infection would help streamline therapeutic decisions in clinical practice and limit the risk of multiresistant bacteria. We hypothesized that polymorphonuclear neutrophil (PMN) phenotype and radical oxygen species (ROS) production in CSF may be specific to the presence of infection.3. Analysis of PMN phenotype was performed using flow cytometry . ROS production was analyzed through luminometry (luminol).This study included 30 patients with suspected CSF infection with ventricular hemorrhage requiring external ventricular drainage, and 13 patients after trauma or surgery. Criteria for evaluating CSF infection included positive culture and > 100 leukocytes/mmInfected CSF exhibited higher production of ROS compared with noninfected CSF. PMNs in CSF exhibited low CD16 and high annexin V expression, suggesting apoptosis.Measurement of ROS production may discriminate infected from noninfected CSF. This simple test would be easy to employ in clinical practice to improve CSF infection management. Diagnosis of meningeal or intracranial infection remains difficult in post-trauma or neurosurgical patients for several reasons. Clinical symptoms often are mild and nonspecific and sometimes are masked by corticoids or therapeutic hypothermia. Additionally, classical criteria based on cerebrospinal fluid (CSF) analysis, such as pleocytosis with a high proportion of polymorphonuclear neutrophils (PMNs), low glucose, and high protein levels, are difficult to interpret soon after bleeding or surgical procedures. Finally, direct bacteriological examination results may be negative because of concomitant antibiotic therapy for another treated infection . BecauseVentriculostomy catheterization, or external ventricular drainage (EVD), is an important and frequently used invasive procedure in neurosurgical and intensive care practice. CSF drainage is a key component in the treatment of acute brain injury and represents the mainstay of emergency treatment for hydrocephalus, especially in cases of ventricular hemorrhage. However, it is an invasive procedure that can expose the patient to CNS infection ; in fact2-) into phagocytic vacuoles. Superoxide production and its reaction products are collectively referred to as ROS; they can be detected and measured by luminescence or by fluorescence after the oxidation of reactive components [PMNs are the first line of defense in the innate immune response to bacterial infection, and their activated phenotype may constitute a hallmark of infection . The kilmponents -9.We hypothesized that PMN phenotype and level of ROS production may be altered in infected CSF compared with noninfected CSF. In the present study, we studied PMN activation markers and ROS production in CSF from patients with EVD according to status of infection. We also applied this test in CSF obtained by lumbar puncture after trauma or surgery. Finally, we studied the stability of ROS measurements under different experimental conditions relevant to clinical practice.This study was approved by the ethical committee of our institution (IRB 00006477). The study included patients who were admitted to the intensive care unit (17 beds) of our teaching hospital during a 1-year period, particularly patients with ventricular hemorrhage requiring EVD. The physician in charge was not aware of the study investigations, and patient management was independent of the results. CSF samples were treated rapidly (within 2 h) in the laboratory of the Department of Critical Care and Anesthesia and analyzed by flow cytometry in the laboratory of hematology, both in the hospital.3; (2) CSF was considered "noninfected" if it contained > 100 leukocytes/mm3 but the results of direct examination and culture were negative after 72 h; (3) CSF was considered "negative" if the leukocyte count was < 100 leukocytes/mm3 and the results of direct examination and cultures were negative after 72 h [3.Four different categories were used to describe CSF infection status: (1) CSF was considered "infected" (ventriculo-meningitis) when it was positive for bacterial infection upon direct examination or in culture and contained > 100 leukocytes/mm3;  CSF was For each patient, we collected data regarding demographic characteristics, indication for EVD, classical clinical signs of inflammation and CSF analyses , treatment regimen at the time of sampling, and outcome. We also noted other concomitant sites of infection and antibiotic regimen, if any.-7 M of phorbol 12-myristate 13-acetate ; each condition was assayed in duplicate. Immediate analysis was conducted during a 20-min period using a luminometer . The signal was recorded for a duration of 1 second every minute and was recorded as relative light units (RLU). Results were expressed as area under the curve (AUC) of luminescence during the 20 min.CSF (250 μl) was diluted in Hanks' Balanced Salt Solution  to a final volume of 1 ml and incubated with luminol  for 10 min at 37°C in the dark. As a functional test, a control was compared with a sample that had been stimulated with 10g, room temperature) and cell washing in HBSS. Measurements obtained in blood PMNs from healthy volunteers with the same protocol (40 μl) were considered reference values for basal PMN parameters.For some samples, we performed several measurements to test reproducibility of the luminometric analysis under different conditions that might be encountered in clinical practice. We chose three different experimental conditions: (1) measurements after a 2-h wait at room temperature; (2) after a 2-h wait at 4°C; and (3) after centrifugation , CSF cells were resuspended in 50 μl of CSF and incubated in the presence of specific antibodies for 30 min in the dark in a 37°C water bath. Anti-CD16-Phycoerythrine-Cyanine 5 (PC5) antibody  was used for PMN selection, and phenotype was assessed using anti-CD11b-Phycoérythrine (PE) or anti-CD62L-PE . Nonspecific binding of antibodies to cell Fc receptors was assessed with a control isotype. Red cells were eliminated after incubation with lysing solution  for 10 min in the dark at room temperature and washing with phosphate buffered saline . Next, the technical procedure was performed according to the manufacturer's recommendations until the flow cytometric analysis . The Quantitative Flow CytoMetry flowmeter was used to convert mean fluorescence intensity into AB/C . The regression slope of correlation between mean fluorescence intensity and AB/C never varied over time and ranged between 0.999 and 1.After centrifugation . This technical procedure was performed according to the manufacturer's recommendations.P < 0.05), 2-by-2 between-group comparison was performed.Quantitative variables were expressed as median and interquartile range (IQR). Analysis was performed using nonparametric Mann-Whitney and Kruskal-Wallis tests; when significant , parenchymal hematoma (n = 6), operated tumors (n = 3), and severe brain trauma (n = 1). Patients' mean age was 46  years, and they presented with a mean SAPSII of 42  at admission. Four patients died during the study of causes related to the severity of their intracranial vascular pathology. During the same period of time, we also tested CSF samples obtained by lumbar puncture from patients with suspected infection a mean of 8  days after trauma or surgery .Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Acinetobacter baumanii, and Corynebacterium. Table According to the protocol, CSF was sampled by EVD in response to fever (> 38°C). Fifteen patients met the criteria for noninfected CSF, seven for infected CSF, and eight for negative CSF. The microorganisms observed in the seven infected CSF cases were At the basal state, ROS production was significantly higher in the infected group than in the noninfected and negative groups Figure . After s3, IQR 1295-2925) than in noninfected  and in negative samples . Noninfected CSF had a lower leukocyte count than negative samples (P = 0.0167). ROS measured in CSF differed significantly among the three groups: negative CSF ; noninfected CSF ; and infected CSF . A similar pattern was observed when the leukocyte counts were normalized to PMN number and after PMA stimulation (data not shown).In CSF obtained by lumbar puncture, the leukocyte count was higher in infected samples  and from blood in healthy volunteers ; the percentages of CD62L-positive PMNs were similar in CSF (32% (6-41)) and healthy blood (12% (7-22)). PMNs in CSF did not present enhanced markers of activation.high cells, high CD11b expression , and a low percentage of CD62L-expressing PMNs (4%). The heightened expression of CD16 and CD11b was probably related to the recruitment of new leukocytes. The presentation of PMNs with high CD11b levels and low CD62L in cases with infection was in agreement with the classical activated phenotype described in the literature [One patient presented with an infection on day 10. The infected CSF was characterized by an increased number of CD16terature .P < 0.0001; PMA-stimulated AUC RLU: 315,592  versus 683,190 , P = 0.0002). Neither the duration of delay before sample treatment (2 versus 4 h) nor the storage temperature (4°C versus room temperature) affected ROS production under basal or PMA-stimulated conditions, as measured by luminometry.Different experimental procedures were conducted to study the impact of various experimental conditions on the results. We performed analyses at different temperatures and with different time delays before measurements. CSF samples collected during patients' follow-up were analyzed following HBSS lavage plus centrifugation, and the results were compared with those obtained without centrifugation. ROS production measured by luminometry was always higher after washing and centrifugation than without  versus 76,336 , Among phenotypic characteristics, ROS production may represent a hallmark of infected CSF. High spontaneous ROS production was related to increased PMN count following cell recruitment to infected tissue. The global increase in ROS production also was related to higher synthesis, because ROS production expressed "per cell" was elevated in cases of infection. ROS measurement in CSF may constitute a rapid test with which to diagnose infection in CSF and may be a suitable tool for use at bedside in clinical practice.ROS production may be related to mitochondria production, especially in the context of apoptotic evolution ,13-15; hWith the objective of developing a test that will diagnose CSF infection, the measurement of spontaneous ROS production by CSF cells seemed to be more discriminating than PMA-stimulated production, because little overlap was observed between the ranges exhibited by noninfected and infected samples. After a threshold is validated in a large-scale study and translated into clinical practice, such a test could drive the administration of antibiotics with few false-positive cases and also could help to schedule catheter changes in cases of infection. The impact of antibiotics on ROS production constitutes a criterion for the validation of the test, and its value during treatment follow-up has been demonstrated in three patients Figure . HoweverWe also obtained preliminary results in CSF obtained by lumbar puncture regarding suspected postoperative or post-trauma meningitis. ROS production had the same profile between groups as was observed with CSF drainage samples, although it reached higher levels. If confirmed in a large-scale study, such overproduction of ROS might be related to the intensity of inflammation induced by surgical products and hemostatic material. We may have to consider clinical conditions when interpreting the results.As observed by other authors, bacterial and aseptic meningitis could not be distinguished by clinical characteristics alone. This distinction was not possible with any of the classical CSF characteristics studied because of large degrees of overlap between the ranges. Lactate level in CSF may be of interest in the differential diagnosis of bacterial and viral meningitis  but has 2O2) with DHR 123 by fluorescence using flow cytometry (data not shown), and (2) the reaction of global ROS production (primarily superoxide anion) with luminol was measured using luminescence [We measured ROS production in CSF using two different methods: (1) the reaction of intracellular hydrogen peroxide . Anne-Claire Lukaszewicz and Didier Payen are holders of the patents EUROPE - n°10290421.6 (2010) and PCT/IB2011/053329 (2011).ACL and GG have equally contributed to the manuscript and are both considered as first author. ACL had the original idea, conceived of the experiments, designed the study, guided the experimental work, analyzed and interpreted the data, and wrote the manuscript. IO and GG performed all of the experimental work. VF participated in study design and conducted the cytometry. DP participated in study design and made major contributions to the intellectual content and writing. All authors read and approved the final manuscript.All authors work in the Department of Anesthesiology and Critical Care, Lariboisière Hospital, University of Paris Diderot, Sorbonne Paris Cité. GG was a student working toward a Master of Science degree. All authors read and approved the final mansucript.


Elevated myocardial energy expenditure (MEE) is related with reduced left ventricular ejection fraction, and has also been documented as an independent predictor of cardiovascular mortality. However, the serum small-molecule metabolite profiles and pathophysiological mechanisms of elevated MEE in heart failure (HF) are still lacking. Herein, we used 1H-NMR-based metabolomics analysis to screen for potential biomarkers of MEE in HF.A total of 61 subjects were enrolled, including 46 patients with heart failure and 15 age-matched controls. Venous serum samples were collected from subjects after an 8-hour fast. An INOVA 600 MHz nuclear magnetic resonance spectrometer with Carr-Purcell-Melboom-Gill (CPMG) pulse sequence was employed for the metabolomics analysis and MEE was calculated using colored Doppler echocardiography. Metabolomics data were processed using orthogonal signal correction and regression analysis was performed using the partial least squares method.P>0.05).The mean MEE levels of HF patients and controls were 139.61±58.18 cal/min and 61.09±23.54 cal/min, respectively. Serum metabolomics varied with MEE changed, and 3-hydroxybutyrate, acetone and succinate were significantly elevated with the increasing MEE. Importantly, these three metabolites were independent of administration of angiotensin converting enzyme inhibitor, β-receptor blockers, diuretics and statins (These results suggested that in patients with heart failure, MEE elevation was associated with significant changes in serum metabolomics profiles, especially the concentration of 3-hydroxybutyrate, acetone and succinate. These compounds could be used as potential serum biomarkers to study myocardial energy mechanism in HF patients. However, this method is difficult and invasive. Some advanced imaging techniques, such as positron emission tomography, single-photon emission tomography and phosphorus-31 magnetic resonance (31P-MR) have allowed the non-invasive measurement of cardiac metabolism Cardiac energy and metabolism are tightly regulated for a high and constant workload, however such regulation becomes compromised in heart failure 1H NMR spectroscopy. However, a metabolomic investigation of serum metabolites associated with different MEE levels in HF patients has not yet been reported. In the present study, we performed NMR-based metabolomic analysis on serum obtained from HF patients, in an effort to determine the serum metabolites differences of patients with different MEE levels.Metabolomic analysis, the systematic study of small-molecule metabolite profiles, has been used to identify potential biomarkers that provide new insights into biological processes This study was carried out in accordance with the Helsinki Declaration. This study was approved by the institutional ethics committee of Nanfang Hospital. All subjects had provided their written informed consent to participate in this study.For this study, 46 HF patients  from the cardiology department of Nanfang Hospital were recruited. All patients were staged in NYHA (New York Heart Association) class II (n = 11), III (n = 18) and IV (n = 17). The exclusion criteria were: (1) type 2 diabetes mellitus and other metabolic disease; (2) acute or chronic inflammatory conditions; (3) malignancies, and (4) significant respiratory pathology. Fifteen age-matched controls with normal cardiac function  were recruited from the health management center in Nanfang Hospital.MEE was measured with a Siemens Sequoia 512 Encompass ultrasound system, using the method described previously −4.MEE   = LV circumferential end-systolic wall stress (cESS) ×LVET×LV stroke volume (LVSV)×HR×4.2×10Venous blood was collected from the antecubital vein on the same day of MEE measurement, coagulated at room temperature for 10 min, and centrifuged at 3000 g at 4°C for 10 min. The resulting supernatant serum was collected and stored at −80°C.2O and 100 µl 3-trimethylsilyl-2H4-propionic acid sodium salt (TSP) in D2O (1 mg/ml). After centrifuged at 13000 g for 10 min, 550 µl aliquots of the supernatant were transferred into 5-mm NMR tubes for analysis.Stored serum samples were thawed prior to NMR analysis. 300 µl of serum was mixed with 200 µl D1H-NMR spectra of the serum samples were acquired on a Varian INOVA 600 MHz spectrometer at 27°C using Carr-Purell-Meiboom-Gill (CPMG) spin-echo pulse sequence with a total spin-spin relaxation delay (2 nτ) of 320 ms. The free induction decays (FIDs) were collected into 32 k data points with a spectral width of 8000 Hz and 64 scans. The FIDs were zero-filled to double size and multiplied by an exponential line-broadening factor of 0.5 Hz prior to Fourier transformation (FT). All serum 1H-NMR spectra were manually phased and baseline corrected using VNMR 6.1C software . Each spectrum over the range of δ 0.4–4.4 was data-reduced into integrated regions of equal width (0.01 ppm). The regions containing the resonance from residual water (δ4.6–5.1) were removed. The integral values of each spectrum were normalized to a constant sum of all integrals in a spectrum. Identification of metabolites in spectra was accomplished based on information in the literature and the Chenomx NMR Suite 5.0 .2 test for discrete clinical variables and by one-way ANOVA for continuous variables. Differences within groups were evaluated using the Bonferroni method if variables were equal; otherwise Dunnett's T3 test was used. The resulting integral NMR data were imported into SIMCA-P  for multivariate analysis. The CPMG data were mean-centered and Pareto-scaled prior to analysis. Principle component analysis was performed to observe the samples distribution. To remove the variations not correlated to the group membership, the data were preprocessed using orthogonal signal correction (OSC) followed by PLS analysis.Clinical, functional data and metabolite concentration are presented as mean ± SD or percentages as appropriate. All the included subjects were divided into three quartile groups according to MEE level. Differences between MEE groups were evaluated using the χ2 = 33.02, P<0.001). Those in the high MEE group had higher fasting glucose, very low density lipoprotein (VLDL), creatinine, uric acid, and BNP levels and a lower LVEF than those in the low MEE group.The mean ejection fraction in HF patients was 39.59±10.94%, 48% showed mildly reduced EF (40%–54%), and 52% had severely reduced EF (≤40%). Medical treatment for the HF patients consisted of diuretics (71.7%), β-blockers (73.9%), angiotensin converting enzyme inhibitors or angiotensin II receptor blocker (69.6%), and statins (28.3%). The mean MEE levels of HF patients and controls were 139.61±58.18 cal/min and 61.09±23.54 cal/min, respectively. All the included subjects (46 HF patients and 15 controls) were divided into three quartile groups according to MEE level. Those with an MEE<72.53 cal/min were included in the low MEE group (Q1); 72.53≤MEE<176.75 cal/min in the intermediate MEE group (Q2+Q3); MEE≥176.75 cal/min in the high MEE group (Q4). Distribution of participants and baseline characteristics of the three groups are shown in 1H-NMR spectra of serum from low, intermediate and high MEE groups groups showed a large numbers of NMR signals, which indicated the complexity of spectral information obtained from the patient donors . Pairwise PCA score plots showed a significant difference between the high and low MEE groups by the score plots of PC1 versus PC2  (2Y = 92.6% and Q2Y = 90.2%). OSC-PLS cross validating parameter are shown in 2Y and Q2Y values of the original model with those of the re-ordered models this type of permutation gauges the statistical significance of the predicting power of the models. It is known that models with a R2Y intercept less than 0.3 and a Q2Y intercept less than 0.05 indicate valid models 2Y (∼0.24) and Q2Y (−0.29) intercept values, which suggested the validity of our OSC-PLS model  , but no LS model . In the LS model . VariablLS model .P>0.05). In addition, the differences of LDL, VLDL, lactate, N-acetyl glycoprotein, choline, phosphocholine, α-glucose, β-glucose, trimethylamine-N-oxide and glycine were not significant among the three groups. Taking the diagnostic criteria of the Framingham Heart Study (FHS) as the benchmark, receiver operating curves (ROCs) of acetone, 3-hydroxybutyrate, succinate and BNP for diagnosis of HF were compared. These results indicated areas under the curve (AUC) were 0.92, 0.90, 0.86 and 0.98, respectively  were shown to rise with increasing MEE. Furthermore, the association of these three metabolites with MEE was independent of all treatment regimes including angiotensin converting enzyme inhibitor, β-receptor blockers, diuretics and statins. Both 3-hydroxybutyrate and acetone are ketone bodies, and mainly synthesized from oxidation of fatty acid catalyzed by acetylcoenzyme A in the liver. In general, myocardial energy acquisition via ketones oxidation pathways is dependent on the concentration. When the ketone bodies level is very low in the serum, they only provide a very small part of the energy required by the myocardium. However, in hungry or diabetes mellitus patients, blood ketone bodies level are greatly increased due to low insulin and high fatty acid levels, and they becomes the major energy supplier of the myocardium et alSuccinate is not only an important intermediate metabolite in the tricarboxylic acid cycle but also an important component of the electron respiratory chain complex II. In our study, the succinate level in the high-MEE group was increased by 1.4-fold compared with the intermediate-MEE group, which was a further 2.4-fold higher than the low-MEE group, suggesting that succinate is closely related to myocardial energy metabolism. Pisarenko Significant differences were found among serum metabolic groups of different MEE in HF patients, and three serum markers  were identified to reflect the myocardial energy metabolism in elevated MEE by using 1H NMR-based metabolic analysis. More advanced technique, such as utility of both 1D noesy and cpmg spectra, may be carried out in future studies to reaffirm the identified metabolites. If these results are confirmed, more studies are needed to investigate the pathophysiological mechanism that dictates elevated levels of these metabolites with increasing MEE in chronic HF.Figure S1PLS score plot of three MEE groups.(DOC)Click here for additional data file.Figure S2PLS score plot of low MEE and intermediate MEE.(DOC)Click here for additional data file.Figure S3PLS score plot of low MEE and high MEE.(DOC)Click here for additional data file.


Hydrocephalus is a central nervous system (CNS) disorder characterized by the abnormal accumulation of cerebrospinal fluid (CSF) in cerebral ventricles, resulting in their dilatation and associated brain tissue injury. The pathogenesis of hydrocephalus remains unclear; however, recent reports suggest the possible involvement of abnormal osmotic gradients. Here we explore the kinetics associated with manipulating CSF osmolarity on ventricle volume (VV) in the normal rat brain.CSF was made hyper-osmotic by introducing 10KD dextran into the lateral ventricle, either by acute injection at different concentrations or by chronic infusion at a single concentration. The induction and withdrawal kinetics of dextran infusion on VV were explored in both contexts.Acute intraventricular injection of dextran caused a rapid increase in VV which completely reversed within 24 hours. These kinetics are seemingly independent of CSF osmolarity across a range spanning an order of magnitude; however, the magnitude of the transient increase in VV was proportional to CSF osmolarity. By contrast, continuous intraventricular infusion of dextran at a relatively low concentration caused a more gradual increase in VV which was very slow to reverse when infusion was suspended after five days.We conclude that hyperosmolar CSF is sufficient to produce a proportional degree of hydrocephalus in the normal rat brain, and that this phenomenon exhibits hysteresis if CSF hyperosmolarity is persistent. Thus pathologically-induced increases in CSF osmolarity may be similarly associated with certain forms of clinical hydrocephalus. An improved understanding of this phenomenon and its kinetics may facilitate the development of novel therapies for the treatment of clinical hydrocephalus. Hydrocephalus is a CNS disorder characterized by the abnormal accumulation of cerebrospinal fluid (CSF) in cerebral ventricles and increased ventricle volume (VV). The pathogenesis underlying the emergence of hydrocephalus is poorly understood -5 and reThe classical view regarding the etiology of hydrocephalus is further challenged by recent reports that chronically increased CSF osmolarity is sufficient to produce hydrocephalus in the normal rat or dog brain -31. It hHere we investigate the kinetics of hydrocephalus following the injection/infusion of 10KD dextran into the lateral ventricle in the normal rat brain. Our results lend additional support to the hypothesis that osmotic effects are sufficient to induce hydrocephalus and reveal that the associated kinetics can have a dramatic effect on the nature and persistence of the hydrocephalic response.ad libitum. Animal procedures were carried out in accordance with NIH guidelines for the care and use of experimental animals and the experimental protocol was approved by the Institutional Animal Care and Use Committee at Wayne State University in Detroit, Michigan.Adult female Sprague–Dawley rats  were used in all experiments. Rats were housed in an AAALAC-accredited animal care facility at Wayne State University and maintained on a 12-h light/dark cycle with food and water provided For this experiment, rats were randomly divided into five groups which received acute injections (15μl) of FITC-labeled dextran (10KD) into the lateral ventricle at different osmolarities via a Hamilton syringe. Group I , Group II , Group III , Group IV  and Group V . Ventricle volumes were assayed by volumetric analysis of MRI image data obtained from scans performed prior to injecting hyperosmotic dextran solution, at 30 minutes post-injection, and at 24 hours post-injection.Rats were anesthetized with Ketamine  and Xylazine  and mounted in an animal stereotactic instrument (Kopf). Using aseptic techniques, 15 μl of fluorescently-labeled 10KD dextran solution (1 μg/μl)15 μl of 10KD (1 μg/μl) fluorescently-labeled 10KD dextran solution was injected (1 μl/s) into the frontal horn of the right lateral ventricle using a Hamilton syringe connected to a stainless steel needle . The needle was inserted through a burr hole positioned 0.9 mm posterior, 1.2 mm lateral to bregma, and lowered perpendicular to the cranial surface to a depth of 3.6 mm ventral to the dorsal surface of the dura mater . After iFor this experiment, rats were randomly divided into 4 groups defined by their intraventricular infusion status (On/Off) during three consecutive 5-day intervals . Groups I and II: On-On-On (n = 10 for both groups); Group III: On-Off-On (n = 9); Group IV: On-Off-Off (n = 10). During ‘On’ intervals, rats received intraventricular infusion at a rate of 0.5 μl/hr with either iso-osmotic  or hyper-osmotic  FITC-labeled dextran (10KD). During ‘Off’ intervals, intraventricular infusion of dextran was suspended with the cannula in place. Pumps were On during interval A for all groups. The infusion status was changed (Groups III and IV) at the beginning of interval B, and again (Group III) at the beginning of interval C. For each of these procedures, rats were anesthetized with ketamine  and xylazine  and a 1.0 cm incision was made on the dorsal surface of the neck to expose the catheter, which was either closed by ligating (‘Off’ condition) or open by unligating (‘On’ condition) the catheter using 3–0 silk suture. The efficacy and reversibility of this ligation procedure in this context was verified in preliminary experiments. Sham procedures were performed for groups where the catheter remained open during intervals A, B and C (Groups I and II). MRI scans were performed prior to pump implantation (baseline) and at the end of each interval to assay ventricular volumes.Rats were anesthetized with ketamine  and xylazine  and mounted in an animal stereotactic instrument (Kopf). Using aseptic techniques, a midline incision was made and the dorsal surface of the skull was exposed completely. A blunt tip 27-gauge needle was inserted through a burr hole positioned 0.9 mm posterior, 1.2 mm lateral to bregma, and lowered perpendicular to the cranial surface to a depth of 3.6 mm ventral to the dorsal surface of the dura mater, placing the tip of the needle in the ventral horn of the lateral ventricle . The nee2, matrix size 256x256, 1 signal average, 1 mm slice thickness, interleaved 24 slices, TA 2 m. Regions of interest (ROI) for all transverse slices were manually outlined and the surface area determined by counting the number of pixels enclosed by the ROI. Segmental volumes were calculated by multiplying slice areas by slice thickness, and total T2-weighted MRI volumes were determined by summing the segmental volumes. Internally developed MR SPIN  software  written in Visual C++ on the Microsoft Windows platform was used for MRI and ventricular volume calculation [Whole head MRI images were acquired under Ketamine  and Xylazine  anesthesia in the 7-Tesla MRI System  at the Magnetic Resonance Research Facility at Wayne State University. For the Experiment 1, MRI scans were performed prior to injection and at 30 minutes and 24 hours post-injection. For experiment 2, MRI scans were performed prior to cannula implantation and at 5, 10 and 15 days post-implantation, immediately prior to initiating surgical procedures to change infusion status. Coronal axial T2-weighted images were acquired using the following parameters: TR 3.53 sec, TE 38 ms, FOV 32x32mmculation . AnimalsP-values between 0.01 and 0.05 were interpreted as significant whereas P-values < 0.01 were considered highly significant.For Experiment 1, descriptive statistics of mean, median and standard deviation were computed for each of the five treatments groups  at each of the three time-points . Because of the wide range of variability among the groups, Wilcoxon two sample tests were performed to assess whether the pairwise group comparisons differed at each time-point. Signed rank tests were done to assess the differences over time within treatment groups. Spearman’s correlation coefficients were computed between the osmolality levels (treatment groups) and the volumes at each time point. P-values between 0.01 and 0.05 were interpreted as significant whereas P-values <0.01 were considered highly significant.For Experiment 2, data for each rat was ‘normalized’ to individual baselines. Log transformations of the data were performed to reduce the influence of variance associated with group means. Descriptive statistics of mean, median and standard deviation were computed for each of the four treatments groups (Groups I-IV) at each of the three time-points . Mixed models methods using repeated measures were used to estimate the rate of change over time (slope), and to test these slopes among the four groups. 2 = 0.8349, P < 0.001), which was not present pre-injection or at 24 hours post-injection. Within group comparisons of pre-injection VVs relative to VVs at 30 min and 24 hours post-injection revealed that VVs were significantly increased for all groups at 30 minutes post-injection (P < 0.037), with highly significant increases for Groups III, IV, and V (P < 0.007); however, these increases in VV had completely resolved by 24 hours post-injection. Between group comparisons of VVs at each time-point revealed that VV was significantly increased in Group V (P < 0.012) at 30 min post-injection relative to Groups I – IV (P < 0.017), with a trend toward increased VVs in Group IV (P <0.061). Significant differences in VV were not observed between groups either pre-injection or at 24 hours post-injection. Moreover, there were no significant differences between pre-injection VVs and VVs at 24 hours post-injection, reflecting that fact that VVs returned to their original pre-injection volumes by 24 hours post-injection.For Experiment 1 . VV in Group II  also increased at each time-point over the course of continuous hyper-osmotic dextran infusion; however, these increases were larger and highly significant . VV in Group III , trended toward increase during interval A (P < 0.069); however, the rate of increase slowed significantly during interval B when infusion was suspended (P < 0.061) and resumed increasing at its original rate and reached significance during interval C when infusion was restored (P < 0.028). VV in Group IV , trended toward increase during interval A (P < 0.081). The rate of increase slowed during intervals B and C when infusion was suspended but the increase in VV reached significance at both time-points . Between group comparisons of VVs at each time-point revealed that, relative to Group I, the increase in VV in Group II was significant during interval A (P < 0.047), and highly significant during intervals B (P < 0.001) and C (P < 0.001). The increase in VV in Group II, relative to Group IV, was also significant during interval B (P < 0.017) and highly significant during interval C (P < 0.001). MRI images (Figure For Experiment 2 (Table Behavioral effects related to these manipulations of CSF osmolarity and VV were not systematically investigated; however, with the exception of transient anesthesia effects, the rats appeared alert and attentive and maintained their weight and hydration status for the duration of the study.The objective of this study was to elucidate the kinetics associated with manipulating CSF osmolarity on VV in the normal rat brain. Our results confirm previous reports suggesting that increasing CSF osmolarity is sufficient to increase VV and include novel observations regarding the influence of concentration and time in this context. Transient increases of CSF osmolarity over a range spanning an order of magnitude are sufficient to cause acute and proportional increases in VV which are completely reversed by 24 hours post-injection. These acute increases in VV are observed primarily within the injected hemisphere at 30 minutes post-injection but encompass the entire rostral-caudal extent of the brain. Sustained increases of CSF osmolarity at the lower end of this range result in more gradual increases in VV that evolve bilaterally over the course of five days of continuous dextran infusion and are much slower to reverse when dextran infusion is suspended. This apparent hysteresis may reflect a persistent saturation of clearance mechanisms which normally regulate CSF osmolarity and/or ventricular volume. Collectively, our results suggest that the induction and withdrawal kinetics of increased VV are directly affected by both the magnitude and duration of increases in CSF osmolarity.Changes in VV produced by manipulating CSF osmolarity may reflect the relatively free movement of water within brain tissues made possible by the presence of aquaporins 1, 4, and 9 -36. ThesThe rapid induction and withdrawal kinetics of VV increases associated with transient increases in CSF osmolarity, suggest that 10 kD dextran is rapidly and efficiently cleared from CSF under these conditions. It remains to be determined whether the associated ipsilateral increase in VV observed at 30 minutes post-injection eventually becomes bilateral or whether the rate of clearance is sufficient to prevent this. By contrast, the much more gradual induction and withdrawal kinetics of VV increases induced by modest increases in CSF osmolarity sustained over several days were unexpected. These may reflect a gradual saturation of endogenous transport mechanisms which normally participate in clearing such solutes from CSF, combined with a long post-saturation refractory period. An analogous influx of solutes into CSF resulting from brain tumors, cerebral trauma, arterio-venous malformations, neurodegenerative diseases and other brain pathologies might, therefore, similarly saturate these putative transport mechanisms and give rise to hydrocephalus displaying similar kinetics. Such solutes could include ions, amino acids, neurotransmitters, proteins and peptides crossing the compromised BBB and other cellular debris associated with apoptotic and/or necrotic cell death.In conclusion, our results suggest that brain pathologies that compromise BBB integrity or which otherwise promote the pathological release of cellular constituents into interstitial fluid and/or CSF may be sufficient to induce hydrocephalus by increasing CSF osmolarity. Moreover, a sustained influx of these constituents at relatively low concentrations and the associated increase in CSF osmolarity may be more pathogenic than an acute influx at higher concentrations. Therapies which prevent or mitigate such increases in CSF osmolarity might therefore prove useful in treating hydrocephalus. Assays of CSF content that affects osmolarity might also prove useful for distinguishing among types of hydrocephalus that may be responsive to such a therapeutic approach.The authors have no competing interests.SK designed the study, was principle investigator and primary author. J L performed the experiments, obtained the MRI scan data, assisted in designing the study and writing the article. LS performed statistical analysis. KAJ served as a consultant for data analysis and interpretation and provided extensive editorial support in drafting the manuscript. All authors have read and approved the final version of the manuscript.


G-CSF has been shown in animal models of stroke to promote functional and structural regeneration of the central nervous system. It thus might present a therapy to promote recovery in the chronic stage after stroke.Here, we assessed the safety and tolerability of G-CSF in chronic stroke patients with concomitant vascular disease, and explored efficacy data. 41 patients were studied in a double-blind, randomized approach to either receive 10 days of G-CSF (10 µg/kg body weight/day), or placebo. Main inclusion criteria were an ischemic infarct >4 months prior to inclusion, and white matter hyperintensities on MRI. Primary endpoint was number of adverse events. We also explored changes in hand motor function for activities of daily living, motor and verbal learning, and finger tapping speed, over the course of the study.Adverse events (AEs) were more frequent in the G-CSF group, but were generally graded mild or moderate and from the known side-effect spectrum of G-CSF. Leukocyte count rose after day 2 of G-CSF dosing, reached a maximum on day 8 (mean 42/nl), and returned to baseline 1 week after treatment cessation. No significant effect of treatment was detected for the primary efficacy endpoint, the test of hand motor function.These results demonstrate the feasibility, safety and reasonable tolerability of subcutaneous G-CSF in chronic stroke patients. This study thus provides the basis to explore the efficacy of G-CSF in improving chronic stroke-related deficits.ClinicalTrials.gov NCT00298597 Stroke is the leading cause of adult disability in the United States and Western Europe. Two thirds of stroke survivors suffer from residual neurological deficits and have to cope with chronic motor and language dysfunctions. So far, we have very limited effective therapies in spite of intensive research efforts and numerous clinical trials Basic science has improved our understanding of the mechanisms underlying recovery of function following brain injury, and started to identify strategies to facilitate these mechanisms, an important one being intensive training Pharmacological agents seem to be attractive tools to facilitate the beneficial effects of training-based interventions on recovery Adverse events (AEs) related to low-dose application of G-CSF in non-oncological individuals generally are only mild to moderate, including bone pain, headache, myalgias, fatigue, nausea, insomnia, and redness at the injection site. However, rare cases of potentially life-threatening conditions like splenic rupture, acute lung injury, vascular events, and exacerbation of autoimmune conditions have been reported In the present study, our main aim was therefore to assess the safety of G-CSF in a cohort of chronic stroke patients. Patients were chosen to reflect the stroke population with regard to age, concomitant diseases like hypertension, diabetes, and generalized atherosclerosis, as well as their cerebral disease sequelae, like white matter hyperintensities (WMH). We also explored the impact of G-CSF on hand motor function for activities of daily living, motor and verbal learning, and finger tapping speed, (secondary efficacy endpoints).The protocol for this trial and supporting CONSORT checklist are available as supporting information; see The study, conducted according to ICH GCP guidelines and to the principles expressed in the Declaration of Helsinki, and approved by the local ethics committee of the University of Münster and the German Federal Institute for Drugs and Medical Products, and subsequently registered on ClinicalTrials.gov (NCT00298597).Fourty-one individuals  gave written informed consent and participated in this double-blind, placebo-controlled and randomized study. All patients received study medication for at least two days. The safety population thus consists of all 41 subjects. From two patients, no efficacy data were obtained. Thus, the full analysis set (FAS) consists of 39 subjects.Patients were recruited at least 4 months after having presented to the local stroke unit with an ischemic stroke, and MR-imaging of the brain had shown the presence of WMH. Severity of WMH was rated according to the validated score of Fazekas and colleagues All patients were evaluated neuropsychologically in a separate session before G-CSF administration, using tests of general intellectual function, attention, verbal fluency, digit spans, and verbal and visuospatial memory. Patients were recruited into the study from June 2006 to August 2008.Study medication, and randomization to either G-CSF or placebo, was provided by the pharmacy of the University of Mainz. Filgrastim  sorbitol, 0.004% Tween 80, ph adjusted to 4.0 with NAOH), or saline as placebo were filled in identical-looking glass vials to ensure blinding. Treatment was then administered by the study nurse. Results of blood analysis and potentially medically necessary actions triggered by elevated leukocyte counts were exclusively accessible to study investigators who did not take part in testing and analysis of efficacy parameters, or rating of adverse events.Patients underwent neuropsychological testing and familiarization in all tasks one to three days before receiving the first dose of G-CSF or placebo. Then, each participant was given either G-CSF (10 µg/kg body weight/day), or saline solution , each administered as daily s. c. injections over 10 days. This dose is in line with literature safety data for healthy human bone marrow donors motor hand functions, the Jebsen Taylor Test (JTT) was employed To assess motor learning, we used the serial reaction time task in a modified version with a probabilistic instead of a deterministic sequence to ensure the procedural task nature To assess verbal learning, a word learning model was employed that mimics vocabulary acquisition in healthy individuals, and is relevant to language reacquisition in stroke patients with aphasia To assess finger tapping speed, patients performed a fast finger-tapping task To assess Sample size was derived from the AXIS I study Additionally, secondary efficacy endpoints were explored, that is, the changes from Day 3 to mean of Day 17 and Day 38 for JTT and finger tapping speed, as well as success in verbal and motor learning during the second week of treatment, as a function of group. Here, the pre-defined primary efficacy endpoint was the JTT. The analyses were performed in the full analysis set according to guideline ICH E9 (FAS) population, which was defined as all subjects who received at least one dose of study medication, had a baseline evaluation, and at least one evaluation after the start of the treatment (n = 39). Last observations were carried forward in the endpoint analysis (LOCF). Analysis of covariance with adjustments for age, time since infarction, location, lesion size, and baseline value was performed for the efficacy variables. Significance for all tests was set at p<0.05. All data are expressed as means ± SEM, unless stated otherwise. All statistical analyses were performed using SPSS 16, JMP 8.01, or SAS 9.1.All 41 subjects originally recruited into the study took at least two doses of study medication (safety population). Two patients that received at least one dose of study medication had to be excluded, one of them due to acute disease unrelated to the study (bronchitis), the other withdrew consent without specific reason. Thus, 39 subjects remained for the efficacy analyses (see All patients (n  = 41) received at least one dose of study medication. In general, the study medication was tolerated well. In three patients receiving verum the dose was halved at day 10 (2) or day 3 (1) due to leukocyte count >50/nl without clinical symptoms. In three patients medication was halted at day 9 (2) or day 8 (1) due to leukocyte count >70/nl without clinical symptoms. In two patients verum medication was stopped at day 3 or day 5 due to bronchitis and headache cum nausea, respectively. One patient received dosing every second day starting at day 4 due to development of bone pain that was manageable under this regimen.Three patients in the G-CSF group 3/21) and one patient in the placebo group (1/20) suffered from serious AEs (SAEs), none of which was judged to be related to study medication. These SAEs were: Pons infarction, bronchitis, bone fracture, and infection with unclear source. The number of patients in each group with at least one AE was 17/21 in the G-CSF and 14/20 in the placebo group . The total number of AEs was 58 for verum, and 37 for placebo (Chi Square Test p = 0.055). AEs in the G-CSF group were more often related to the treatment itself: 33/58 (59%) events reported in the G-CSF group were classified as likely related to the treatment, versus 18/37 (49%) in the placebo. This effect revealed a strong trend for more related events in the G-CSF group when comparing absolute numbers (Chi Square Test p = 0.054). All treatment-related events were rated in their severity as mild or moderate, and were within the known side effect profile of G-CSF treatment, most often headache (n = 9), fatigue (n = 9), malaise (n  = 6), back pain (n = 5), limb discomfort (n = 3), and abdominal pain (n = 1). Nausea (n  = 4), and sleep disorders (n = 7) were rated as not treatment-related; other side effects occurred with a frequency of<2. Most frequent side effects in the placebo group included headache (n = 7), fatigue (n = 8), and dizziness (n = 5). For further information on adverse events, please refer to /21 and oAnalysis of blood pressure, heart rate, and body temperature revealed no significant difference between groups at baseline, at Study Days 6–10, Study Day 17, or 38.We measured G-CSF values at day 0, days 4 or 5; days 8 or 9, and day 12/13. G-CSF levels in the verum group rose from 9.26±2.42 pg/ml serum at baseline to 306.07±106.81 pg/ml at day 4, and were already back to 37.83 ±13.68 pg/ml at day 12/13 .As expected, leukocyte count showed a significant increase in the verum group, from mean 7.5/nl at baseline to a mean value of 41.9/nl on day 8. Highest leukocyte count, reached in one patient on day 8, was 75.4/nl. At day 17 (7 days after last administration of G-CSF/placebo) values were back to normal . Numbers of all AEs, and of likely related AEs, the primary endpoint aimed at safety, were more frequent in the G-CSF group although this difference failed formal significance. However, this is not unexpected given the known side effect spectrum of G-CSF, especially with the longer time frame of administration in comparison to the recent AXIS trial. Importantly, AEs with likely relationship to study medication were always rated as mild to moderate, and all involved well-known side effects from G-CSF. In two patients, likely related adverse events (bone pain and headache cum nausea) led to intermittent dosing or to discontinuation of study medication.A rise in leukocytes is the main expected effect of G-CSF outside the central nervous system. Leukocytes reached an average maximum of 42/nl at day 8, and dropped back to baseline levels less than one week after end of treatment. Although no clinical data exist that ascribe harmful effects to an isolated, G-CSF-triggered rise in leukocytes, we halved dosing at leukocyte counts >50/nl, and halted dosing at values >70/nl.Similar to the study in acute stroke (AXIS), the rise in leukocytes did not lead to an increase in thromboembolic events. It is reassuring that so far no clinical evidence suggests a G-CSF treatment-related adverse effect on the cerebral microvasculature assessed in almost hundred stroke patients Apart from the rise in leukocytes, a lowering in platelets was noted, which is a known side effect of G-CSF treatment also in other patient populations. Thrombocyte counts did rapidly recuperate to normal levels 7 days after end of G-CSF treatment. However, in future studies with chronic G-CSF treatment this parameter needs to be closely monitored, especially in patients receiving anticoagulation or platelet aggregation blockers.In summary, the present study suggests safety and reasonable tolerability of G-CSF application over the course of 10 days, even in the vulnerable population of elderly stroke patients with concomitant diseases like hypertension, diabetes, and WMH, known to increase morbidity and mortality However, it needs to be clarified if higher elevation of leukocyte count than 50 – 70/nl can be tolerated in chronic application, and lowering of platelets needs to be closely monitored.We have conducted a broad array of tests in view of demonstrating feasibility of such a trial approach, but also with the aim to explore possible efficacy signals. The tests explored did not show a meaningful difference between G-CSF and placebo, including the Jebsen-Taylor test (JTT), chosen as primary efficacy parameter.Several reasons may be responsible for the failure to detect clearer signals of efficacy in our study. First, the current small study is certainly underpowered to show effects in a highly heterogeneous population as the one examined here. This is underlined by a retrospective power analysis: We had a 25% chance of detecting a 25% effect size in the main outcome parameter, change in JTT from day 3 to mean of day 17 and day 38 (population change = 6.1 points±3.6 SD), and an 8% chance to detect an effect size of 10%. A sample size of 176 is needed to detect a 25% effect size with a power of 80%. Second, we chose patients that had almost completely recovered from their initial deficits, and were therefore able to participate in the experimental tasks. However, some patients were more than a year away from their stroke, and larger effects of G-CSF may have been present if G-CSF had been given at an earlier stage Concomitant white matter lesions might also have complicated benefit assessment in our population: Efficient communication in the human brain relies on the integrity of white matter tracts Apart from particular limitations of the study cohort and power issues, questions of dosing and timing are evident. Higher doses should be included in the next trial, as an increase of effect size seems to occur with dose in acute stroke patients (AXIS). Also, a longer time period or repeated dosing intervals may be needed to see sustained effects.In summary, the present study is a further indication that G-CSF administration is safe and reasonably well-tolerated, even in elderly chronic stroke patients with concomitant vascular disease. It has to be kept in mind though that only a small number of patients (n = 21 in the G-CSF group) were examined. However, this safety analysis is the first step on the road to introducing a novel therapeutic approach to the clinical realm Given the long-time experience with G-CSF in other indications, and the strong rationale for G-CSF as a recovery-enhancing drug it is now the time to initiate adequately powered trials examining the potential efficacy of this drug in improving functional outcome in the subacute or chronic stage after stroke.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.Protocol S1Trial Protocol.(DOC)Click here for additional data file.


A 20-fold excess of unlabeled LDL had no effect on 125I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect 125I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of  Circulating low density lipoprotein (LDL) is the major carrier of cholesterol in the blood, and its level can predict the risk of developing atherosclerosis. Atherosclerosis progression involves accumulation of cholesterol in arterial macrophages to form foam cells. Since uptake of cholesterol-rich LDL by macrophages is a critical step for foam cell formation, targeting this pathway may be beneficial in the treatment of atherosclerosis.Initially, investigators focused on modification of LDL as a mechanism to promote macrophage LDL uptake and foam cell formation Previous studies from our laboratory demonstrate that human macrophages take up native LDL by fluid-phase pinocytosis forming foam cells independently of receptors Macrophage colony-stimulating factor (M-CSF) is expressed in atherosclerotic lesions Pharmacological targeting of the phosphoinositide 3-kinase (PI3K) family of kinases with the pan-PI3K inhibitors wortmannin and LY294002 has previously been shown to inhibit macropinocytosis in murine M-CSF-differentiated bone marrow-derived macrophages Bone marrow from wild-type mice was differentiated with M-CSF to generate macrophages. These macrophages showed an elongated morphology and numerous cytoplasmic vacuoles that we determined were macropinosomes by time-lapse, phase-contrast microscopy , thus imWe next incubated LDLR−/− M-CSF-differentiated macrophages with increasing concentrations of LDL. Macrophages showed progressive accumulation of cholesterol . The tot125I-LDL  alone or with a 20-fold excess of unlabeled AcLDL. A 70% reduction of macrophage 125I-AcLDL uptake was observed when macrophages were incubated with 125I-AcLDL and a 20-fold excess of unlabeled Ac-LDL compared with macrophages incubated with 125I-AcLDL alone. This finding of competitive inhibition was consistent with the known scavenger receptor-mediated uptake of this modified form of LDL . All PI3K kinase-dead KI macrophages showed an elongated morphology and macropinosomes, similar to wild-type macrophages. No significant difference in LDL-derived net cholesterol accumulation was observed comparing macrophages derived from wild-type, PI3K gamma kinase-dead KI, and PI3K delta kinase-dead KI mice . Surpris125I-LDL uptake approximately 70%  contains high concentrations of LDL (0.7–2.7 mg/ml) In an earlier study Although the M-CSF receptor is the only known receptor for M-CSF, and withdrawal of M-CSF inhibited both macropinocytosis and cholesterol accumulation, we cannot conclude that M-CSF signaling directly mediates macropinocytosis and cholesterol accumulation in these macrophages. This is because the M-CSF receptor tyrosine kinase inhibitor, GW2580, did not decrease the absolute level of M-CSF receptor tyrosine kinase activity. Possibly, GW2580 is inhibiting some other kinase that mediates macropinocytosis in these macrophages. Regardless, we show in this study that GW2580 inhibits macropinocytosis, which could explain why this inhibitor impedes the development of atherosclerosis In the present study, we observed inhibition of macrophage uptake of LDL, cholesterol accumulation, and macropinocytosis by dynasore. Dynasore also may have inhibited fluid-phase micropinocytosis because the total inhibition of LDL uptake was substantially greater than the amount of uptake inhibited by the macropinocytosis inhibitors, LY294002 and wortmannin (discussed below). In this regard, dynamin 2 mediates fluid-phase micropinocytosis in epithelial cells The molecular components mediating macropinocytosis by human M-CSF-differentiated monocyte-derived macrophages is different than that of murine M-CSF-differentiated bone marrow-derived macrophages, because macropinocytosis in mouse macrophages was blocked by bafilomycin, an inhibitor of vacuolar type H(+)-ATPase, while this was not the case for human macrophages in our earlier study Macropinocytosis is induced by transfection of Rac and Pak1 signaling molecules in non-macrophage cell types Also, macropinocytosis in human M-CSF-differentiated monocyte-derived macrophages is constitutive and does not depend on the presence of M-CSF, whereas as discussed above, murine macrophage macropinocytosis did depend on the presence of M-CSF. It is possible that these differences represent a species difference in the regulation of macropinocytosis, or are due to the different origins of the macrophages, blood monocyte-derived compared with bone marrow-derived. In any case, all the above findings underscore the importance of not generalizing fluid-phase macropinocytosis signaling components found in one cell type to all cell types.Previous reports show that inhibition of the PI3K family with wortmannin or LY294002 inhibits murine M-CSF-differentiated bone marrow-derived macrophage macropinocytosis In summary, we demonstrate that murine M-CSF-differentiated bone marrow-derived macrophages take up native LDL by fluid-phase pinocytosis to become macrophage foam cells, and identify fluid-phase pinocytosis signaling components that may be targeted to limit macrophage accumulation of cholesterol accumulation within atherosclerotic plaques.g for 5 min at 4°C. Next, bone marrow cells were resuspended in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, and 50 ng/ml M-CSF . Bone marrow from one mouse was plated into five 6-well CellBIND culture plates . After a 24 h-incubation at 37°C with 5% CO2/95% air, macrophage cultures were rinsed 3 times with 3 ml RPMI 1640 to remove non-adherent cells and cultured with 3 ml of RPMI 1640 containing 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM L-glutamine, and 50 ng/ml M-CSF, hereafter referred to as complete medium. Cell culture medium was subsequently replaced every 2 days with fresh complete medium. After 1 week of culture, experiments were performed with 1 ml serum-free RPMI 1640 containing 50 ng/ml M-CSF and the indicated additions. M-CSF differentiation of murine bone marrow cells required 1 week to achieve a near uniform culture of macrophages with an elongated shape and numerous vacuoles. All inhibitors were obtained from EMD Millipore  except NSC23766 , AS605240 , dynamin specific inhibitory peptide , dynamin specific inhibitory peptide control , and Y-27632 . DMSO was used as a vehicle for all inhibitors. All animal procedures were pre-approved by the NHLBI Animal Care and Use Committee.Male C57BL/6 wild-type and LDL receptor null (LDLR−/−) (C57BL/6 background) mice were obtained from the Jackson Laboratory . PI3K-β, γ and δ KI mice were generated as previously described 125I-LDL and 125I-AcLDL  was dialyzed 24 h against RPMI 1640 medium containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine . All dialysis was carried out with 10,000 molecular weight cut-off Slide-A-Lyzer cassettes . After dialysis, lipoproteins were filtered through a 0.45-µm (pore-size) Acrodisc low protein-binding filter . 125I-LDL and 125I-AcLDL  specific activities were adjusted to 2.25×10−5 µCi/ng by adding unlabeled LDL and AcLDL, respectively.Human LDL and AcLDL  was dialyzed at 4°C for 24 h against 1 liter of RPMI 1640 medium containing 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM L-glutamine, with a medium change at 12 h. Human 2+ and Mg2+ (DPBS), lysed with 1 ml/well of ultrapure water, and then detached with a cell scraper. Lipid was isolated using the Folch method et al.Following incubation of macrophage cultures with 1 mg/ml of LDL (unless indicated otherwise) in serum-free RPMI 1640 containing M-CSF (50 ng/ml) and the indicated additions, macrophage cultures were rinsed three times in Dulbecco’s phosphate-buffered saline containing CaMacrophages were incubated with 4 mg/ml LDL for 24 h, rinsed three times with DPBS, and then fixed 30 min at room temperature with 4% paraformaldehyde in DPBS. Then, the fixed macrophage cultures were rinsed three times with DPBS, and cultures were stained 15 min at room temperature with filtered Oil Red O staining solution. First, Oil Red O stock solution was prepared by dissolving Oil Red O in isopropanol to obtain a 3% (w/v) solution. Next, Oil Red O solution was diluted by adding 2 parts ultrapure water to 3 parts Oil Red O stock solution and then filtered (0.45 µm pore-size). Following Oil Red O staining, macrophage cultures were rinsed three times with ultrapure water, stained two min with Modified Mayer’s hematoxylin QS , rinsed three times with ultrapure water, and then coverslipped with glycerol-gelatin mounting media .125I-LDL or 125I-AcLDL. 125I-LDL or 125I-AcLDL macrophage uptake was assessed by measuring cell-associated and degraded 125I-lipoprotein according to the method of Goldstein et al.g for 10 min and trichloroacetic acid-soluble organic iodide radioactivity was measured to quantify lipoprotein degradation. Cell-associated 125I-lipoprotein was assessed as follows. Macrophages were rinsed first three times with DPBS with 0.2% bovine serum albumin (BSA), followed by three rinses with DPBS containing Ca2+ and Mg2+, all at 4°C. Macrophages were dissolved overnight in 0.1 N NaOH at 37°C and then assayed for 125I radioactivity with a γ counter. 125I radioactivity values for wells incubated with 125I-lipoprotein without macrophages were subtracted from 125I radioactivity values obtained for macrophages incubated with 125I-lipoprotein. Values were <1% of cell-associated for 125I-lipoprotein. For protein quantification, a small aliquot of cell lysate was measured using the Lowry method 125I-Lipoprotein uptake is presented as the sum of cell-associated 125I- lipoprotein and degraded 125I-lipoprotein.Macrophages were pretreated with various drugs for one hour and subsequently incubated with the indicated concentration of Macrophage cultures were observed by time-lapse, phase-contrast digital video microscopy with a 20X long working distance PanFluor phase-objective lens (0.3 N.A.) mounted on an Olympus L70 inverted microscope. Cultures were maintained in an enclosed LiveCell™ chamber  containing 5% CO2/95% hydrated air at 37°C. Images were acquired every 10 s for 30 min. The acquired 180 images were converted into digital movies created using IP Lab software . When viewed at standard rates , movies are 100X real-time.3, 3% BSA, and anti-Rac1 mouse monoclonal antibody supplied by the manufacturer’s kit . After incubation with primary antibody, membranes were washed and incubated 1 h with anti-mouse IgG-HRP-conjugate  in TBST. Detection of proteins was determined by chemiluminescence using Western Blotting Luminol Reagent .For assessment of Rac1 activation, macrophages were incubated for 1 h with 100 µM NSC23766 or vehicle (DMSO) and then lysed with lysis buffer supplied by the manufacturer’s kit . Then, GTP-bound Rac1 was immunoprecipitated with Pak1 as described by the manufacturer. Lysates were diluted with reducing sample buffer and loaded onto a 4–15% Tris-HCl polyacrylamide gel . After SDS-PAGE, protein was transferred to nitrocellulose membranes and then incubated overnight at 4°C in Tris-Buffered Saline containing 0.1% Tween (TBST)-20, 0.1% NaNFor assessment of Src-family kinase activation, macrophages were incubated 1 h with 20 µM PP2 or vehicle (DMSO) and then lysed with RIPA Lysis buffer . Lysates were diluted with Laemmli Sample Buffer and loaded onto 4–15% Tris-HCl polyacrylamide gradient gels (BioRad). After SDS-PAGE, protein was transferred to nitrocellulose membranes and then incubated overnight at 4°C in TBST, 3%BSA, and rabbit Phospho-Src Family (Tyr416) Antibody . After incubation with primary antibody, membranes were washed and incubated 1 h with anti-rabbit IgG-HRP-conjugate  in TBST. Detection of proteins was determined by chemiluminescence using Western Blotting Luminol Reagent . As a loading control for NSC23766 and PP2 experiments, macrophage lysates were treated with Laemmli Sample Buffer and loaded onto 4–15% Tris-HCl polyacrylamide gradient gels (BioRad). Protein was then transferred to nitrocellulose membranes and incubated overnight at 4°C in TBST, 3%BSA, and mouse anti-GAPDH Antibody . Membranes were washed and incubated 1 h with anti-rabbit IgG-HRP-conjugate  in TBST and detection of proteins was determined by chemiluminescence using Western Blotting Luminol Reagent.3, 5% BSA, and 4G10 mouse monoclonal antibody . After incubation with primary antibody, membranes were washed and incubated 1 h with anti-mouse IgG-HRP-conjugate  in Tris-Buffered Saline containing 0.1% Tween-20, 0.1% NaN3, and 5% BSA. Detection of proteins was determined by chemiluminescence using Western Blotting Luminol Reagent .For tyrosine phosphorylation assessment of the M-CSF receptor, macrophages were incubated 1 hour with 5 µM GW2580 or vehicle (DMSO) and then lysed with RIPA Lysis Buffer  containing Halt Protease and Phosphatase inhibitor . The lysates were immunoprecipitated with anti-CSF-1R , then protein A agarose beads were added to lysates and incubated overnight. The lysate/bead mixture was then pelleted and resuspended with reducing sample buffer and loaded onto a 4–15% Tris-HCl polyacrylamide gel . After SDS-PAGE, protein was transferred to nitrocellulose membranes and then incubated overnight at 4°C in Tris-Buffered Saline containing 0.1% Tween-20, 0.1% NaNp value less than 0.05 was considered significant.Data are presented as the mean ± SEM. The means were determined from three culture wells for each data point. Data was analyzed by the 2-tailed unpaired Student’s t test for statistical significance. A Figure S1Assessment of M-CSF receptor tyrosine kinase inhibitor activity. Wild-type macrophages were incubated for 1 h with 5 µM GW2580 or vehicle (DMSO). M-CSF was present in the culture medium before and after GW2580 or vehicle treatment. GW2580-treated and vehicle-treated lysates were probed with M-CSF receptor antibody, GAPDH antibody, or immunoprecipitated with M-CSF receptor antibody and then probed with anti-phosphotyrosine (4G10) for Western blot analysis.(TIF)Click here for additional data file.Figure S2M-CSF withdrawal decreases macrophage accumulation of LDL-derived cholesterol. Wild-type macrophages were incubated 6 h with 1 mg/ml LDL without or with M-CSF, and then cholesterol accumulation was assessed.(TIFF)Click here for additional data file.Figure S3Native LDL induces macrophage foam cell formation. A. LDLR−/− macrophages were incubated with increasing concentrations of LDL for 24 h and then total cholesterol accumulation was assessed. B and C. LDLR−/− macrophages were incubated without (B) or with 4 mg/ml native LDL (C) for 24 h, and then were stained with Oil Red O to label neutral lipid. Scale bar in B = 75 µm and also applies to C.(TIF)Click here for additional data file.Figure S4The scavenger receptors CD36 and SRA do not mediate LDL uptake. Wild-type (WT), CD36 KO (ΔCD36), and SRA KO (ΔSRA) macrophages were incubated 6 h with 200 µg/ml 125I-LDL. Incubations were performed in serum-free medium containing 50 ng/ml M-CSF. Uptake values represent the sum of cell-associated and degraded 125I-LDL. The range of cell-associated and degraded 125I-LDL was 8–9% and 91–92%, respectively. There was no statistical difference between macrophage groups.(TIF)Click here for additional data file.Figure S5Macropinocytosis in LDLR−/− macrophagesis inhibited by PI3K and actin polymerization inhibitors. LDLR−/− macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (A), 4 µg/ml cytochalasin D (B), or 50 µM LY294002 (C). Following treatment, macrophages were examined by phase-contrast microscopy. Arrows indicate macropinosomes. Bar in C = 75 µm and applies to all.(TIF)Click here for additional data file.Figure S6Assessment of Rac1 and Src-family kinase inhibitor activity. Wild-type macrophages were incubated for 1 h with 100 µM NSC23766, 20 µM PP2, or vehicle (DMSO). (A) NSC23766-treated and vehicle-treated lysates were probed with GAPDH antibody or immunoprecipitated with Pak1 and then probed with anti-Rac1 for Western blot analysis. (B) PP2-treated and vehicle-treated lysates were probed with phospho Src-family antibody or GAPDH antibody. Incubations were performed in serum-free medium containing 50 ng/ml M-CSF. Phosphorylated Src Family Kinase = p-SFK.(TIF)Click here for additional data file.Videos S1Macrophage macropinocytosis is M-CSF dependent. Wild-type macrophages differentiated with M-CSF were pretreated 30 min either with 5 µM GW2580 or vehicle without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of DMSO vehicle (Video S1) or 5 µM GW2580 (Video S2). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S2Macrophage macropinocytosis is M-CSF dependent. Wild-type macrophages differentiated with M-CSF were pretreated 30 min either with 5 µM GW2580 or vehicle without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of DMSO vehicle (Video S1) or 5 µM GW2580 (Video S2). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S3M-CSF withdrawal inhibits macrophage macropinocytosis. Wild-type macrophages differentiated with M-CSF were incubated (Video S3) with or (Video S4) without M-CSF (50 ng/ml) for 30 min, and then macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy. Incubations were performed in serum-free medium.(MP4)Click here for additional data file.Videos S4M-CSF withdrawal inhibits macrophage macropinocytosis. Wild-type macrophages differentiated with M-CSF were incubated (Video S3) with or (Video S4) without M-CSF (50 ng/ml) for 30 min, and then macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy. Incubations were performed in serum-free medium.(MP4)Click here for additional data file.Videos S5Macropinocytosis in wild-type macrophagesis inhibited by PI3K and actin polymerization inhibitors. Wild-type macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S5), 100 nM wortmannin (Video S6), 4 µg/ml cytochalasin D (Video S7), or 50 µM LY294002 (Video S8). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy. Note that LY294002-treatment of macrophages induced small cytoplasmic vacuoles that were not formed by macropinocytosis.(MP4)Click here for additional data file.Videos S6Macropinocytosis in wild-type macrophagesis inhibited by PI3K and actin polymerization inhibitors. Wild-type macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S5), 100 nM wortmannin (Video S6), 4 µg/ml cytochalasin D (Video S7), or 50 µM LY294002 (Video S8). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy. Note that LY294002-treatment of macrophages induced small cytoplasmic vacuoles that were not formed by macropinocytosis.(MP4)Click here for additional data file.Videos S7Macropinocytosis in wild-type macrophagesis inhibited by PI3K and actin polymerization inhibitors. Wild-type macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S5), 100 nM wortmannin (Video S6), 4 µg/ml cytochalasin D (Video S7), or 50 µM LY294002 (Video S8). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy. Note that LY294002-treatment of macrophages induced small cytoplasmic vacuoles that were not formed by macropinocytosis.(MP4)Click here for additional data file.Videos S8Macropinocytosis in wild-type macrophagesis inhibited by PI3K and actin polymerization inhibitors. Wild-type macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S5), 100 nM wortmannin (Video S6), 4 µg/ml cytochalasin D (Video S7), or 50 µM LY294002 (Video S8). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy. Note that LY294002-treatment of macrophages induced small cytoplasmic vacuoles that were not formed by macropinocytosis.(MP4)Click here for additional data file.Videos S9Effect of GTPase, tubulin, and vacuolar type H(+)-ATPase inhibitors on macrophage macropinocytosis. Macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, the macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S9), 80 µM dynasore (Video S10), 500 nM bafilomycin A1 (Video S11), 100 µM NSC23766 (Video S12), 10 µM nocodazole (Video S13), or 40 µM Y-27632 (Video S14). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S10Effect of GTPase, tubulin, and vacuolar type H(+)-ATPase inhibitors on macrophage macropinocytosis. Macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, the macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S9), 80 µM dynasore (Video S10), 500 nM bafilomycin A1 (Video S11), 100 µM NSC23766 (Video S12), 10 µM nocodazole (Video S13), or 40 µM Y-27632 (Video S14). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S11Effect of GTPase, tubulin, and vacuolar type H(+)-ATPase inhibitors on macrophage macropinocytosis. Macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, the macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S9), 80 µM dynasore (Video S10), 500 nM bafilomycin A1 (Video S11), 100 µM NSC23766 (Video S12), 10 µM nocodazole (Video S13), or 40 µM Y-27632 (Video S14). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S12Effect of GTPase, tubulin, and vacuolar type H(+)-ATPase inhibitors on macrophage macropinocytosis. Macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, the macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S9), 80 µM dynasore (Video S10), 500 nM bafilomycin A1 (Video S11), 100 µM NSC23766 (Video S12), 10 µM nocodazole (Video S13), or 40 µM Y-27632 (Video S14). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S13Effect of GTPase, tubulin, and vacuolar type H(+)-ATPase inhibitors on macrophage macropinocytosis. Macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, the macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S9), 80 µM dynasore (Video S10), 500 nM bafilomycin A1 (Video S11), 100 µM NSC23766 (Video S12), 10 µM nocodazole (Video S13), or 40 µM Y-27632 (Video S14). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S14Effect of GTPase, tubulin, and vacuolar type H(+)-ATPase inhibitors on macrophage macropinocytosis. Macrophages differentiated with M-CSF were pretreated 30 min with the drugs indicated below but without serum and M-CSF. Then, the macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of either drug vehicle (Video S9), 80 µM dynasore (Video S10), 500 nM bafilomycin A1 (Video S11), 100 µM NSC23766 (Video S12), 10 µM nocodazole (Video S13), or 40 µM Y-27632 (Video S14). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S15Dynamin is a mediator of macrophage macropinocytosis. Wild-type macrophages differentiated with M-CSF were pretreated 30 min either with 20 µM myristoylated dynamin inhibitory peptide (control) or 20 µM dynamin inhibitory peptide without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of 20 µM myristoylated dynamin inhibitory peptide (control) (Video S15) or 20 µM dynamin inhibitory peptide (Video S16). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.Videos S16Dynamin is a mediator of macrophage macropinocytosis. Wild-type macrophages differentiated with M-CSF were pretreated 30 min either with 20 µM myristoylated dynamin inhibitory peptide (control) or 20 µM dynamin inhibitory peptide without serum and M-CSF. Then, macrophages were treated 30 min with fresh serum-free medium containing M-CSF (50 ng/ml) in the presence of 20 µM myristoylated dynamin inhibitory peptide (control) (Video S15) or 20 µM dynamin inhibitory peptide (Video S16). During the treatment, macrophage macropinocytosis was visualized by time-lapse, phase-contrast digital microscopy.(MP4)Click here for additional data file.


Haploidentical related donors are alternative stem cell sources for patients without human leukocyte antigen (HLA)-matched related or unrelated donors. Immediate access to the donor, availability for patients with rare haplotypes, ease of stem cell procurement, and lack of a requirement for a physical cord blood bank or an extensive HLA database render this type of hematopoietic stem cell transplantation particularly attractive despite the high histoincompatibility barrier between the recipient and the haploidentical graft. In this review, we answer the following questions: 1) What are the current transplant strategies used to overcome the histoincompatibility barrier in haploidentical stem cell transplantation and their clinical results? 2) How should we choose the donor when there is more than one available haploidentical donor? 3) How does transplantation from haploidentical donors compare to that from umbilical cord blood? Conflict of interest:None declared. Two-thirds of patients who require allogeneic hematopoietic stem cell transplantation (SCT) do not have a human leukocyte antigen (HLA)-matched related donor available . A matchWhat are the current transplant strategies used to overcome the histoincompatibility barrier in haploSCT and their clinical results? For successful haploSCT, both the patient’s and the graft’s immunity should be suppressed or modified to prevent graft failure and graft-versus-host disease (GVHD). Various strategies have been devised to achieve the required suppression without substantially increasing treatment-related mortality (TRM) arising from immunosuppression. These strategies may be studied in 2 groups: those utilizing ex vivo T cell-depleted grafts and those utilizing T cell-replete grafts. With currently available magnetic selection methods, 3 to 5 logs of ex vivo T cell depletion (TCD) of the stem cell graft is possible . The risWithout TCD of the graft, a higher-intensity GVHD prophylaxis regimen or selective inhibition of graft T cells becomes necessary to prevent GVHD after haploSCT. While Chinese researchers chose to intensify immunosuppression and prime the BM graft with granulocyte colony-stimulating factor (G-CSF) , researcOverall, while TCD results in lower GVHD incidence with acceptable engraftment rates when a “mega-dose” of CD34+ cells is used, a relatively high TRM rate primarily due to infections remains an issue. Furthermore, TCD requires an initial investment in facilities employing good manufacturing practice with cell selection instruments, i.e. CliniMACS, and expertise to run such facilities. The initial investment cost may be difficult to attain in developing and under-developed countries where haploSCT would be particularly valuable since residents of such countries are generally under-represented in international HLA databases. While haploSCT with T cell-replete grafts may lead to higher GVHD incidence, it allows the intensity of conditioning regimens to be reduced through host immunity suppression utilizing engraftment. However, the reduced intensity conditioning regimen used in most studies of post-SCT cyclophosphamide may lead to high relapse incidence in acute leukemic patients. At the MD Anderson Cancer Center, we compared the outcomes of haploSCT with TCD peripheral blood grafts to that with unmanipulated BM grafts after an identical ablative conditioning regimen  . Early rHow should we choose the donor when there is more than one available haploidentical donor?Most patients requiring SCT have more than one haploidentical donor. The presence of recipient antibodies against donor-specific HLA, killer immunoglobulin-like receptor (KIR) mismatch predicting NK cell alloreactivity, mismatch for non-inherited maternal vs. paternal alleles, degree of HLA mismatch between donor and recipient, vdonor age, and ABO-match should be taken into account while deciding on the donor among available haploidentical candidates. Transplant recipients may have developed anti-HLA antibodies against donor HLA antigens  during pregnancy or after blood product transfusions. The presence of DSAs is associated with increased risk of primary graft failure after SCT ,17,18,19NK cells primarily attack hematopoietic cells, sparing solid organs . In reciAlthough a progressive increase in TRM with increasing genetic disparity has been historically reported, contemporary transplant strategies may negate this correlation by overcoming larger histoincompatibility barriers. In fact, Kasamon et al. reported no increased incidence of acute GVHD (aGVHD) and non-relapse mortality (NRM) after haploSCT from full-haplotype mismatched donors compared to those with better-matched donors . MoreoveImmunologic tolerance may develop between mother and fetus during pregnancy ,29, leadThe immune system is subject to senescence with advancing age. Although no data exist on an association between donor age and outcomes after haploSCT, the findings of higher GVHD incidence and shorter survival after unrelated donor transplants from older donors compared to younger donors would probably apply for haploSCT, as well. Older multiparous women may be the least preferred donors for male recipients .Studies have demonstrated that infusion of larger numbers of CD34+ cells improved outcomes after SCT ,34,35. SAn in-depth review of donor selection in haploSCT is available from Ciurea and Champlin  and the How do transplants from haploidentical donors compare to those from umbilical cords?For patients lacking an HLA-matched related or unrelated donor, umbilical cord blood (UCB) is another alternative stem cell source. UCB is more immune-plastic than peripheral blood and bone marrow grafts; therefore, 2 or 3 out of 6 HLA mismatches are allowed for UCB transplants. However, use of UCB as a stem cell source has been limited until recently by the delayed engraftment and relatively high rate of primary graft failures due to the low volume and low CD34+ cell content. Use of double, instead of single, UCB has partially overcome these issues ,37.The advantages and disadvantages of haploSCT and UCB SCT are outlined in Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included. With our current knowledge, it is difficult to recommend one stem cell source over another for patients without matched donors. Until a large-scale randomized prospective study shows one’s superiority, transplant centers will and should choose an alternative stem cell source based on their own expertise. However, T cell-replete haploSCT is clearly advantageous for countries and centers without the financial backing to invest in and maintain an umbilical cord bank. Despite these advantages and recent advances, haploSCT is a risky procedure with additional perils of late-onset chronic GVHD and infections due to the histoincompatibility barrier, late immune reconstitution, and intensified GVHD prophylaxis limiting its use to experienced centers.


LFY and AP1 to initiate flowering transition[AP1, responds of diverse signals during flower development remains largely unknown. Here we have obtained an inbred line exhibiting an abnormal stem/inflorescence and flower development.Bolting is regarded as the initiation of reproduction stage in the growth and development of an Arabidopsis plant, when a set of floral integrator genes activate the expression of floral meristem identity genes ransition. HoweveThe morphological variations were carefully observed visually or with a Zeiss SteREO Discovery V8 stereomicroscope. Hybridization of GeneChip arrays was done in an Affymetrix Hybridization Oven 640 following the manufacturer’s protocol (Affymetrix) and expression levels in seedlings and flower buds were calculated from Affymetrix intensity data. Real-time PCR was performed on an ABI 7500 Real-Time PCR System  with SYBR GreenPCR Master Mix (Applied Biosystems) as the fluorescence source.ap1, such as homeotic conversion of sepals (first whorl) to leaf-like, petals often absent (second whorl) and complete flower-to-inflorescence conversions , OVEREXPRESSION OF CONSTANS(SOC1), AGAMOUS-like 24 (AGL24), SEPALLATA (SEP) and upstream genes FLOWERING LOCUS M (FLM) were increased in flower buds .Microarray and quantitative real-time PCR analysis revealed that the expression of AP1 sequence analysis showed that the promoter, coding sequences and intron splice sites of AP1 genomic sequence in this inbred line were unchanged comparing to that in wildtype, suggesting the complexity in the regulation of AP1 in the line. Therefore these synthetic contributions caused the development of this unique phenotype. For instance, TFL1 was found to be highly expressed, and this gene can negatively regulate AP1 and specify inflorescence meristem identity leading to a delay of floral meristem formation[AGL24 and SOC1, which promote inflorescence fates rather than flower formation in the meristem and result in more abundant and longer inflorescences[AP1) to specify a floral or a stem/inflorescence meristem.formation. On the ap1 mutants with noticeable deviations. The abnormal stem/inflorescence of the inbred line was mainly caused by significant downregulation of AP1, but also is attributable to crosstalk among key genes like TFL1, AGL24, SOC1, etc. to control the transition of vegetative growth to the flowering phase. The inbred linemerits further molecular characterization to understand better the regulatory molecular network.The inbred line identified in this study is phenotypically similar to