Previously there have been no methods for directly tracing the flow of cerebrospinal fluid (CSF) under physiological conditions, and the circulation of CSF has therefore been studied and visualized by injecting a radioactively labeled tracer or contrast medium visible in x-ray images. The newly developed Time-Spatial Inversion Pulse (Time-SLIP) method makes it possible to directly visualize the flow of CSF using magnetic resonance imaging (MRI), permitting CSF dynamics to be depicted in a certain time frame. The CSF dynamics visualized using Time-SLIP has been found to differ markedly from the classical CSF circulation theory described in medical textbooks. It can be said that research on CSF dynamics has advanced to the next stage with the use of this innovative imaging method. Obtaining a more accurate understanding of normal CSF physiology and pathophysiology should lead to improved diagnostic accuracy, permit the identification of new etiological factors in a variety of diseases, and promote the development of new therapeutic approaches. Descriptions of the physiological circulatory dynamics of cerebrospinal fluid (CSF) can be found mainly in neurosurgery textbooks. More than a hundred years ago, it was only natural that the circulation of CSF was considered to be similar to the circulation of blood. It was naturally thought that the physiology of CSF followed the same pattern as that of blood, which is a typical example of a physiological phenomenon in the body. Harvey Cushing, who was a pioneer in the field of neurosurgery, referred to the CSF as the third circulation ,2. SinceAlthough the newly developed Time-Spatial Inversion Pulse (Time-SLIP) method makes it possible to visualize the flow of CSF using MRI, the imaging procedures and examination time are fundamentally different from those of the phase contrast technique. Therefore, most of the information obtained using these methods cannot be directly compared with each other. In the Time-SLIP method, the CSF itself serves as an endogenous tracer when radiofrequency (RF) pulses are applied. The flow of CSF can be observed for a period of approximately 5 seconds until the effects of the RF pulses diminish and are no longer visible. The Time-SLIP method makes it possible to depict CSF dynamics in a time frame that is not possible with any other method.The CSF dynamics visualized using Time-SLIP differ markedly from the descriptions of CSF flow that have been given in medical textbooks. CSF is clear like water in the absence of diseases such as meningitis, and it has been demonstrated that it undergoes turnover. However, whether or not the CSF flows like a river from the sites where it is produced to the sites where it is absorbed requires further verification ,9.A detailed explanation of the principles of Time-SLIP can be found in other reports . BrieflyThe fact that CSF motion varies in response to cardiac pulsation and respiratory motion can be observed as a fluid level fluctuation in the ventricular external drainage tube from the patient cerebral ventricle, observing the changes in fluid level of the CSF during surgery and the lumbar puncture. In other words, CSF dynamics exhibit various types of motion according to the combination of cardiac pulsation and respiratory motion, which means that the data must be acquired in real time . This drUsing Time-SLIP, the flow of CSF from the third ventricle into the lateral ventricles has been observed . CSF floActually, it was previously thought that this type of CSF flow does not occur in healthy persons , but onlWhen we consider this finding more deeply, it is clear that we are unable to answer the simple question “Where is the CSF produced in the case of hydrocephalus in which ventricular reflux is observed?” Nevertheless, this theory was widely accepted as the truth for nearly 50 years until CSF dynamics could be clearly visualized using Time-SLIP. No CSF reflux from the third ventricle to the lateral ventricles was observed in a patient with hydrocephalus using Time-SLIP MR imaging . Since sThe CSF motion visualized using Time-SLIP does not show unidirectional backflow of CSF into the lateral ventricles, but rather demonstrates that the CSF in the lateral ventricles and the CSF in the third ventricle are actively exchanged through the foramen of Monro in the normal brain. On the other hand, there is virtually no flow (or only extremely slow flow) of CSF in the body of the lateral ventricles, except in the area adjacent to the foramen of Monro . At leasIn examinees who are unable to remain still and therefore move their heads during MRI scanning, the CSF in the body of the lateral ventricles is strongly agitated . ConsideThe circulation of CSF is clearly not like that of blood, which flows through tubular blood vessels at high speed. A more accurate model may be that of placing the brain and spinal cord in a glass container, immersing them in water, and then shaking the container. Even though CSF motion can only be visualized in real time for a period of several seconds, it is clear that these observations are totally different from the classical concept of CSF circulation.CSF motion in the third and fourth ventricles is swirling vortex-type flow even when the head is stationary . The areWe can easily imagine that the function of the CSF is surely not only to protect the brain from external forces  or to serve as a means for eliminating wastes from the brain -26 althoThe standard method for determining the amount of CSF is based on an ingenious ventriculo-cisternal perfusion experiment described by Pappenheimer et al ,28. ArtiBrain interstitial fluid has the same composition as CSF, and there is no barrier between them like the blood-brain barrier. Normally, free exchange occurs between the CSF and brain interstitial fluid ,36 throuAccording to medical textbooks, the CSF is produced by the choroid plexus. But this raises an important question: When the cerebral aqueduct or the outlets of the fourth ventricle are obstructed, where is the CSF downstream from the point of occlusion produced? This is another example of a situation in which the CSF physiology described in current medical textbooks is unable to fully explain CSF flow under physiological or pathological conditions.At present, Time-SLIP is unable to depict the correlated dynamics of the CSF and brain interstitial fluid. Although this may be a bit of a digression from the main topic, which is the visualization of CSF using Time-SLIP, the production and absorption of CSF mediated by brain capillaries is one of the most important issues in the reassessment of CSF physiology ,39. ThisTime-SLIP allows the dynamics of CSF to be depicted under the most physiological conditions because it does not require the injection of a tracer. The CSF itself serves as an endogenous tracer. The most important finding related to the absorption pathways of CSF obtained using Time-SLIP is that no flow or pulsation of the CSF is observed over the cerebral convexity . Even thWhen the flow of CSF in the subarachnoid space of the spine is visualized, the CSF is seen to pulsate along the ventral side of the spine in the supine position, but hardly any CSF flow is seen along the dorsal side of the spine. However, the CSF begins to pulsate along the dorsal side of the spine when the same examinee is placed in the prone position . On the The spinal subarachnoid space is a cylindrical structure with its tip at the caudal end and it does not contain any internal partitions. It is very difficult to imagine that CSF flows in opposite directions on the dorsal side and ventral side. The observations underlying this theory of CSF flow in the subarachnoid space were reported by Di Chiro , who surCSF pulsation observed by Time-SLIP was traced using semi-automated tracing software called Dynatracer . The movIt can be said that research on CSF dynamics has advanced to the next stage with the use of the innovative imaging method known as Time-SLIP. Obtaining a more accurate understanding of normal CSF physiology and pathophysiology should lead to improved diagnostic accuracy, permit the identification of new etiological factors in a variety of diseases, and promote the development of new therapeutic approaches.


The use of midazolam for dental care in patients with intellectual disability is poorly documented. The purpose of this study was to determine which method of premedication is more effective for these patients, 0.15 mg/kg of intramuscular midazolam or 0.3 mg/kg of oral midazolam.This study was designed and implemented as a non-randomized retrospective study. The study population was composed of patients with intellectual disability who required dental treatment under ambulatory general anesthesia from August 2009 through April 2013. Patients were administered 0.15 mg/kg of midazolam intramuscularly (Group IM) or 0.3 mg/kg orally (Group PO). The predictor variable was the method of midazolam administration. The outcome variables measured were Observer’s Assessment of Alertness/ Sedation (OAA/S) Scale scores, the level of cooperation when entering the operation room and for venous cannulation, post-anesthetic agitation and recovery time.p=0.034). There were no differences in demographic data and other variables between the groups.Midazolam was administered intramuscularly in 23 patients and orally in 21 patients. More patients were successfully sedated with no resistance behavior during venous cannulation in Group PO than in Group IM (The results of this study suggest that oral premedication with 0.3 mg/kg of midazolam is more effective than 0.15 mg/kg of midazolam administered intramuscularly, in terms of patient resistance to venous cannulation. If both oral and intramuscular routes of midazolam are acceptable in intellectually disabled patients, the oral route is recommended.Key words:Premedication, midazolam, intellectual disability. The number of people with special needs who require oral health services is increasing . PatientAlthough several drugs, such as ketamine , clonidiTo address the research purpose, the investigators designed and implemented a retrospective study to evaluate the effectiveness of intramuscular and oral administration of midazolam as sedative premedication before general anesthesia in intellectually disabled patients. This study was performed according to the Declaration of Helsinki and approved by the institutional review board and ethics committee of Osaka University Dental Hospital (H26-E50).- PatientsThe study population was composed of patients with intellectual disability receiving dental treatment under ambulatory general anesthesia from August 2009 through April 2013. To be included in the study, patients had to be American Society of Anesthesiologists Physical Status (ASA-PS) class I or II, and aged over 16 years old. Patients were excluded from participation if their body weight was over 67 kg, because the oral dose of midazolam was limited to a maximum of 20 mg in this study. Patients who could not walk normally, declined measurement of their BP, or whose perioperative records were incomplete, were also excluded. All study variables were collected from the medical or anesthesia records.- Standard ambulatory anesthesia procedurePatients were admitted to the hospital by 8:30 a.m. after more than 8 hours of fasting. If patients regularly took a morning dose of tranquillizers or anti-epilepsy medication, they were asked to take these drugs between 6:00 and 7:00 a.m. with a little plain water. After entering the day surgery preparation room, patients were administered midazolam  intramuscularly (0.15 mg/kg: Group IM) or orally (0.3 mg/kg: Group PO) as premedication. Orally administered midazolam was limited to a maximum dose of 20 mg  scale . OAA/S sAfter evaluation of the sedation level, the patients entered the operation room from the day surgery preparation room and lay down on the operating table. Then, a peripheral 22-gauge catheter was inserted into a dorsal hand vein. Patient cooperativeness during transfer and during venous cannulation was closely recorded. The patient’s condition during transfer and when entering the operation room was evaluated by an original score: 1, sleeping; 2, cooperative; 3, fighting. The patient’s condition during venipuncture was also evaluated as: 1, no body movement; 2, cooperative; 3, fighting.Anesthesia was usually induced with 5 mg/kg of thiamylal sodium , followed by 0.6 mg/kg of rocuronium bromide  to facilitate nasotracheal intubation. After intubation, a nasogastric tube was inserted to withdraw gastric contents. Anesthesia was maintained with 66% nitrous oxide and sevoflurane at an end-tidal concentration of 1 to 2% in oxygen .When the procedure was expected to be painful, 2% lidocaine with 1:80,000 epinephrine was injected for local anesthesia. When the patient underwent tooth extraction, 1 mg/kg of flurbiprofen axetil , up to a dose of 50 mg, was administered intravenously before the end of the operation.Immediately after the end of the dental procedure, administration of sevoflurane was discontinued. After extubation, agitation score was evaluated according to the scoring system for emergence delirium as : 1, sleeThereafter, patients were shifted from the operating room to the post anesthesia care unit (PACU). Blood pressure, pulse rate, arterial oxygen saturation (SpO2), body temperature, and modified post anesthetic discharge scoring system (mPADSS) scores ,10 were - Statistical analysesIn this study, the primary predictor variable was the administration method of midazolam. The outcome variables measured were OAA/S Scale score, transfer condition while entering the operation room, venipuncture condition, post-anesthetic agitation and recovery time. The third category of variables studied included gender, age, height, weight, anesthesia time, operation time and coexisting disabilities.P < 0.05), the Mann-Whitney test was used for analysis. These data are expressed as median (interquartile range). Parameters with normal distributions (P > 0.05) and equal variance (P > 0.05), such as age, height, body weight and recovery time, were analyzed by the Levene and unpaired t tests. These data are expressed as mean ± standard deviation. Patient gender, medications, coexisting disabilities, OAA/S scale score, transfer condition, venipuncture condition and agitation score were compared by the chi-square test. Statistical analysis was performed with the SPSS software package . A P value less than 0.05 was considered statistically significant.Distributions of patients’ values were tested for normality using the Shapiro-Wilk test. Since distribution of anesthesia time and operation time were non-normal . More patients in Group PO were more deeply sedated than patients in Group IM during venous cannulation. However, 3 patients exhibited resistant behaviors in each group.The patients’ condition at the time of transfer to the operating room is shown in Agitation scores after extubation are shown in The main finding of this study was that 0.3 mg/kg of oral midazolam was more effective in facilitating intravenous cannulation than 0.15 mg/kg of intramuscular midazolam. Although there were no differences in OAA/S scale scores after sedation and in the post-anesthesia transfer condition between the two groups, 0.3 mg/kg of oral midazolam could reduce venipuncture-related anxiety.The optimal intramuscular and oral dose of midazolam for patients with intellectual disability is not known. In normal adult patients, the optimal premedication dose of intramuscular midazolam was reported to be 0.08 mg/kg . Howeveret al. used a small dose of oral midazolam (an average of 0.042 mg/kg) before induction of general anesthesia when intravenous cannulation was difficult , and demonstrated the improvement in patient cooperation  during venous cannulation in about three-quarters of patients . The proportion of patients with a cannulation score of 3 was 61.9% (13/21) in Group PO and 26.1% (6/23) in Group IM. Our results suggest that 0.3 mg/kg of oral midazolam provides adequate sedation during intravenous cannulation. In the present study, there was no difference in recovery times between the groups. The effect of oral midazolam on recovery time is still controversial.et al. (et al. (et al. reported that 0.5 mg/kg of oral midazolam premedication did not delay discharge of children undergoing general anesthesia for dental treatment (et al. also demonstrated that premedication with oral midazolam 0.5 mg/kg has minimal effect on recovery time (In pediatric patients, Bevan et al.  showed p (et al.  reportedreatment . The sysery time .et al. demonstrated that oral midazolam was associated with delayed recovery after dental sedation (Maeda sedation  and ambusedation . Howeveret al. (As an administration route for midazolam, the oral route is more comfortable than other routes, such as intramuscular, intranasal and intrarectal routes. Some investigators compared the administration routes of midazolam. One study compared intramuscular and intranasal administration of midazolam . The intet al.  comparedAlthough our results showed the superiority of 0.3 mg of oral midazolam, oral administration has two disadvantages. First, onset time through the oral route is longer than that with the intramuscular route. It takes about 30 min to achieve the maximum sedative effect based on the peak effect ,17. Secoet al. (There are some limitations to the present study. First, most patients were on regular medication, such as anticonvulsant and anxiolytic drugs, which might affect the effectiveness of midazolam. However, it was neither possible to omit these drugs before anesthesia, nor to implement a study using these drugs, because of the variety of drugs and doses received by the patients. Second, the main biases of our study are that it was a retrospective, uncontrolled and unblinded study with a small sample size. We could not implement a randomized controlled prospective protocol for this study because many patients’ parents or caregivers requested the oral route of midazolam administration when providing informed consent, and hence, we could not randomly allocate patients to the two groups. The sample size of this study was small because many patients were excluded due to insufficient information on outcome variables. Moreover, the administration route was not blinded to the evaluator. Therefore, the possibility of selection and observer bias exists in the present study. Third, the study patients had different pathologies and degrees of intellectual disability. Asahi et al.  demonstrAs premedication for intellectually disabled dental patients, 0.3 mg/kg of oral midazolam was more effective than 0.15 mg/kg of intramuscular midazolam in terms of patient cooperation for venous cannulation, without resultant prolongation of recovery time. If both oral and intramuscular midazolam are acceptable, oral administration is recommended due to less resistant behaviors by the patient for venous cannulation.


Scenedesmus dimorphus has better potential as biofuel feedstock. Two of the isolates of Scenedesmus dimorphus performed better than other isolates with respect to important growth parameters with lipid content of ~30% of dry biomass. Scenedesmus dimorphus was found to be more suitable as biodiesel feedstock candidate on the basis of cumulative occurrence of five important biodiesel fatty acids, relative occurrence of SFA (53.04%), MUFA (23.81%) and PUFA (19.69%), and more importantly that of oleic acid in its total lipids. The morphological observations using light and Scanning Electron Microscope and molecular characterization using amplified 18S rRNA gene sequences of microalgae species under study were also performed. Amplified 18S rRNA gene fragments of the microalgae species were sequenced, annotated at the NCBI website and phylogenetic analysis was done. We have published eight 18S rRNA gene sequences of microalgae species in NCBI GenBank.In current study isolates of two native microalgae species were screened on the basis of growth kinetics and lipid accumulation potential. On the basis of data obtained on growth parameters and lipid accumulation, it is concluded that  Scenedemus dimorphus and Scenedesmus quadricauda collected and isolated previously [Microalgae are among the most promising, renewable, non-food crop based alternative biofuel feedstocks due to several characteristics such as non-competition with food and feed crops, high oil content and growth rate . Microaleviously  for growScenedesmus sp. as one of them. Griffiths and Harrison 2009 [Scenedesmus dimorphus was 26%. Many of the scientific reports claim very high accumulation of lipid content (>70%) in some microalgae species but under special nutrient starvation conditions that adversely affect the overall lipid productivity. The gain due to higher lipid content is counteracted by the lower productivities attained under nutrient shortage. This is one of major technical obstacle in realization of the algal biofuel and research efforts are required to explore and develop algal strains which defy this general principle. At the same time research is also needed to be done to devise and develop new methods to provide stress to algae culture which do not exert adverse effects on the growth. There is a need to isolate, screen, select, test and improve algal strains, for both higher oil content and overall productivity.There are many reviews and reports available in the literature in which different species of microalgae have been discussed as potential biofuel feedstocks , 9 incluson 2009  reviewedTraditionally taxonomic classification and subsequent identification often depended on morphological description of cell and colony features. Such observations being subjective sometimes result in double classification of the same organism and generates mistakes in the taxonomic assignation and identification –12. LateScenedesmus dimorphus and Scenedesmus quadricauda and Chlorella sp. isolated previously from various locations of Himachal Pradesh, India  was also recorded using a haemocytometer. When the stationary phase of growth reached, the cultures were centrifuged to harvest cell biomass, which was freeze dried to determine dry biomass content. Growth rate parameters of each isolate were calculated on the basis of cell count during exponential phase of growth and cell biomass. Following growth parameters were calculated from the observed data [The isolates of microalgal species under study were characterized for growth kinetics and biomass production potential to identify those which grew fast and accumulate higher biomass. 900 ml of BG-11 medium was inoculated with 100 ml inoculum of each isolate (10% v/v), adjusted to a cell density of 10–12 ×10ved data .μ = ln (Nt/No)/Tt-To where Nt is the number of cells at the end of log phase, No is the number of cells at the start of log phase, Tt is the final day of log phase and To is starting day of log phase.Specific growth rate (μ): Tt = 0.6931/μ.Doubling time: -1 day-1).Biomass productivity (Pdwt): as the dry biomass produced per day (g lLp = Pdwt×Lc×1000/100 and expressed as milligrams per liter per day (mg L-1 day-1) [Lipid accumulation estimation in microalgae biomass was done using modified Bligh and Dyer method  and indi1 day-1) .−4 mol L–1 Nile Red (Sigma) dissolved in acetone according to Elsey et al [For indirect estimation 15 ml of the culture from samples was centrifuged for 15 minutes at 7000 rpm, supernatant was discarded and the pallet was stored at –80°C. 2 ml of chloroform and 4 ml of methanol were added to the vials, which were then incubated for 45 minutes in a water bath at 45°C with intermittent mixing on vortex shaker for extraction of lipids. This solution was not disturbed for some time so as to separate it in two phases. 1.5 ml of light fraction (lipids in chloroform) was taken for staining with 5μL of 10ey et al . The fluFatty acid profile analysis of species wise pooled total lipids isolated from different isolates of a microalgae was carried out using gas chromatography mass spectroscopy (GC-MS) at CIL/SAIF at Panjab University Chandigarh, India. Fatty acid methyl esters were prepared using following procedure. 30 mg of total lipid dissolved in 1 ml of methanol was mixed with 1 ml of 12% solution of KOH prepared in methanol. To this solution equal volume of 5% HCL in methanol was added and heated at 75°C for 15 min. This solution was allowed to cool and 1 ml of distilled water was added and shaken. Upper organic layer containing fatty acid methyl esters was carefully transferred to a new clean vial. GC-MS analysis of FAMEs was performed using diethylene glycol succinate capillary column 30m × 0.25 × 0.25μm). 100μl of methyl ester sample solution was injected for each analysis. Helium was used as a carrier gas. The injector temperature was 180°C and detector temperature was 230°C which was increased to 300°C at a temperature gradient of 15°C/min. Identification of fatty acid methyl ester was done by comparing the mass spectra with NIST database library [ × 0.25μmData were expressed as mean of three independent parallel experiments. ANOVA was used to assess the differences amongst the treatments. Standard error of means values were calculated at <0.05 level of significance. The superscripts in tables represent the positions based on Tukey HSD Multiple Range test and different alphabets denote significantly different values as analyzed using SPSS 17. Five percent error is depicted in data presented as bar diagrams.Morphological observations on cells and colonies of microalgae species were carried out using an OLYMPUS 100× light microscope (LM) and digital images were captured. Scanning Electron micrograph (SEM) observations of representative samples of microalgae were also carried out to visualize three dimensional shape and size of cells. For SEM, 1 ml cultures were harvested  and fixeSd12, Sq2, Chl1, from S. dimorphus, S. quadricauda and Chlorella sp. were included randomly in this analysis. 50 ml of microalgae cultures were centrifuged at 7000 rpm for 10 minutes and pellet crushed in cold mortar and pestle to a fine powder by using liquid nitrogen. Subsequently microalgal DNA was isolated by using the Pure Link Plant Total DNA Purification Kit (Invitrogen), following the manufacturer's instruction. The quality of DNA was checked by visualization of genomic DNA after gel electrophoresis in 0.8% agarose in gel doc system. The 18S rRNA gene fragment of each microalgae species was amplified using green microalgae specific primer pairs previously described in literature  of the three microalgae under study was annotated with genus specific sequences from NCBI GenBank database using the basic local alignment search tool (BLAST). Ten genus specific sequences showing the highest similarity scores with each query sequence were retrieved from NCBI GenBank database. Multiple sequence alignment was carried out among retrieved and query sequences using the CLUSTAL-X program and phylogenetic tree was constructed using the software Mega 5.0.2 . The unwScenedesmus dimorphus and Scenedesmus quadricauda measured as optical density at 730 nm and cell density on 18th day of culture is presented in Sd1, Sd12 & Sd20 of Scenedesmus dimorphus and Sq16, Sq19 of Scenedesmus quadricauda displayed better performance compared to others within the same species. All the isolates of both the species were further characterized for the growth rate, biomass and lipid accumulation potential. Biomass productivity and lipid content are two most studied parameters in search of the prominent isolates for large-scale cultivation of microalgae for biofuel production [Scenedesmus dimorphus and Scenedesmus quadricauda is shown in Tables Scenedesmus dimorphus isolates ranged from 0.113 to 0.148; isolate Sd1, followed by Sd20 displayed the highest. Both these isolates performed better than others with respect to important growth parameters viz. biomass productivity, lipid content and lipid productivity as well  [Scenedesmus dimorphus and found that lipid content in the examined strain was 21.6% in BBM and 18.5% in 3N-BBM medium. Jena et al (2012) [Chlorococcum sp., Chlorella sp. and Scenedesmus sp.) of Odisha coast for the suitability for biodiesel production. They found Scenedesmus sp. to be the best one for high lipid productivity biomass yield. Ren et al (2013) [Scenedesmus sp. strain R-16 with high total lipid content. Compared to autotrophic situation, the strain R-16 grew well heterotrophically without light and the accumulated total lipid content and biomass reached 43.4% and 3.46 g L-1, respectively. Under nitrogen starvation the total lipid content was as high as 52.6%. From the data obtained and literarature reports so far on the Scenedesmus species, it can be concluded that isolates Sd1 and Sd12 of Scenedesmus dimorphus clearly diverge from the rest and thus can be considered for the further research on standardization of cultivation methods and strain improvement.The growth response of isolates of  as well . The lip biomass , Fig 2A.l (2013)  conductel (2012)  screenedl (2013)  reportedScenedesmus quadricauda, and isolates Sq12, Sq16 and Sq19 performed better than other isolates of this species. Highest lipid content of 14.99% and fluorescence intensity was recorded in case of isolate Sq19 , linolenic acid (12.68%), palmitic acid (10.14%), linoleic (10.11%) and 2-methy tetracosane (6.83%). Scenedesmus quadricauda lipids contained palmitic acid (16.36%), oleic acid (15.60%), linoleic acid (11.67%). 3, 7, 11- trimethyl-2, 4-dodecadiene (9.67%) and linolenic acid (6.21%). Similarly palmitic acid (19.97%), hexadecane, 2, 6, 11, 15- tetra methyl (C20:2), (9.85%), heptadecane-2, 6, 10, 15 tetramethyl (8.15%), Heneicosane, 11 (1-ethylpropyl) (6.27) and Phytol (5.95%) were most abundant fatty acids in the total lipids of Chlorella sp. Many of the parameters of biodiesel such as cetane number, kinematic viscosity, oxidative stability and cold flow properties depend on the fatty acid profile of biodiesel oil [Scenedesmus dimorphus contained highest amount of desirable fatty acids (55.15%) followed by Scenedesmus quadricauda (51.54%) among the microalgae under study. The relative occurrence of saturated fatty acid (SFA), mono unsaturated fatty acid (MUFA) and poly unsaturated fatty acid (PUFA) is also depicted in S. dimorphus 38.03, S. quadricauda 53.04%) followed by PUFA  occurred in higher proportion in both of Scenedesmus species, whereas MUFA was observed highest in Chlorella sp. (48.2%). The relative degree of unsaturation and saturation of fatty acids in biodiesel feedstock influence the biodiesel properties [Scenedesmus species oils was comparable or slightly better to that of soybean oil  [Scenedesmus dimorphus (16.12%) and Scenedesmus quadricauda (15.6%) compared to Chlorella sp. (4.58%)  isolated from Odisha coast of India. Their study suggested that Scenedesmus sp. is appropriate for bio-diesel production for its high lipid content and selected this strain for higher scale studies. Similarliy Prabakaran and Ravindran (2012) [Scenedesmus sp. to conatain highest amount of oleic acid (11.77 mg g–1dry wt) among different microalgal cultures  isolated from six different water bodies from Gandhigram, Tamil Nadu. Their results suggested that Scenedesmus sp. is useful for producing biodiesel, based on its high lipid and oleic acid contents. Thus on the basis of cumulative occurrence of five important biodiesel fatty acids, relative occurrence of SFA, MUFA and PUFA and more importantly oleic acid, and literature reports it is concluded that Scenedesmus dimorphus is one the of suitable candidate as biodiesel feedstock.The list of fatty acids and their relative proportion in term of area represented by the specific peak in GC-MS profile of total lipids of three microalgae species is given in esel oil . Very sh acid 16.%, oleic operties . Cetane operties , 38. Simoperties . The levA 59.1%) ), which  (4.58%) . Rodolfi workers , 40, 41  workers  found Scn (2012)  found ScA number of microalgae species have been investigated for growth and lipid production potential previously and as such there is no clear cut model species which can be included in experimental set up as standard species to make comparisons. In such a scenario it was thought of comparing the results with similar type of studies from the literature. Such comparison has been also made by Ren et al 2013 and Rodolfi et al., 2009 , 34, 39.Sd1 and Sd12 can be considered for the further research and productivity can be improved on standardization of cultivation methods and strain improvement.The specific growth rate and lipid productivity figures in most of studies are higher from the our isolates. It may be attributed to the fact that in most of these studies, algal culture were flushed with carbon dioxide which may be responsible for higher growth rates and biomass. It may be noted that this is a preliminary study in which primary focus was to screen the best performing isolates of the indigenous microalge species. The isolates Sd12 and Sd1 (~30%) was found to be higher as compared that of in all of such studies  were spherical where as that of Scenedesmus quadricauda and Scenedesmus dimorphus were near to spherical cuboidal and spherical lanceolate respectively. Scenedesmus quadricauda and Scenedesmus dimorphus formed typical colonies consisting of four or eight cells although two cell formation and single cells were also observed. The cells of Chlorella sp.were mostly free living sometimes aggregating into numerous cells. The length of longer axis of Scenedesmus quadricauda and Scenedesmus dimorphus ranged from 6.21 to 8.3 μm and 8.28 to 8.74 μm respectively whereas diameter of Chlorella sp. cells ranged between 3.47–3.62μm.The cell morphology of three of microalgae species as observed under the LM and SEM is depicted in Figs Scenedesmus dimorphus while Chloro F-R amplified from Chlorella sp. only. The amplified 18S rRNA gene fragments ranged between 300–1000 bp in size DNA was extracted in sufficient amount and the quality suitable for PCR amplification from three microalgae using Pure Link Plant Total DNA Purification Kit . The 18S rRNA specific primer pairs P73-P47 and CV3- CV4 amplified 18S rRNA gene fragments from all three microalgae. Primer pair CV1- CV2 did not amplify the same from Scenedesmaceae [Scenedesmus quadricauda, Scenedesmus quadricauda and Chlorella sp. constructed using the longest 18S rRNA query sequence and ten sequences from database showing the highest scores and maximum query coverage are depicted in Scenedesmus quadricauda strongly supported the branch points with bootstrap values 53–100% showing closest relationship with Scenedesmus abundans having minimum branch point distance of 0.0172. The bootstrap values range of phylogenetic tree of Scenedesmus dimorphus and Chlorella sp. were 18–71% and 33–100%, respectively. Scenedesmus dimorphus showed closest relationship with Scenedesmus sp. LG2VF16 and Scenedesmus incrassatulus with branch point distance of 0.0243 whereas Chlorella sp. was found to be phylogenetically nearest to Chlorella emersonii with branch point distance of 0.0347.The ITS1 and ITS2 sequences are significant alternative markers for studying the phylogenetic relationship within the esmaceae . The phyScenedesmus dimorphus is a better potential biofuel feedstock compared to S. quadricauda. Scenedesmus dimorphus was also found to be suitable as biodiesel feedstock candidate on the basis of fatty acid profile analysis of total lipids. Two Scenedesmus dimorphus isolates viz. Sd12 and Sd1 out performed the rest with respect to important growth kinetic parameters. The lipid content of these isolates was found to be higher than most other species as reported in similar studies. These isolates can further be used in scale up experiments, improvements of culture and cultivation methods and even in genetic improvement studies. Molecular characterization of microalgae species under study was also performed using amplified 18S rRNA gene sequences. Amplified 18S rRNA gene fragments of the microalgae species were sequenced, annotated at the NCBI website and phylogenetic analysis was done. We have published eight 18S rRNA gene sequences of microalgae species in NCBI GenBank.On the basis of data obtained on growth parameters and lipid accumulation and profile, it is concluded that S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 TableSimilarity scores and accession numbers of ten most close sequences with query sequence of microalgae under study which were used for phylogenetic analysis are shown.(DOCX)Click here for additional data file.


Future multicentric, longitudinal, prospective studies with long-term follow-up and neuropathological confirmation would be useful in establishing appropriate biomarkers for PD.The blood-brain barrier supplies brain tissues with nutrients and filters certain compounds from the brain back to the bloodstream. In several neurodegenerative diseases, including Parkinson's disease (PD), there are disruptions of the blood-brain barrier. Cerebrospinal fluid (CSF) has been widely investigated in PD and in other parkinsonian syndromes with the aim of establishing useful biomarkers for an accurate differential diagnosis among these syndromes. This review article summarizes the studies reported on CSF levels of many potential biomarkers of PD. The most consistent findings are: (a) the possible role of CSF urate on the progression of the disease; (b) the possible relations of CSF total  The diagnosis of Parkinson's disease (PD) in live patients is fundamentally clinical, and is based on the presence of its cardinal signs , and the absence of atypical data for idiopathic PD. The final confirmation of the diagnosis is made by post-mortem neuropathological analysis. To date, there are no definitive biomarkers to make an accurate differential diagnosis with other parkinsonian syndromes.Because the cerebrospinal fluid (CSF) is in close contact with the extracellular space of the brain, it is believed that many of the biochemical modifications in the brain should be reflected in the CSF. Therefore, CSF has been widely investigated in PD and in other parkinsonian syndromes with the aim of acquiring knowledge on the pathogenesis of this disease. This article summarizes the data on analyses performed in the CSF of patients diagnosed with PD compared with controls, with regard to: (1) concentrations of neurotransmitters (mainly monoamines and their metabolites), neuromodulators, and related substances as possible biological markers of the disease itself or its complications; (2) concentrations of endogenous neurotoxins; (3) status of oxidative stress markers or substances which could be related with the induction of oxidative stress or with “neuroprotection” against it; (4) status of inflammation and immunological markers, neurotrophic and growth factors, and (5) concentrations of proteins related with the pathogenesis of PD or other compounds.The aim of this review is to provide an extensive descriptive overview of studies published on this issue .Parkinson's disease” was crossed with “cerebrospinal fluid” and “blood brain barrier,” and the related references were selected. Table References for this review were identified by searching in PubMed from 1966 until June 20, 2014. The term “Because the main neurochemical finding in PD is the depletion of dopamine (DA) in the nigroestriatal system  . It is to be expected that low CSF HVA levels should be a reflection of DA depletion in the nigroestriatal system. However, CSF DA metabolite levels are not useful to distinguish between different parkinsonian syndromes and could be normal in early stages of the disease. To our knowledge, no studies have been published regarding the correlation of CSF DA metabolite levels and brain DA levels, although the observation of a correlation between CSF HVA levels and striatal uptake of DA markers in PET imaging , the main metabolite of 5-HT, in PD patients , an enzyme involved in NE synthesis, has been found decreased in PD patients when compared with controls , the main enzyme involved in Ach degradation, has been reported to be similar in PD patients and controls  have been found to be similar in PD patients and controls  amino acids are even more controversial. CSF levels of neutral and basic amino acids have been reported to be both increased . The most important are cyclic adenosine 3′5′ monophosphate (cAMP) and cyclic guanosine 3′5′ monophosphate (cGMP). Belmaker et al.  reportedPARK8 mutation , has also been found decreased , a neurotoxin that induces a parkinsonism resembling PD.Moser et al. . Zinc has antioxidant activity and is a constituent of Cu+2/Zn+2-SOD  1-β levels were found to be normal in one study  and factor H (FH) levels were reported to be normal in one study  levels have been found to be similar in PD patients with major depression to those in patients with major depression without PD in one study  brains. CSF tau protein levels are increased in AD patients, and so are a useful marker for this disease. The high risk of PD patients of developing cognitive impairment or dementia patients makes measurement of CSF tau reasonable as a possible marker of this disease.Because tau and phosphorylated tau (phosphotau) in PD patients to controls  and PSP have shown higher CSF total and phospotau levels   gene are related with early-onset monogenic familial PD and are associated with increased risk for sporadic PD  is a 140 amino acid-long presynaptic protein, which is the major component of the Lewy bodies , and has been implicated in the pathogenesis of PD and in synucleinopathies such as MSA and DLBD. Mutations of the α-Aerts et al.  found siLRRK2 gene. CSF neurosin (a protease that degrades α-synuclein) levels have been found to be decreased  are a group of different lengths peptides resulting from the enzymatic cleavage of the amyloid precursor protein (APP). The most common is the 42 amino-acid long Aβ42. These peptides have a differential trend toward aggregation  to form amyloid plaques, one of the pathological hallmarks of AD and DLBD. The increased risk for developing cognitive impairment and dementia of PD patients in comparison with the general population makes it reasonable to link AD markers such as Aβ42 to PDD. Several studies have shown similar , members of the cytoskeleton proteins expressed by neurons, have been detected in neurodegenerative diseases including AD, MSA, DLBD, and PD. CSF levels of neurofilament light (NFL) proteins have been found normal in PD patients  levels have been found increased when compared with controls and decreased when compared with AD patients in one study . The results on DJ-1 are, therefore, inconsistent and should not be considered as a marker of PD.Defects in the gene encoding DJ-1 protein cause an autosomal recessive early-onset PD, PARK7  cause familial PD, PARK5. A recent study found decreased CSF UCH-L1 levels in PD, MSA, and PSP compared with controls  found decreased  , remains to be elucidated.CSF Aβ1-42 levels could be considered as a useful marker of the presence of further cognitive decline in PD patients.CSF NFL protein levels should be useful for the differential diagnosis of PSP, MSA, CBD, and PDD from PD, but not to discriminate between PD and healthy controls.While possible biomarkers for PD in classical studies have been hypothesis-driven, attempts to develop effective procedures for the differential diagnosis of PD in its early stages have led to the performance of CSF multianalyte methods including systematic measurements of patterns of variation in proteins (proteomics) or small molecules (metabolomics). These methods have led to the identification of possible unexpected biomarkers of diseases involved in neurodegenerative processes. However, the results of these types of studies, which are briefly described below, are not clearly established and await replication.4 when compared with controls, while Pigment epithelium-derived factor (PEDF or serpin F1) was not significantly increased, and complement C3 and haptoglobin were similar in PD patients and controls.Guo et al. , in a prZhang et al.  performeMaarouf et al.  analyzedTrupp et al.  reportedIdeally, future studies should fulfill the following conditions: (a) a multicenter and prospective design; (b) inclusion of patients diagnosed with PD and other types of parkinsonism according to standardized criteria; (c) measurement of multiple potential biological markers in the CSF; (d) a very long-term follow-up period (till death as end-point), with assessment of both clinical features and serial determinations of the biological markers; and (e) final neuropathological confirmation by examination of the brains of patients at death (this is lacking in most of the studies published).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Magnetic resonance (MR) techniques used to detect lesions of the ligament complex for articulation of the ankle lack the desired accuracy for the study of the calcaneofibular ligament (CFL). The lack of sensitivity of the conventional techniques is due to variations in the dimensions of the CFL. The best results are obtained when the image plane is oriented parallel to the ligament. This study aims to develop a model that addresses the width, length and angle parameters of the CFL and the orientation of the MR image plane, and thus determine a technique in the oblique transversal plane with the foot in anatomical flexion, that is adequate for the majority of patients.To determine this orientation and adapt it to the majority of people, images of the articulation of the ankle in the 3D isotropic, volumetric, sagittal plane of 100 volunteers were taken using the MR technique. None of the volunteers had a clinical history of ligament lesions, serious pathologies, or surgeries. A measurement of the length, width, and angle of the CFL relative to the sole of the foot was performed using the MR tools. A virtual model was developed that simulated the visualization of the CFL in the oblique transversal image plane from 35° to 45° using the CFL dimensions of 100 volunteers. The comparison of the simulations with the reconstructed images validated the model and permitted the calculation of the agreement and sensitivity of each technique in the detection of the complete CFL.Using the simulator, it was possible to obtain the limit angle for complete CFL visualization as a function of its dimensions for any angle of the oblique transversal image plane of the MR.The results suggest that a single image acquisition technique in the oblique transversal plane at 38° with the foot in anatomical flexion would serve the majority of patients. The ligament complex of the ankle is composed of anatomical structures of various ligaments that are difficult to analyze. Among these structures, the calcaneofibular ligament (CFL) is frequently injured in young adults, especially individuals involved in sports such as basketball and football. This ligament is also the most vulnerable during inversion of the ankle. AccordiMagnetic Resonance (MR), a non-invasive and well accepted imaging technique, has beeThe lack of sensitivity of these techniques can be explained by variations in the dimensions of the CFL. In fact, Mayerhofer et al. proved tThus, in this investigation, to consider the influence of the width, length, and angle of the CFL, a model that addresses these parameters and the orientation of the MR image plane was developed. A technique was thereby determined, using the oblique transversal plane with the foot in an anatomical flexion that evidenced the whole CFL for the majority of healthy subjects.Male and female adult volunteers with ages between 21 and 50 years were selected by the doctors of the Department of Radiology of the Lumen Diagnostics Centre.The inclusion criteria were volunteers without a history of ankle sprains and without CFL lesions, without fractures, congenital diseases, or orthopedic surgeries involving the ankle and foot. Cases where fracture of the CFL was detected in the volumetric acquisition of the images were excluded from the sample. The acquisition of volumetric isotropic images of the volunteers in the sagittal plane was performed. The images of these volunteers were reported and sent to the researchers (CAE 03556912.0.0000.5497).The volumetric isotropic MR images of the 100 volunteers were first used to determine the dimensions of the CFL using the tools of the supervisory system of the Achieva Magnetic Resonance device. The measurements were performed 3 times by two technicians specializing in the musculoskeletal system.Next, a computational model was developed that represents the CFL relative to the image plane and determines whether there is complete visualization of this ligament. The model can consider all of the dimensions of the ligament with the foot in the anatomical position and simulates the visualization of any orientation of the oblique transversal image plane.To validate the computerized model, the CFL and its visualization were initially simulated considering the oblique transversal image plane at 35°. This technique was chosen because 35° is the CFL angle with the highest prevalence in the studied population. The CFL morphometric data from the volunteers were used for the simulation.As follow, the images were acquired in oblique plane at 35° and the isotropies images from the 100 volunteers were reconstructed in the same image plane. The accuracy of image detection was obtained directly in the oblique plane at 35° and compared with the reconstructed images accuracy. This comparison was also accomplished with the simulated images using the C contingency test.After validating the model, simulations were performed considering the oblique image plane at 36°, 37°, 38°, 39°, 40°, 41°, 42°, and 45°. The accuracy of detection was calculated to determine the best technique.To test the method, reconstructions of the 100 volumetric isotropic images were performed in the oblique transversal plane at 38°, which was considered to be the most efficient by the simulator, and at the 45° angle used by many clinics. These images, without technical information, were evaluated at work stations by 2 Radiology Technicians specialized in Magnetic Resonance of the musculoskeletal system. The examiners analyzed the images and, in consensus, evaluated which images visualized the entire length of the CFL.An analysis of agreement of the accuracy obtained across the various techniques was performed, both of the reconstructed images and of the simulations using the C contingency test. The reports from the doctors of the Lumen Diagnostic Centre were considered to be the gold standard.3; matrix 300 × 250; number of excitations 2; and acquisition time 3 minutes and 10 seconds)  weighted turbo spin echo (PDW-TSE) sequence without fat saturation in the sagittal plane The volunteers were positioned in the supine position with the arms extended along the body, and the sequences were performed with free breathing. During the exams, the position of the foot relative to the ankle was maintained at an angle of 90°, using immobilizers and special foams to maintain an anatomical position.The 3D sagittal volumetric images of the 100 volunteers permitted multiplanar reconstructions. For these reconstructions, the multiplanar reconstruction (MPR) commands were used, followed by the orientation of the oblique transversal plane and then the line setting function to select an angle of 35°, 38° and 45° Figure .Figure 2Before to start this research with the specific, this research project was approved by the Ethics Committee advice in Research involving humans at the University of Mogi das Cruzes .All participants were informed about the aims, and methodology of the study, as well as about the privacy of research subjects and confidentiality of their personal information, and institutional affiliations of the researcher.Images were used in the volumetric sagittal plane to measure the average angle, length, and width of the CFL of each volunteer.To measure the angle, using the “Line Setting” tool with angle option, a straight line was traced in each image in the sagittal plane, parallel to the horizontal flat surface, and another line was traced parallel to the plane of the ligament investigated Figure . After tThe programming and graphic development module of MatLab® was used to simulate the limits of complete visualization of the calcaneofibular ligament (CFL) as a function of the angle of the image plane.The variable dimensions  are input into the model by the user. The application considers the CFL as a rectangle represented by continuous red lines. Initially, the central axis of the rectangle is aligned with the image plane, represented by a dotted blue line Figure . The recF = 50 , with YF depending on the angular coefficient of the line  until it achieves the biometric position provided by the user. The matrix of rotation M is given by equation The rectangle is rotated around the central point , using equation The “Processing” menu allows the user to choose the direction (clockwise or counter clockwise) of rotation of the CFL as a function of the image plane. The application calculates the new coordinates as a function of the ordinate axis  of the representation of the CFL can be found. The expressions used in this phase are equations From YAs a result, the simulator shows the representation of the CFL (width and length), the angle of the image plane (α), the angle of the CFL (γ), the maximum angular variation as a function of the image plane (β), and whether complete visualization of this ligament occurred, as shown in Figure Another characteristic of the developed tool is the possibility of exporting the input and output data to a MS Excel® spreadsheet. This application also permits the printing of the graphical controls in conjunction with the data panels.The averages of the measurements of the width (W), length (L), and angle of the CFL (γ) were calculated, thus the prevalence of these values Figures  and11.FThe average angle of the CFL from the sample varied from 29° to 50°, with the highest prevalence being at 35° and with 80% of the sample between 35° and 41°. The width of the CFL varied from 1 to 10 mm with the highest prevalence between 4 and 7 mm. The length varied from 18 to 30 mm, and the most frequent measurements were 20 mm (17 cases) and 24 mm (25 cases).The reconstructed images and the simulations, both with the technique at 35°, where the complete CFL was not visualized, are in Table The accuracy of the images reconstructed at 35° was 96%, and the simulation accuracy at 35° was 95%. The 1% difference between the accuracy of the simulation and the accuracy of the reconstructed images shows that the model is valid for predicting the efficiency of the technique in the detection of the complete CFL.The position of the CFL that was not visualized in the simulation using the oblique transversal technique at 35°, shown in Figures Using the simulator, it was possible to obtain the maximum angular variation that allows the complete CFL visualization as a function of its dimensions for any angle of the oblique transversal image plane of the MR. Table Figure Table The C contingency test was applied to relate 2 variables: the GS with the multiplanar reconstruction (MPR) and later the GS with the simulated data (Sim) for the 35°, 38°, and 45° angles. Both, considering a degree of freedom of 99 and p = 1, showing that the technique at 38° provides the closest result to the report from the doctors of the lumen clinic Table .Table 6Additionally, the C contingency test was applied to relate 3 variables: the GS with the MPR and the Sim images for the 35°, 38°, and 45° angles, considering one degree of freedom = 198 and p = 1. Table The average values obtained for the length, width, and angle of the CFL in this study are similar to the values obtained in the studies with cadaver samples performed by Golanó et al., Taser eA study evaluateWhen a ligament is not completely visualized in the image, a lesion is diagnosed. However, the reliability of this discovery does not depend only on the lesion but also on the technical aspects of the magnetic resonance exam and the anatomical variations of the ligament. The use of an inappropriate image plane can provide images that lead to an incorrect diagnosis. Other pVarious researchers have sought to improve the complete detection of the CFL. Initially, the studies investigated the best image plane and foot position to avoid false positive. Beltran et al. used theA study performed by Boonthathip et al. showed tThis study did not address the cases of false negatives in patients with suspicions of lesion of the CFL or cases of CFL partially torn.This MR technique is proposed to improve the diagnosis for the majority of patients with suspicions of CFL ankle lesions, minimizing the possibility of false positives. This technique determine the most appropriate image plane. The choice of MR as a tool to measure CFL is due to the lack of protocols for CFL ankle lesions. In addition, this approach provided better sensitivity to investigate correlates impairment such as degenerative, traumatic, and ligament diseases of the ankle.In future studies we will analyze the oblique transversal technique of the ankle at the angle of 38° for patients with CFL lesions.


GFAP, the gene encoding glial fibrillary acidic protein, the major intermediate filament protein of astrocytes in the CNS. A key step in pathogenesis appears to be the accumulation of GFAP within astrocytes to excessive levels. Studies using mouse models indicate that the severity of the phenotype correlates with the level of expression, and suppression of GFAP expression and/or accumulation is one strategy that is being pursued as a potential treatment. With the goal of identifying biomarkers that indirectly reflect the levels of GFAP in brain parenchyma, we have assayed GFAP levels in two body fluids in humans that are readily accessible as biopsy sites: CSF and blood. We find that GFAP levels are consistently elevated in the CSF of patients with Alexander disease, but only occasionally and modestly elevated in blood. These results provide the foundation for future studies that will explore whether GFAP levels can serve as a convenient means to monitor the progression of disease and the response to treatment.Alexander disease is a rare, progressive, and generally fatal neurological disorder that results from dominant mutations affecting the coding region of  Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is predominantly expressed in astrocytes of the CNS. Although typically confined to the cytoplasm, GFAP is released at low levels into the extracellular space, and can appear at measurable levels in CSF and blood. Here we show that the fluid levels of GFAP increase markedly in individuals with Alexander disease, a genetic disorder that results from mutations in GFAP itself, particularly in the CSF. CSF analysis may therefore offer a relatively noninvasive means for indirectly monitoring the levels of GFAP in brain and assessing the efficacy of future experimental treatments that are designed to reduce these levels.Alexander disease (AxD) is a progressive and generally fatal neurogenetic disorder, with ages of onset ranging from fetal through late adulthood, resulting from heterozygous dominant mutations in the astrocyte intermediate filament protein glial fibrillary acidic protein GFAP; . The halHow one might assess the efficacy of any potential treatment is a major question. Although AxD is genetically homogeneous, the clinical presentations are quite varied, with age of onset ranging from fetal through late adulthood. The most widely used classification system is based simply on age of onset, and divides patients into early, juvenile, and adult categories . More reOne potential biomarker is GFAP itself. GFAP, despite being a cytoplasmic protein, is normally present at low levels in CSF and blood, and the levels in these fluids are increased in the context of a wide variety of injuries and diseases of the CNS . FollowiGFAP levels could conceivably serve as a biomarker in AxD in two distinct ways. First is the known association of GFAP release with injury or damage, as cited above; and, indeed, the lesions in AxD range from very subtle, without evident leukodystrophy, up to large cavitating lesions with loss of nearly all tissue elements . Second GFAP. Informed consent for studies of CSF was obtained following protocols approved by the institutional review boards (IRBs) at the University of Wisconsin-Madison, the Children’s National Medical Center, and the Mayo Clinic. Only leftover samples from a previous clinical use were permitted for study. Informed consent for studies of blood was obtained following protocols approved by IRBs at the University of Wisconsin-Madison, Children’s National Medical Center, Massachusetts General Hospital, Washington University in St. Louis, and the Mayo Clinic. Again, consents were obtained either through in-person interview or by telephone, with written confirmation. For samples collected in The Netherlands, Italy, and Germany, the principles outlined in the Declaration of Helsinki were followed.The sole inclusion criterion for participation in this study was genetic confirmation of the diagnosis by sequencing of Table 1. Controls for blood were obtained from apparently healthy adults of both sexes , who were asked to exclude themselves if they had specific conditions  or were taking specific medications within the past 3 months . The exclusion criteria for plasma control samples were based on a literature review of conditions known or hypothesized to influence GFAP levels in CSF and blood (Controls for CSF studies consisted of 24 deidentified samples collected by lumbar puncture (LP) for various purposes but considered within the range of normal for protein, glucose, and cell counts. The CSF controls were exempted from the requirement for consent. Additional information about the CSF control group  is given in nd blood  and a stnd blood .2-EDTA as an anticoagulant to allow the preparation of plasma. The samples were centrifuged within 60 min of collection at 2500 relative centrifugal force for 15 min at room temperature, and the supernatant was immediately placed in a polypropylene tube and stored either on dry ice for shipping or in a −20°C freezer until shipping could be arranged. Upon arrival at the central laboratory, the samples were then thawed, divided into aliquots, and stored at −80°C until further analysis. Three blood samples were collected as serum rather than plasma, and so were considered nonstandard. Statistical analyses were repeated with or without these samples, and the results were the same.Fresh samples of venous blood were collected into lavender-topped tubes that contained KTgGFAP mice that overexpress wild-type human GFAP, was 11%. CSF and plasma samples from Gfap-null mice gave readings that were below the BLD in this assay (data not shown), thus validating its specificity. In addition, plasma samples from Gfap-null mice were spiked with known concentrations of purified bovine GFAP to verify that the 1:1 dilutions of plasma used here did not interfere with the sensitivity of the assay (data not shown). All animal procedures were approved by the Animal Care and Use Committee for the Graduate School at the University of Wisconsin-Madison.GFAP levels in CSF and blood were quantitated using a sandwich ELISA, as previously described . The capp values were two sided, and p < 0.5 was used to determine statistical significance. Statistical analyses were conducted using SAS version 9.3 software (SAS Institute).GFAP levels obtained from CSF and blood samples were summarized in terms of medians and ranges. To determine a reference range for GFAP levels in blood samples from healthy control samples, we took into account the BLD of the assay (46 ng/L). Samples yielding values lower than this limit were treated as censored values at 46 ng/L in the analysis. Due to the presence of censoring, semiparametric quantile regression was conducted . OverallGFAP. Those patients for whom leftover clinical CSF samples were available included five females and seven males, ranging in age from 3.7 to 46 years. Those patients from whom blood samples were collected included 26 females and 22 males, ranging in age from <1 to 65 years of age. Both blood and CSF samples were available for 10 of these patients. Information for each patient regarding gender, specific mutation, age of first symptom, age at collection of samples, and age at death (if relevant) is provided in Table 2.Samples were collected from AxD patients with confirmed mutations in p < 0.001a). Considered as individuals, GFAP levels in 9 of the 10 samples from AxD patients were elevated compared with those from the control population. Because of the paucity of samples, this experiment was replicated once, with similar results. Two additional samples were subsequently received (from patients 35 and 50) and analyzed separately, and yielded values of 2721 and 1749 ng/L, respectively. Individual GFAP values for each AxD patient in relation to age, gender, genotype, and duration of illness are shown in Table 3.We established a reference range for CSF controls in our assay using a set of 24 samples, and found the mean GFAP level to be 249 ng/L . The highest values in the control group came from one child with lymphoma (site unspecified) and one child with an “acute life-threatening event” (type unspecified). CSF samples were initially available for 10 AxD patients, and in a single assay all of the AxD patient samples were run alongside 12 samples from the control subjects  and juvenile (p = 0.025c) onsets compared with those in the control subjects . Considered as a group, GFAP levels in the AxD patients were significantly elevated in the AxD case patients with infantile , plasma values ranged from 64 to 1314 ng/L, only patient 36 being higher than the 95% CI established for control subjects.Subsequent to the experiments described above on the large set of AxD patients, 12 additional samples were received, which were analyzed on three separate occasions . In these latter patients (Table 3). For each of these patients, CSF values were significantly higher than the blood values. Although the number of patients was too low for valid statistical analysis, it appears that the blood levels may only exceed the 95% CIs of control subjects when the CSF values are above a certain threshold , and two samples were available for the one genotype that is generally considered severe (R239H); but the values for GFAP were all highly variable in these groups. Hence, there does not appear to be any direct connection between genotype and the levels of GFAP detected in these assays. In addition, when gender was considered as a variable, there was no significant difference between males and females.We analyzed the individual blood and CSF values of GFAP in relation to several aspects of the clinical and genetic histories. Additional clinical information about each AxD patient is given in p = 0.001d; type II patients vs control subjects, p = 0.203e). Additional information about the statistical tests used throughout and the confidence intervals for each set of comparisons is given in Table 5.The number of CSF samples was so small that we could not establish any particular connection to age of onset of illness, age at sample collection, or duration of illness . For blood, the GFAP values were significantly correlated with the age of illness onset and age at sample collection (which is likely linked to the age of onset), but not duration of illness. Four blood samples were from patients sampled within months of their deaths , but only one of these had a value that was above the 95% CI. If the data are analyzed according to the clinical classification scheme of In an earlier study of three AxD patients, Why GFAP levels in CSF  rise in AxD patients is not clear. Previous work in mouse models suggests a linear relation between the levels in parenchyma (presumably largely intracellular) and that in the fluids , and it Individuals with AxD experience several associated conditions that plausibly could influence GFAP expression and levels in biofluids. Seizures are seen in most infantile or type I AxD patients . NeverthWe recognize a number of limitations to our study. First is the problem of age matching in our samples, particularly with respect to blood. Our IRB protocol limited the collection of blood from healthy control subjects to those >18 years of age, whereas 27 of our 48 AxD patient samples were below this age. However, previous studies of GFAP in CSF and blood found no relation to age or sex , though Ultimately, a panel of biomarkers may prove more informative than focusing on just one. Several candidates for consideration in such a panel could include markers of neuronal or oligodendroglial injury . A recent proteomic study of CSF in one mouse model of AxD  suggeste


Background. In 2010, mass vaccination with a then-new meningococcal A polysaccharide–tetanus toxoid protein conjugate vaccine  was undertaken in 1- to 29-year-olds in Bamako, Mali. Whether vaccination with PsA-TT effectively boosts tetanus immunity in a population with heterogeneous baseline tetanus immunity is not known. We assessed the impact of PsA-TT on tetanus toxoid (TT) immunity by quantifying age- and sex-specific immunity prior to and 2 years after introduction.Methods. Using a household-based, age-stratified design, we randomly selected participants for a prevaccination serological survey in 2010 and a postvaccination survey in 2012. TT immunoglobulin G (IgG) antibodies were quantified and geometric mean concentrations (GMCs) pre- and postvaccination among all age groups targeted for vaccination were compared. The probability of TT IgG levels ≥0.1 IU/mL (indicating short-term protection) and ≥1.0 IU/mL (indicating long-term protection) by age and sex was determined using logistic regression models.Results. Analysis of 793 prevaccination and 800 postvaccination sera indicated that while GMCs were low pre–PsA-TT, significantly higher GMCs in all age–sex strata were observed 2 years after PsA-TT introduction. The percentage with short-term immunity increased from 57.1% to 88.4%  and with long-term immunity increased from 20.0% to 58.5%  pre- and postvaccination.Conclusions. Significantly higher TT immunity was observed among vaccine-targeted age groups up to 2 years after Mali's PsA-TT mass vaccination campaign. Our results, combined with evidence from clinical trials, strongly suggest that conjugate vaccines containing TT such as PsA-TT should be considered bivalent vaccines because of their ability to boost tetanus immunity. Neisseria meningitidis, Haemophilus influenzae type b (Hib), Streptococcus pneumoniae, and other pathogens [In recent years, conjugate vaccines that link the polysaccharides on the outer surface of bacterial pathogens to protein carriers such as tetanus toxoid (TT) have been developed to protect against athogens . Conjugaathogens . Conjugaathogens . In addiathogens . The 201athogens . In addiathogens , a levelH. influenzae, and hepatitis B vaccine at 6, 10, and 14 weeks, with additional TT vaccination during pregnancy [Currently, the World Health Organization (WHO) recommends that 3 doses of a TT-containing vaccine be administered in the first year of life as part of routine immunization programs with 2 booster doses of TT-containing vaccines given during childhood followed by vaccination of pregnant women to prevent neonatal tetanus . Meetingregnancy .Tetanus remains an ongoing challenge in some low- and middle-income countries , and an We conducted 2 cross-sectional serologic surveys in Mali's capital, Bamako, and villages nearby immediately prior to and 2 years after a PsA-TT mass vaccination campaign. The PsA-TT campaign was launched in December 2010 and targeted all individuals 1–29 years of age during a 4-week period in the south of the country, including the areas we surveyed. In January 2011, an evaluation of the PsA-TT campaign vaccination coverage level was undertaken in several districts of Bamako. Overall, 93%  of eligible participants were vaccinated with PsA-TT, with somewhat higher coverage both among children aged 1–4 years  and adolescents aged 5–14 years  compared with adults aged 15–29 years  .The prevaccination seroprevalence survey was conducted as part of the African Meningococcal Carriage Consortium (MenAfriCar) research program, which aimed to assess the impact of the 2010 introduction of PsA-TT on meningococcal carriage in Africa. MenAfriCar's methodologies have been detailed previously . In brieParticipants were eligible if they were aged ≥6 months, had no acute or chronic illness, and if they or their parent or guardian provided consent. Participants responded to a questionnaire and provided up to 5 mL of blood.The postvaccination seroprevalence survey was conducted as part of the US National Institutes of Health–funded PsA-TT (MenAfriVac) Antibody Persistence (MAP) study, which was designed to assess the impact of the 2010 introduction of PsA-TT on population-level immunity and the duration of protection against meningococcal disease provided by the vaccine . ParticiIn both surveys, blood samples were collected and allowed to clot before being stored in a cool box (2°C–8°C). Samples were transported to the laboratory at CVD-Mali in Bamako within 4–6 hours where the serum was extracted, aliquoted, and stored at −20°C for the first survey and −80°C for the second survey prior to shipment on dry ice to the Vaccine Evaluation Unit, Public Health England  for TT IgG antibody level assessment.6 microspheres/mL; Luminex, Texas) using a 2-step carbodiimide reaction [N-hydroxysulfosuccinimide . Activated microspheres were then coupled to TT  and briefly vortex-mixed for 10 seconds, prior to incubation with shaking at 50 rpm at room temperature for 1.5 hours in the dark. Following this, the microspheres were washed 3 times with PBS (pH 7.3) by centrifugation (13 000 rpm for 2 minutes). On the final wash, microspheres were resuspended in 1 mL PBS and transferred to protein LoBind Eppendorf tubes  for storage in the dark at 4°C–8°C.Tetanus toxoid  was coupled to carboxylated microspheres  was prepared in serum diluent . Each dilution of standard, serum samples and quality control serum (25 µL of each) were diluted 1:100 in serum diluent and run in duplicate on the plate. A concentration of 5000 microspheres/region/well (25 µL) and test and control sera were incubated in a 96-well MV Multiscreen filter plate  for 20 minutes at RT with shaking at 500 rpm in the dark. Plates were washed twice by vacuum filtration and then 100 µL of a 1:200 dilution of R-phycoerythrin conjugated antihuman IgG  in PBS was added to each well and plates were incubated for 20 minutes with shaking . Following another wash, the beads were resuspended in a final volume of 125 µL PBS-T and shaken for 5 minutes prior to reading on a Bio-Plex system . Data were acquired using Bio-Plex Manager version 4 (Bio-Rad). Data for unknown test sera were generated from a 5-parameter logistic standard curve for the relevant reference serum and converted to international units (IU/mL).In the prevaccination survey, field and laboratory data were recorded on specially designed paper forms and converted to an electronic format automatically using the TeleForm system . In the postvaccination survey, field and laboratory data were entered directly into specially designed Microsoft Access (Microsoft Office) databases on small laptops. TT IgG data were reported in Microsoft Excel (Microsoft Office). All data were merged, managed, and analyzed using Stata SE 13.1 .t tests on the log-transformed variable. Point estimates and CIs were back-transformed for presentation.Age was defined as age at enrollment, which occurred between September and December 2010 for the prevaccination survey and age on 1 December 2010 for the postvaccination survey to assess cohort effects because we are interested in whether age-specific immunity, which would be expected to wane over time, was boosted by PsA-TT in cohorts of individuals targeted for PsA-TT vaccination. Age- and sex-specific pre- and postvaccination geometric mean concentrations (GMCs) and 95% CIs for TT IgG were calculated. To correct for the skewness of the distribution of TT IgG concentrations, the variable was log (base 10) transformed and differences were assessed using Data were analyzed to determine the proportion of participants before and after PsA-TT introduction with TT IgG response of ≥0.1 IU/mL and ≥1.0 IU/mL. The former is a marker of short-term immunity , 21, andThe prevaccination study was approved by the ethics committees of the University of Bamako and the London School of Hygiene and Tropical Medicine. The postvaccination study was approved by the ethics committee of the University of Bamako and the institutional review boards at Princeton University and the University of Minnesota.P = .30, 2 sample t test); these data are combined in the following analyses. All but 4 (0.5%) of the postvaccinated survey participants self-reported having received PsA-TT. Because we were interested in population-level changes among targeted age groups following the mass vaccination campaign, data from all participants, regardless of self-reported vaccination status, are included in the analyses.In the prevaccination survey, 800 participants were enrolled between September and December 2010 prior to the mid-December 2010 PsA-TT mass vaccination campaign, which lasted approximately 4 weeks; sera from 793 of these participants was available for analysis. In the postvaccination survey, 800 participants were enrolled between December 2012 and January 2013, 2 years after the PsA-TT mass vaccination campaign, and sera from all participants were available for analysis. The proportion of females in the pre- and postvaccination surveys was similar (54% vs 57%), as was the mean household size , the distribution of the number of rooms per household , and the average number of residents per room (2.5 vs 2.7) (Table P < .0001), with the highest prevaccination GMCs observed among 18- to 29-year-old women (Table P = .1).The distribution of pre- and postvaccination GMCs by age and sex indicated that the minimum and maximum TT IgG concentrations observed were similar regardless of age, sex, or time of survey Figure . PrevaccP < .0001; Table The percentage of participants with TT IgG levels indicative of short-term protection (≥0.1 IU/mL) increased from 57.1% prior to the vaccination campaign to 88.4% postvaccination, a 31.3-point increase . All age groups experienced highly statistically significant changes in the percentage of participants who were putatively protected long-term 2 years after PsA-TT introduction , accounting for household clustering. After accounting for age group, sex, and their interaction , the adjusted odds of demonstrating short-term protection against TT 2 years after PsA-TT introduction vs prior was 7.3 . The results for long-term protective concentrations ≥1.0 IU/mL were similar. The unadjusted odds of having a TT IgG level ≥1.0 IU/mL 2 years post–PsA-TT introduction vs prior was 5.6 , accounting for household clustering. After also accounting for age group, sex, and their interaction , the odds of demonstrating long-term protection against tetanus 2 years after PsA-TT introduction vs prior was 9.0 .The 1345 participants aged 1–29 years at the time of PsA-TT introduction were included in a multivariate logistic regression model. These participants were drawn from 625 households. The unadjusted odds of having a TT IgG level ≥0.1 IU/mL 2 years post–PsA-TT introduction vs prior was 5.7  and long-term protection . The probability of short-term protection decreased with increasing age across these groups prevaccination. The probability of both short-term and long-term protection was significantly higher among women compared with men aged 18–29 years. The probability of reaching both short-term and long-term protection was statistically significantly greater in all age–sex strata after the PsA-TT mass vaccination campaign (Figure We also estimated the probability that an individual from each age–sex strata would have evidence of short-term and long-term immunity before and after PsA-TT introduction from the final models. In 2010, prior to PsA-TT introduction, both males and females <18 years of age had very similar age-specific probabilities of achieving short-term protection Figure A and lonn Figure B. The prn Figure .Figure We found significantly higher TT immunity 2 years following the PsA-TT mass vaccination campaign in Mali across all age groups targeted for vaccination. Before PsA-TT introduction, TT immunity was low across all age groups, although higher in females than males and highest in women aged 18–29 years, who are likely to have received TT vaccination during pregnancy. Two years after PsA-TT introduction, TT GMCs and the proportion with both short-term and long-term protection against tetanus increased significantly across all groups targeted for PsA-TT vaccination. After accounting for age and sex, the odds of having at least short-term protection were 7.3 times higher and the odds of long-term protection were 9 times higher after PsA-TT introduction, indicating significant boosting of tetanus immunity in the population, which persisted 2 years after the campaign ended.Our results are consistent with evidence from PsA-TT clinical trials that have demonstrated significant boosting of TT immunity among vaccine recipients 4 weeks after vaccination . CriteriA limitation of our study is that we were unable to obtain information about individual-level history of vaccination with TT-containing vaccines. Thus, we cannot be certain that the higher immunity observed after the vaccination campaign indicates an anamnestic response or whether it is indicative of a combination of anamnestic responses and prolonged response to primary immunization in some ages. A key question is whether the boosting of tetanus immunity observed is causally related to the introduction of PsA-TT or whether vaccination with other TT-containing vaccines could account for the increase in immunity observed. Examining our findings in light of routine immunization coverage data suggests that coverage with TT-containing vaccines remained relatively constant during this 2-year period and supports our claim that PsA-TT boosted tetanus immunity. WHO-UNICEF estimates that in 2010, 18% of children aged 12–23 months in Mali had received any doses of tetanus-containing vaccine and 24% had not received all 3 recommended EPI doses , which iOur evidence suggests that polysaccharide-protein conjugate vaccines containing TT such as PsA-TT boost tetanus immunity and should be considered bivalent vaccines. Introduction of these vaccines in widespread population campaigns can help increase tetanus immunity across age groups not typically targeted for TT-containing vaccines or when coverage with TT vaccines is suboptimal. Recognition of this added benefit of PsA-TT could inform future vaccination strategies in resource-limited settings where maintaining high tetanus immunity, in addition to preventing meningococcal disease, is a priority.


Influenza viruses (IVs) circulate seasonally and are a common cause of respiratory infections in pediatric and adult patients. Additionally, recurrent pandemics cause massive morbidity and mortality worldwide. Infection may result in rapid progressive viral pneumonia with fatal outcome. Since accurate treatment strategies are still missing, research refocuses attention to lung pathology and cellular crosstalk to develop new therapeutic options.Alveolar epithelial cells (AECs) play an important role in orchestrating the pulmonary antiviral host response. After IV infection they release a cascade of immune mediators, one of which is granulocyte and macrophage colony-stimulating factor (GM-CSF). GM-CSF is known to promote differentiation, activation and mobilization of myeloid cells. In the lung, GM-CSF drives immune functions of alveolar macrophages and dendritic cells (DCs) and also improves epithelial repair processes through direct interaction with AECs. During IV infection, AEC-derived GM-CSF shows a lung-protective effect that is also present after local GM-CSF application. This mini-review provides an overview on GM-CSF-modulated immune responses to IV pneumonia and its therapeutic potential in severe IV pneumonia. Respiratory viral infections are often seen in pediatric patients . They maantigenic drift, while the reassortment of viral gene segments is called antigenic shift [IVs are enveloped single-stranded negative-sensed RNA viruses, divided into three genera A, B, and C . They caic shift . The creic shift .Primary target cells for IV infection in humans are respiratory epithelial cells . The curAs soon as the infection spreads from the upper to the lower respiratory tract, alveolar epithelial cells (AECs) become primary targets for productive IV replication . Pro-infRapid and effective viral clearance from the distal lung by immune effector cells and the initiation of epithelial repair processes including expansion of local epithelial progenitor cells and resealing of the epithelial layer are crucial to recover from IV-induced lung injury , 12. TheMyeloid cells like monocytes, macrophages, dendritic cells (DCs), and their common precursor cells, summarized as mononuclear phagocytes, are crucial in driving IV clearance . CombiniThe growth factor granulocyte and macrophage colony-stimulating factor (GM-CSF) is widely recognized to promote proliferation, differentiation, and activation of monocytes, granulocytes, macrophages, and DCs in vivo and plays an important role in immunity , 17. Inv− cell population in the distal lung parenchyma that upregulates GM-CSF upon IV infection, and produces high levels of GM-CSF in the alveolar lining fluid [Pulmonary GM-CSF is mainly expressed by AEC type II and released under inflammatory conditions . Other cng fluid . New finng fluid .+ DCs were found to be key players for the GM-CSF-dependent lung protective effect by our group [+ DCs are expanded and activated and their migration and antigen (Ag) presentation to the draining mediastinal lymph nodes is mediated by GM-CSF. This is associated with a better viral clearance and Ag-specific T cell recruitment, suggesting improved resident lung DC host defense capacities during IV infection by AEC GM-CSF. Min et al. [+ T lymphocytes to activate DCs during priming in lymphoid tissue, providing a positive feedback loop in the stimulation of CD8+ T cell proliferation. Accordingly, Greter et al. [+ T cell immunity. Chen et al. [It is well established that AEC-released GM-CSF improves innate immune responses of myeloid cells, in particular alveolar macrophages. Alveolar macrophages of GM-CSF-deficient mice show abnormalities in morphology, maturation, and function, depending on transcription factor PU.1 , 26. Sevur group . After In et al.  identifir et al.  revealedn et al.  also fouDuring IV pneumonia, GM-CSF furthermore has a lung barrier-protective effect and improves survival, also after local application , 32, 36.Figure + DCs in inflamed lungs [In adult patients with acute respiratory distress syndrome (ARDS), elevated GM-CSF levels in the bronchoalveolar lavage fluid (BALF) were shown to be associated with antiapoptotic effects and with improved epithelial barrier integrity and survival , 41. In ed lungs . These fed lungs , highligCurrent treatment strategies for IV infection focus on vaccines and antiviral agents. Due to its rapid genetic modification capacity, it will be a challenge to keep up with therapeutic targets as resistance rapidly occurs against current antivirals. Immune mediators that drive pulmonary host defense function like GM-CSF propose appealing alternative treatment strategies, especially if they do not only protect against IV infection itself but also against common complications like secondary bacterial pneumonia and severe damage of alveolar epithelium.A clinical trial for low-dose intravenous GM-CSF treatment in adult patients with severe sepsis and respiratory dysfunction led to the conclusion that GM-CSF treatment was associated with improved gas exchange and might play a homeostatic role . AnotherFor patients with PAP, inhaled GM-CSF therapy displayed encouraging results , 48. WitThis review focuses on GM-CSF-mediated effects in IV pneumonia and associated ARDS. The clinical trials mentioned above investigate GM-CSF treatment not exclusively in IV pneumonia but in  pneumonia-associated ARDS in general. While in the mouse model, specific conditions can be studied in detail, clinical conditions are different. In the clinical setting, the underlying germ of infection is not always clearly defined when patients present with pneumonia/ARDS and therapy needs to be initiated. Furthermore, bacterial superinfection of primary viral pneumonia is a common complication. GM-CSF-mediated protective effects are not limited to IV infection as they result in improvement of host defense capacities and epithelial repair in general, both critical processes in ARDS patients. GM-CSF might be working similarly in other respiratory viral infections as well. It was reported to prevent RSV-exacerbated airway hyperresponsiveness by alveolar macrophage maturation , for exaLocal therapeutic application of GM-CSF increases mononuclear phagocyte-mediated innate and adaptive host defense and accelerates epithelial repair processes during severe IV pneumonia in pre-clinical models. There is evidence that it might be a powerful therapy in viral  pneumonia and associated ARDS and eventually even other forms of ARDS in children and adults. Current data suggest that local application by the inhalative route seems to be most promising.AEC, alveolar epithelial cell; Ag, antigen; ALI, acute lung injury; AM, alveolar macrophage; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage fluid; c-Met, hepatocyte growth factor receptor; DC, dendritic cell; EGFR, epithelial growth factor receptor; FcɣR, Fcɣ receptor; GI-HOPE, clinical trial on GM-CSF treatment in ARDS patients; GM-CSF, granulocyte and macrophage colony stimulating factor; HA, hemagglutinin; HGF, hepatocyte growth factor; ICU, intensive care unit; IFNγ, interferon-γ; iGRASP, clinical trial on GM-CSF treatment in children with virus-associated ARDS; IL, interleukin; IV, influenza virus; miRNA, microRNA; mRNA, messenger RNA; NA, neuraminidase; PAP, pulmonary alveolar proteinosis; PARDS, pediatric acute respiratory distress syndrome; PU.1, transcription factor PU.1; ROS, reactive oxygen species; RSV, respiratory syncytial virus; TGF α, transcriptional growth factor α