Vascular calcification is a prominent feature of atherosclerosis. The mineral composition and quantity within calcified arterial plaques remains unelucidated; therefore, the aim of this study was to analyze the mineral composition of such plaques. Calcified arterial plaques were obtained from patients with abdominal aortic aneurysms (AAAs) and carotid artery stenoses. Calcified aneurysmal plaques were obtained during the routine open repair of AAAs, while calcified carotid plaques were collected from patients who underwent carotid endarterectomy (CEA). Following the appropriate preparation of each sample, inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to analyze the calcium and phosphate levels, while flame atomic absorption spectrometry (FAAS) was used to analyze the levels of iron and zinc. The levels of these mineral components were evaluated. In the aortic and carotid plaques, the mean calcium concentration was 9.83 and 11.94 wt.%, respectively, and the mean phosphate concentration was 4.31 and 6.08 wt.%, respectively. It was not possible to analyze the absolute concentration of iron in the carotid plaques due to the concentration being below the measurement limit. The zinc concentration was variable between samples. In conclusion, the main components of aortic and carotid plaques are calcium and phosphate. The mineral concentrations of the plaques in the present study may be used as reference values for further studies on vascular calcification. More studies are required to elucidate the correlation between the mineral components and vascular calcification. Vascular calcification, the deposition of calcium phosphate in the vessel wall, is a prominent feature of atherosclerosis. Calcification may occur in the intima and media of the vessel wall with aging, diabetes or uremia .in vitro studies and molecular biological techniques, unanswered questions remain, particularly with respect to the correlation between bone formation and vascular calcification. Bone formation is an active process involving the production of an extracellular matrix conducive to mineralization. Arterial calcification was previously considered to be a degenerative, end-stage process of vascular diseases, and early studies of arterial calcification described an association with tissue necrosis . Calcified arterial plaques were obtained from patients with abdominal aortic aneurysms (AAAs) and carotid artery stenoses. Calcified aneurysmal plaques were obtained during the routine open repair of AAAs using conventional techniques. The conventional repair of the AAAs included closing the AAA sac over the graft to protect the prosthetic graft from eroding into the bowel and from exposure to intraperitoneal contamination. Prior to the closure of the AAA sac, a piece of calcified aneurysmal plaque was obtained. Calcified carotid plaques were collected from patients who underwent carotid endarterectomy (CEA). CEA was performed using conventional surgical techniques. Briefly, the carotid artery was incised and the plaques were removed from the lumen as a single specimen. A piece of specimen was obtained from the most calcified portion of the endarterectomized plaques. Patients with carotid artery stenoses or AAAs whose lesions demonstrated heavy calcification when analyzed by preoperative computed tomography (CT) scanning were selected for the study. The informed consent was obtained from either the patients or the patients’ family.All plaques were immediately rinsed with normal saline to remove the blood components, prior to digestion being conducted for each sample. This process was performed in a closed vessel with high-pressure at 1,200 psi, heated at 200°C by microwave energy. The 5 ml of 0.1 M nitric acid was used to digest 1 g of each sample.Certain mineral components of the plaques, specifically, calcium, phosphate, iron and zinc, were investigated. Inductively coupled plasma atomic emission spectrometry  was used to analyze the calcium and phosphate levels. The inductively coupled plasma was an electrically conductive gaseous mixture of argon, argon ions and electrons. Plasma was produced from a stream of argon gas, which was then energized by a high-energy, radio-frequency field. The sample was subsequently introduced as an aerosol mixed with argon into the plasma, where high temperature caused efficient desolvation, volatilization, atomization, excitation and ionization of the samples .Flame atomic absorption spectrometry  was used to analyze the iron and zinc levels. In the FAAS, a flame was used to atomize a compound in solution. The air-acetylene flame was used in atomic absorption spectrometry. FAAS has been indicated to be the simplest and easiest analytical technique for element analysis ,10.A total of 11 pieces of aortic plaque were obtained from 11 patients during the routine open repair of AAAs. Six (n=6) pieces of carotid plaque were obtained from six patients during CEA.The levels of the mineral components calcium, phosphate, iron and zinc, were analyzed in each sample. et al . Furtheret al(et al reported that low serum Zn levels have been associated with increased cardiovascular mortality (The levels of zinc and iron measured in this study were variable. Sullivan  first suet al. Endotheet al. Leone eortality .In conclusion, although the mineral components were analyzed in relatively few samples of aortic and carotid plaques in the present study, the concentration of calcium and phosphate in the aortic plaques was lower than that in the carotid plaques. The mineral concentrations of the plaques in the present study may be used as reference values for further studies on vascular calcification. An enhanced understanding of the complex mechanism regulating vascular calcification and the clinical correlation with vascular calcification may have therapeutic potential in the reduction of cardiovascular disease-associated morbidity and mortality. Further studies are required to elucidate the correlation between the mineral components and vascular calcification.


In addition, numerous varicose β-END-immunoreactive fibers are situated close to the ventricular surfaces. In the present paper we surveyed the evidence that volume transmission via the CSF can be considered as an option for messages to reach remote brain areas. Some of the points discussed in the present review are: release mechanisms of β-END, independence of peripheral versus central levels, central β-END migration over considerable distances, behavioral effects of β-END depend on location of ventricular administration, and abundance of mu and delta opioid receptors in the periventricular regions of the brain.There is increasing evidence that non-synaptic communication by volume transmission in the flowing CSF plays an important role in neural mechanisms, especially for extending the duration of behavioral effects. In the present review, we explore the mechanisms involved in the behavioral and physiological effects of β-endorphin (β-END), especially those involving the cerebrospinal fluid (CSF), as a message transport system to reach distant brain areas. The major source of β-END are the pro-opio-melano-cortin (POMC) neurons, located in the arcuate hypothalamic nucleus (ARH), bordering the 3 Beta- in 1977 , in comb in 1977 . The molThe behavioral effects of β-END were soon recognized and vary from prolonged muscular rigidity  to generInterestingly, β-END is produced in the pituitary for release into the peripheral systemic circulation, and by hypothalamic POMC neurons for release inside the central nervous system (CNS). Since it has been observed that peripheral administration of β-END does not necessarily induce the same effects as intracerebroventricular (icv) administration, this suggests the existence of two functionally different β-END systems, one for the central effects and one for the peripheral effects. The present review explores the existence of a special central and brain-directed β-END system and the possibility that the cerebrospinal fluid (CSF) plays a special role in the propagation of these brain-directed β-END messages., from brain to blood makes it possible that CSF peptides like β-END can gain access to peripheral systems at the brain capillaries [The evidence discussed in the present review has led to the conclusion that CSF-levels of β-END are not a reflection of the peripheral levels, but are controlled and regulated by separate inputs and by specific mechanisms that are functionally separate from the pituitary release mechanisms involved in the plasma levels. This conclusion does not necessarily indicate that central CSF and peripheral plasma levels of β-END are totally unrelated. Peripheral β-END may be able to access the CNS from the periphery via the circumventricular organs (CVO’s) lacking in a blood–brain barrier (BBB), and the choroid plexus ,39. In aillaries -46. ThesThe flowing CSF serves as a medium to transport neuropeptides or other substances to distant receptive brain areas. This type of transport has been described as long-distance volume transmission (VT) ,39,47-56On the basis of the evidence presented here, we propose the following: There are two functionally different systems for the release of β-END, one for the peripheral effects via the systemic circulation and one directed to the central nervous system. The latter system uses synaptic communication and additional volume transport mechanisms provided by the flowing CSF. It is on the second part of this hypothesis that our present paper is focused.in situ hybridization studies have confirmed the existence of a main population of β-END-immunoreactive (IR) neurons in the mediobasal hypothalamic region, most of them located in the arcuate hypothalamic nucleus (ARH) [Xenopus, β-END neurons have been described as contacting the CSF directly [rd ventricle or of the subarachnoid space [Numerous immunocytochemical as well as us (ARH) -73  ,71. At tus (ARH) ,68,73-76directly ,78. Thesid space , borderiid space . Such coet al[The process of cleaving POMC molecules, followed by additional processing during axonal transport (see below), controlled release and extrinsically controlled levels of receptors (see below), provides the POMC system with a high degree of plasticity. We mention a few aspects: Firstly, while ACTH, β-END and α-MSH are fully co-localized in the ARH POMC neurons ,70,72, tet al-86 have et al,86-88. Set al, these mThe extrinsic projections of the hypothalamic POMC neurons, also known as opiocortin projections, have been mapped extensively ,76,90-96. There is a prevalence of POMC projections in brain areas such as the amygdala , hypothalamus, periventricular thalamic nuclei and the periaqueductal gray (PAG). In the hypothalamus the densest innervations are provided to the parvocvellular, paraventricular, preoptic, periventricular and arcuate nuclei, which are all involved in anterior pituitary functions, via the median eminence [There are a number of aspects of the POMC circuitry that deserve special attentioneminence . The releminence ,97-101 aeminence ,102, sugeminence ,93 receiStudies combining retrograde tracers with POMC staining techniques have shown that subpopulations of the POMC neurons project to different destinations . The β-EIn 1983 an additional group of opiocortin neurons was described in the caudal brainstem, within the commissural division of the nucleus tractus solitarius (NTS) ,105. TheIn addition to the POMC neurons in the brain, the pituitary contains large numbers of POMC-producing cells. These cells are located in the intermediate as well as in the anterior lobe. Interestingly, however, the processing of the large POMC molecule seems to vary in different parts of the pituitary. In the anterior pituitary cells, ACTH is one of the main products of the POMC fragmentation, while in the intermediate lobe β-END and α-MSH predominate as the main fragments of POMC processing ,108-110.In summary, there are three sources of POMC and its derivatives including β-END: the ARH, NTS and pituitary. The first two are directed towards the CNS including the spinal cord, the last one towards the systemic circulation and peripheral organs.. Another complicating factor is that β-END levels show diurnal fluctuations in plasma as well as in the CSF [Early experiments showed that β-END in brain changed very little, or not at all, after hypophysectomy, while the peripheral concentration decreased dramatically and it was concluded that brain and peripheral β-END were regulated independently ,110,115. the CSF ,135. Theversus peripheral regulatory mechanisms [The functional correlation between CSF- and peripheral β-END is clear. With only one exception , all papchanisms ,137-140.chanisms ,141,142,chanisms .et al[Despite the general conclusion about separate central and peripheral control mechanisms, we have to keep in mind that the plasma and CSF compartments of β-END are not completely independent. Early experiments in the rabbit  already et al have diset al, or backet al or uptaket al. In addiet al. Such meThe strongest evidence that there is a separate central mechanism for β-END comes from the following experimental manipulations that induced elevated CSF levels without affecting peripheral concentration: electrical and chemical stimulation (10 Hz) of the ARH -148; obeIn conclusion, for the purpose of the present review, we assume that the effects of peripheral levels of β-END on the CSF-levels are too limited and too slow to explain any of the direct brain and behavioral effects of central β-END. The extracellular pathways involved have been discussed by Banks and may possibly play a role in long-term treatment of Alzheimer’s disease or stroke ,156.Summarizing, we conclude that the available data suggest that central β-END effects on the CNS may occur in three successive stages using different time intervals. First, in a matter of milliseconds, β-END released from terminals and varicosities of the β-END-IR fibers may have immediate action on the neighboring receptive neuronal elements. Second, β-END, arriving with the flow of the CSF after seconds or only a few minutes, may influence a larger number of receptive brain areas for a longer period of time, partially sustaining the earlier neuronal release effects. Third, elevated peripheral levels of β-END slowly penetrate the CSF compartment and induce after a delay of 30 min or more, some increase in CSF concentration. However, these slow effects are perhaps modulatory and do not seem to play a role in the behavioral or pain-suppressing effects of β-END discussed below.From the earliest reports, it has been recognized that POMC-fibers closely surround the ventricular spaces, traverse the subependymal layers and contain many varicosities. These may release their contents by non-synaptic release or exocytosis, either into the CSF or into the neighboring extracellular fluid (ECF) where it can influence receptive neurons locally ,157,158.Strategic location of the ARH: The median eminence of the hypothalamus is open to the portal vessels connecting the hypothalamus to the pituitary, without a BBB-ensheathment, but closed to the CSF. On the other hand, the ARH is open to the CSF in the third ventricle and in the subarachnoid space. Along the dorsolateral and ventromedial borders, the ARC is completely surrounded by relatively impenetrable barriers [barriers ,167.Thesbarriers , which ibarriers . This dibarriers ,170,171,barriers . The POMbarriers . On the barriers . The exibarriers ,174 suppNatural conditions rapidly increase central β-END levels: There are behavioral conditions under which the β-END level increases rapidly in the CSF. Many animal and clinical studies have measured CSF and peripheral levels of β-END but observations were taken at 15 minutes after the experimental challenge [, hypothalamic release of β-END started within a few minutes [hallenge ,175. In hallenge ,177. Leahallenge ,164. Aft minutes ,178. The minutes ,178. The minutes , which i minutes ,151.Artificially raised β-END in the CSF induces rapid effects: Periventricular brain stimulation for pain relief in humans, resulted in an increase in β-END levels in the third ventricle CSF to a maximal value up to 20 times the basal level, within five minutes [et al.[ minutes ,177. Aft minutes . These r minutes ,181. Yads [et al. transplas [et al., genes ws [et al..Uptake of β-END from the CSF: For our hypothesis concerning long-distance VT, we also need to establish that specific ependymal and other cells, partially remote from the ventricular surface, are able to take up specific substances from the CSF. Such ependymal and neuronal elements are abundantly present throughout the ventricular system, including the lateral and fourth ventricles, and have been located in both forebrain  and a variety of brainstem areas, especially the raphe nuclei [e nuclei ,184-186.e nuclei .The strategic situation of the POMC system shows striking similarities to that of the oxytocinergic (OT) system, which in mammals has two separate nuclei, the paraventricular hypothalamic nucleus bordering and freely accessing the third ventricle, and the supraoptic hypothalamic nucleus bordering and freely accessing the subarachnoid space, reviewed in . The magIn 1982, O’Donohue and Dorsa  mentioneFrom these data, we conclude that plasticity in the peptide cocktail of the POMC neurons does indeed occur. In addition to tolerance effects, hormonal factors, so far mainly controlling energy homeostasis, may induce changes in the composition of the peptide cocktail which may have serious consequences for the different activation/inhibition patterns in the projection areas of the opiocortin neurons. Interestingly, such plastic changes are even region-specific to some extent, because certain hormonal conditions induced changes in local hypothalamic β-END levels only, without affecting other hypothalamic nuclei . In addiet al[Interestingly, many POMC neurons participate in a dense local network resulting in POMC-POMC synapses inside parts of the ARH ,90,92,95et al , such cet al, for insAnatomically, the population of POMC neurons does not form a single functional unit. As mentioned, there are two groups of neurons, one located in the ARH and other in the NTS. These sources have widely diverging projections: the ARH mainly to mesencephalic, hypothalamic, limbic and forebrain areas, and the NTS mainly to the caudal brainstem and spinal cord ,107. Oveet al.[et al.[But even the POMC neurons in and around the ARH do not seem to be a single homogeneous group either anatomically or functionally. Retrograde tracer studies combined with POMC-immunocytochemical staining are relatively scarce but where available they show that only a limited portion of the POMC neurons become labeled after each tracer injection, with several indications for a topographical organization of the origins. Such retrograde double-labeling studies showed that only about 20% of the POMC neurons project to the preoptic area ,103. Labet al. showed tl.[et al. found thEstrogen receptivity is another factor differentiating between 4-20% of the β-END neurons from the majority of the neuronal population -208. The, as well as topographical organization in the distribution of the fibers originating from different parts of the ARH. Additional experiments, combining various retrograde tracers with immunological or in situ hybridization techniques, are required to elucidate the functional topographical organization of the mediobasal hypothalamic β-END neurons in more detail.In summary, it is clear that β-END neurons ‘never walk alone’, anatomically and functionally, but operate in subgroups distributed over different regions of the ARH. Projections to specific brain regions may arise from different subgroups of β-END neurons, since there are multiple indications for functional differentiation, e.g. estrogen receptivityFrom the preceding sections we conclude that β-END can be released into the CSF in sufficient quantities to induce physiological and/or behavioral effects. The β-END neurons are located in the mediobasal hypothalamus with, on the one hand, interconnections which permit them to function as sub groups and on the other an internal topographical organization, enabling them to send messages to specific targets. Dendritic release from POMC neurons in the ARH probably occurs but its contribution relative to axonal release from varicosities and terminals, remains to be investigated. Production and release of POMC and its derivatives is under control of external factors such as leptin, for energy homeostasis. This implies that differential messages can be released into the CSF and/or neuronal target areas participating in the involved neuronal circuitry. From this summary, it is clear that quite a few questions remain to be answered. What are the input–output relationships among and within the subgroups of POMC neurons? Do all POMC subgroups contribute to the release into the CSF? Which external factors influence the activity and productivity of POMC neurons? Do all POMC neurons show the same sensitivity for factors like leptin or GABA-ergic inhibition via the preoptic region?While several additional questions can be raised, those mentioned can all be studied by combinations of readily available techniques.β-END reacts with at least two types of opioid receptors: mu and delta . The locet al[There is a so-called ligand-receptor mismatch, where the distribution of immuno-labeled terminals is different to the distribution of the relevant receptors ,230. In et al, studiedet al. The perIn the human brain, specific endorphinoreceptive neurons have been described in the deeper layers of the cingulate and frontal cortices, which are densely ensheathed by β-END terminals . These lReceptor density alone may not be the best indicator for the size of the expected effect, behaviorally or metabolically, because of possible indirect effects. Ableitner and Schulz  measuredIt has been shown by Herbert and his coworkers -235 thatrd ventricle stimulated female sexual behavior in rats [rd ventricular administration [versus cisternal administration of lysine-vasopressin [rd or the 4th ventricle, combined with or without occlusion of the cerebral aqueduct. It is beyond the scope of the present review to discuss these in detail, but it is clear that factors flowing in the CSF will reach the 4th ventricle region and influence brainstem areas like area postrema and the solitary complex that control food intake. Hypothalamic and hindbrain melanocortin receptors play an important role [The findings discussed in the preceding sections raise interesting possibilities. If POMC neurons function in separate groups, then messages may be released into specific areas of the ventricular system and affect particular CNS sites responsible for physiological/behavioral reactions. There is evidence to support this. Experiments show that when a specific messenger is released into the ventricular system, the location plays an important role in the resulting effects. For example, administration of 5-HT into the lateral but not the 3 in rats  and oxyt in rats . On the stration . Similaropressin ,270. Theopressin ,272. Theant role ,274 and ant role -279.In addition to the contributions of the hypothalamo-medullary projections, however, the flowing CSF deserves more attention as a medium for sending messages into the brainstem. ARH-activation induces more than 15-fold increases in β-END in the CSF , and therd ventricular CSF in amounts at least 10 times larger than the peptide derivatives [Finally, since POMC seems to be released into the 3ivatives ,197, it rd ventricle, surrounded by the arcuate nucleus. Maybe the use of larger mammals, like sheep or even cows, is necessary to obtain experimental evidence for an addressing system with specific behavioral effects. Using sheep, has turned out to be very fruitful in studies concerning release and circulation patterns of melatonin [There is no direct experimental evidence available that β-END can be released by specific opiocortin terminals at other ventricular sites than the lower part of the 3elatonin ,64,66 anelatonin ,283,284.rd ventricle, to reach the cerebral aqueduct via the CSF flow and to influence the periaqueductal gray (PAG) region of the mesencephalon [rd ventricle will arrive with the flow of the CSF to the PAG. When local levels start to diminish after the instantaneous release, the elevated CSF levels arriving from the ARH start diffusing into the surrounding tissue to sustain and prolong the local behavioral effects of elevated β-END levels. This enables the target circuitry to be programmed in a flexible way: a different cocktail of neuropeptides could induce different functionalities of the circuit, by modifying simultaneously a relevant set of parameters.The population of POMC neurons is apparently able to produce massive amounts of β-END which is released into the 3cephalon ,148,176.cephalon ,93,210. In summary the evidence reviewed here suggests that the combination of axonal release of β-END in specific brain areas and its transmission via the CSF vary according to brain region. The varicose fibers surrounding the ventricular system allow short- as well as long-distance VT. The axonal messages of subgroups of β-END neurons are targeting specific destinations. The CSF-messages, released from the mediobasal hypothalamus, will arrive with some delay at the downstream brain areas, supporting and extending the effects of axonal messages.We conclude that β-END, released into the flowing CSF, may have effects on distant brain regions where they affect a variety of behaviors relating to reward mechanisms and motivational and mental states, and lead to stress-reduction and homeostatic balance.5-HT, Serotonin; ACTH, Adreno-corticotropic hormone; α-MSH, α-melanocyte-stimulating hormone; ARH, Arcuate nucleus of the hypothalamus; β-END, Beta endorphin; BBB, Blood–brain-barrier; CNS, Central nervous system; CRH, Corticotropin releasing hormone; CSF, Cerebrospinal fluid; GABA, γ-aminobutyric acid; GnRH, Gonadotropin releasing hormone; icv, Intracerebroventricular (administration); NTS, Nucleus of the solitary tract; OT, Oxytocin; PAG, Periaqueductal gray region of the midbrain; POMC, Pro-opio-melano-cortin containing neurons or fibers; VT, Volume transmission.The authors declare that they have no competing interests.JGV: 80%, composition, contents, writing; HB: 15%, composition, discussion, improvements and corrections; POG: 5%: discussion and general improvements; All authors have read and approved the final version of the manuscript.


Enterococcus and Enterobacter spp.) were found after no handwashing in 44% of samples. Handwashing with water alone reduced the presence of bacteria to 23% (p < 0.001). Handwashing with plain soap and water reduced the presence of bacteria to 8% (comparison of both handwashing arms: p < 0.001). The effect did not appear to depend on the bacteria species. Handwashing with non-antibacterial soap and water is more effective for the removal of bacteria of potential faecal origin from hands than handwashing with water alone and should therefore be more useful for the prevention of transmission of diarrhoeal diseases.Handwashing is thought to be effective for the prevention of transmission of diarrhoea pathogens. However it is not conclusive that handwashing with soap is more effective at reducing contamination with bacteria associated with diarrhoea than using water only. In this study 20 volunteers contaminated their hands deliberately by touching door handles and railings in public spaces. They were then allocated at random to (1) handwashing with water, (2) handwashing with non-antibacterial soap and (3) no handwashing. Each volunteer underwent this procedure 24 times, yielding 480 samples overall. Bacteria of potential faecal origin (mostly  Diarrhoeal diseases are one of the leading causes of child death around the world . The WorPromotion of improved hand hygiene has been recognised as an important public health measure but it is unclear how much hand hygiene is required to interrupt transmission of diarrhoea pathogens. In particular it has not been conclusively shown whether use of soap is essential to remove pathogens from hands. Recent hygiene promotion campaigns especially in low income settings have not been unanimous in recommending soap use .A number of studies have compared different hand hygiene methods in hospital settings . In contLuby and colleagues found that a simple microbiological method with three fingers directly imprinting a MacConkey agar for thermotolerant coliforms was unable to distinguish between households who were given soap during a large randomized handwashing trial and control households . They coWe conducted a randomised controlled trial to determine whether non-antibacterial soap is better at reducing bacteria of potential faecal origin than water only. A further goal was to clarify whether a simple microbiological test that can be applied to large groups in a relatively short time ,10 wouldThis study was carried out between July and August 2009. Overall, 20 volunteers were taken to a large, frequently visited British museum, or asked to travel on a bus or the underground. They were asked to deliberately wipe their hands over hand contact surfaces such as handrails, door handles and seats with the aim of contaminating their hands with whatever bacteria were present. Using a pre-determined random sequence, not known to the participants during self-contamination, participants were then asked to wash their hands with soap, to use water only or not to wash at all. Each volunteer underwent this sequence 24 times, 8 times for each of the three hand hygiene approaches . Participants assigned to handwashing were asked to wash their hands as they would normally do, without instructions on length of time or thoroughness. The volunteers allocated to handwashing were then provided with a paper towel to dry their hands. A wet NaCl-soaked charcoal swab was then wiped across the fingers of the dominant hand of the participant. The participants were finally given an alcohol gel to clean their hands . The swabs were returned to the laboratory within 5 hours of being taken. In total, 480 samples were collected; 160 after handwashing with plain soap, 160 after handwashing with water alone and 160 with no handwashing. During the experimental phase we measured the amount of time taken to conduct handwashing with and without soap, once for each volunteer.Enterococcus spp. and group D Streptococcus and inhibition of other gram positive bacteria. These plates were incubated at 37 °C for 18–24 hours. Enterococcus and Group D Streptococcus spp. are able to hydrolyse the aesculin to form aesculetin, producing a brown/black complex. Any white colonies on Bile Aesculin agar were presumed to be Staphylococcus spp. and any black colonies were tested with Lancefield group D antisera. Agglutination indicated a positive result for Enterococcus spp.Upon arrival at the laboratory the swabs were immediately cut into a universal tube containing 10 mL of Purple MacConkey broth using aseptic techniques. The swabs were incubated at 35 °C for 48 hours. All samples were then streaked onto the MacConkey agar No.3 and Bile Aesculin agar. MacConkey agar No. 3 is a selective media which can differentiate between coliforms and non-lactose fermenters, whilst inhibiting gram-positive cocci. These plates were incubated for 18–24 hours at 35 °C. For all other colonies produced on MacConkey agar No. 3 and those which were spot indole negative, a gram stain, catalase and oxidase test was carried out followed by an API 20E biochemical test to determine the identity of the bacteria. Bile Aesculin agar is a differential medium for the isolation of The prevalence of bacterial contamination in the three study arms  was compared using logistic regression. Since the same volunteers repeatedly underwent testing, within-subject correlation was accounted for by the use of generalised estimating equations (GEE) with robust standard errors. If the cell numbers were too low for conducting regression analysis, Fishers exact test was used instead, ignoring clustering .Enterococcus spp. were the most common bacteria found, followed by Enterobacter spp. Enterococcus spp. There was a trend that handwashing with soap was also more effective in reducing the prevalence of other species and of multiple isolates, but the statistical support was low  ranged from 1.2 to 1.3 (depending on the comparison group).Participants were asked to wash their hands as long and as thorough as they would normally do. The length of time required to carry out handwashing was measured once for each method in all volunteers. Participants took on average 12 seconds (standard deviation 2.8) to wash their hands with water alone, and 14 seconds (standard deviation 2.3) to wash their hands with water and soap (p = 0.02).Thus, handwashing with soap took them only slightly longer than handwashing with water alone. It seems unlikely that this small difference can explain the large difference in the removal of bacteria. Soap on its own appears to have an effect on the removal of bacteria of potential faecal origin, independent of the possibility that soap use may cause people to wash their hands longer.Unlike the study by Hoque and colleagues our trial was conducted in an experimental  setting, where volunteers deliberately contaminated their hands. Additional testing showed that this approach increased the prevalence of contamination from around 10% to over 40% of individuals. It also improved control over the conduct of the experiment, but may affect generalisability, as the study primarily aimed at providing a proof of principle. However, we believe that the superior effect of soap on the removal of bacteria compared to water alone as the principal finding of our study is unlikely to depend on the setting.Not all of the bacteria isolated in our study are known to cause disease in humans. Surprisingly, we found few E. coli on hands which may be due to their short survival time in the environment. Overall, the effect of soap appeared to be independent of the type of bacteria , a view We used plain non-antibacterial soap for the experiment. Future studies could address whether antibacterial soap is more effective in removing pathogens from hands. However, Luby and colleagues conducted a large double-blind randomised trial in Pakistan and found antibacterial soap no more effective in reducing diarrhoea than normal soap . It is sThe bacteriological methods used in this study provide no quantification of bacterial load, unlike a study by Hoque and colleagues . QuantifIt seems reasonable to assume that handwashing with soap is also more effective in reducing bacterial load compared to water alone. Future studies could address the effect of different hand hygiene procedures on removing gastro-intestinal or respiratory viruses such as influenza A. Hands have been implicated especially in the spread of Norovirus . Viral sThe results demonstrate that handwashing with non-antibacterial soap is much more effective in removing bacteria from hands than handwashing with water only. Although handwashing with water alone reduced the presence of bacteria on hands substantially, the study supports the policy of many current hand hygiene campaigns promoting the use of soap ,19. The Hygiene behaviour is difficult to measure because people tend to change their behaviour under observation or over-report desired practices ,20. We h


After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and in vitro expansion capacity after SC mobilization. Most importantly, in vivo G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after in vitro expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4 Currently, clinical approaches worldwide aim to maximize the benefits and to overcome the challenges and risks of Treg cell therapy Prior to sample collection approval was given by the institutional ethics committee of Hannover Medical School. After obtaining signed written informed consent forms from 86 stem cell donors peripheral blood withdrawals were taken before  and after G-CSF administration . HSC mobilization was performed by the subcutaneous administration of 10 µg/kg/d G-CSF  for 4 consecutive days.+ T cells was performed using the CD4 T-cell isolation Kit II and the AutoMACS separating system (Miltenyi Biotec) according to manufacturer’s instructions. Subsequently, CD4+ T cells were stained with monoclonal antibodies against CD14 APC , CD4 FITC , CD25 PE  and CD127 Alexa647 . CD14−CD4+CD25high Tregs were isolated by fluorescence activated cell sorting using a FACSAria Cytometer. Isolated target Treg populations were CD127− and displayed a purity >96%.Heparinized blood samples of 40 ml were obtained from stem cell donors before and after G-CSF administration. Peripheral mononucleated cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Tregs were separated in two steps because of massive increase of granulocytes after G-CSF mobilisation. First, separation of CD4Phenotype of Treg cells was determined by direct immunofluorescence with monoclonal antibodies against the antigens with following fluorochromes: CD4 PerCP, CD127 Alexa647, CD45RA PE, Foxp3 Alexa488, CD25 PeCy7, CD8 APC-H7, CD39 APC, CD45RA PE, CD45RO APC, CD31 PE, CD62L PE, ICOS PE, CCR5 PE, CCR7 A647, CD69 APC-Cy7, CXCR3 PE , LAP APC (R&D Systems), LAG3 FITC (enzo), 4-1BB APC, CD44 PE, Helios PE, GZMA A647 and Ki67 A647 . In some experiments CCR4 APC (Biolegend) and CXCR4 PE (Biolegend) have been used. Isotype and FMO controls were included for the respective antibodies. Fixation and permeabilization for staining with antibodies against Foxp3 were performed with Foxp3 Buffer set (BD Pharmingen). After surface staining Treg cells were incubated with Fixation Buffer A for 15 min and washed twice. Fixed cells were permeabilized by incubating with Buffer C for 30 min and stained with Foxp3 antibody. Cells were analyzed using FACS Canto (BD) and FACS Diva 6 software.in vitro expansion and stored at −80°C until analysis. In selected experiments CD4+CD25highCD127− Tregs were isolated before G-CSF treatment (n = 5) and cultured for 14 days with daily 100 ng/ml G-CSF  stimulated with CD3/CD28 Dynabeads and 500 U/ml IL2. For overnight culture, freshly cell sorted CD4+CD25highCD127− Tregs were washed twice with PBS, transferred into a 96-well u-bottom plate and cultured overnight in RPMI 1640 containing Glutamax medium (Gibco) supplemented with 10% FCS (Biochrom), 50 µg/ml gentamycin and 50 µg/ml penicillin/streptomycin at 37°C.Cell culture was performed under aseptic conditions in 96-well u-bottom plates (Greiner bio-one). Isolated Treg cells were cultured in RPMI 1640 containing Glutamax medium (Gibco) supplemented with 10% FCS (Biochrom) for 14 days at 37°C. To avoid contaminations, 50 µg/ml gentamycin (Sigma Aldrich) and 50 µg/ml penicillin/streptomycin (Sigma Aldrich) were added to the medium. Tregs were stimulated by IL-2 500 U/ml  and CD3/CD28 dynabeads (Invitrogen) in a 4∶1 ratio. Dynabeads were changed after seven days and removed after eleven days of culture. Aliquots of supernatants were frozen after overnight culture (d1) and 3, 7, and 14 days 6 PBMCs were stained with anti human CD4 PerCp (BD Pharmingen) and anti human CD25 PeCy7 (Biolegend) antibodies. Stained cells were washed, resuspended in cell cuture media and prewarmed at 37°C for 30–60 minutes. Subsequently 300 ng IP-10 was added quickly and incubated for 30 seconds. Cells were fixed, permeabilized and stained with anti human Foxp3 A647 antibody (Biolegend) and Phalloidin FITC (Sigma). Cells were analyzed using FACS Canto (BD) and FACS Diva 6 software.Phalloidin assay was performed using a modified protocol described elsewhere . Briefly, 5×10+CD25highCD127− T cells isolated from SC donors before and after G-CSF treatment were quantified by ‘Bio-Plex, Human Cytokine 17-Plex Panel’ and ‘Bio-Plex, TGFβ 3-Plex’ protein arrays according to the manufacturer’s instructions (BioRad Laboratories). A 2-laser array reader (BioRad Laboratories) simultaneously quantifies all cytokines and chemokines of interest. Standard curves and concentrations were calculated with Bio-Plex Manager 4.1.1 on the basis of the 5-parameter logistic plot regression formula. The detection sensitivity of all analyses was between 0.5 and 78000 pg/ml.Cytokine and chemokine concentrations in supernatants of cultured and expanded CD4+CD25− T responder cells  were stained with 0.5 µM CFSE (Invitrogen) for 15 minutes at 37°C according to the manufacturer`s protocol. These CFSE stained Tresp were cultured with 5×104 irradiated (30 Gy) allogeneic PBMCs and autologous Treg cells in the presence of 1 mg/ml purified anti CD3 and anti CD28 antibodies (both from Biolegend) in RPMI 1640 containing Glutamax medium (Gibco) supplemented with 10% FCS (Biochrom), 50 µg/ml gentamycin (Sigma Aldrich) and 50 µg/ml penicillin/streptomycin (Sigma Aldrich). The final suppressor to responder T cell ratios were: 2∶1, 1∶1, 1∶2, 1∶10 and 0∶1 (ctrl). After 7 days, cells were analyzed by FACS Canto (BD). Percentage of suppression was calculated as followed: 100 - (percentage of proliferated Tresp x 100/percentage of proliferated Tresp). For suppression assays after in vitro expansion frozen autologous PBMCs were thawed one day before usage and kept under cell culture conditions overnight. CD4+CD25− T cells were isolated from thawed autologous PBMCs by depletion of CD4− T cells and positive selection for CD25+ T cells using the CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotech) and stained with 0.5 µM CFSE (Invitrogen) as described for freshly isolated Tresp.CD4foxp3 was carried out as previously described Genomic DNA was isolated from >150.000 freshly sorted or expanded regulatory T cells using the AllPrep DNA/RNA Micro Kit (Qiagen) according to manufacturer’s instructions. Concentrations of genomic DNA were measured by Nano Drop (Thermoscientific). DNA demethylation analysis of GraphPad Prism software has been used for interpretation of data and statistical analysis. For the analyses two-tailed paired Students’ t-test and two-tailed Mann-Whitney t-test were performed. P values with p>0.05 were indicated as not significant (n.s.) and significant p values with p<0.05 as *, p<0.01 as **, p<0.001 as *** and p<0.0001 as ****.+ T cells were not significantly increased in the peripheral blood of the stem cell donors and leukapheresis products  and in the leukapheresis products revealed significantly increased numbers of PBMCs after G-CSF mobilization . AlthougFoxp3 locus (TSDR) specifies the committed state of regulatory T cells +CD25highCD127− Tregs after stem cell mobilization is comparable to Tregs before G-CSF stimulation of the stem cell donors  after overnight culture  compared to 52-fold (mean 33-fold) before G-CSF mobilization  showed a spontaneous secretion of IL-10, IL-17, IFNγ, TNFα and TGFβ1,-2 and -3. However, no significant difference could be observed for cytokine secretion by Tregs before and after G-CSF application of the SC donors. After 3 days of ex vivo culture an increase of cytokine production could be observed for IL-10, IL-4, IL-5, IL-12p70, IL-13, IL-17, IFNγ, and MCP1, while the concentration of TNFα decreased. However, significant differences between donor Tregs before and after G-CSF mobilization were only detected at day 3 for IL-12p70  and do not show signs of activation/proliferation arguing for homeostatic trafficking from the bone marrow (BM) as natural reservoir of Tregs to the peripheral blood G-CSF application leads to a numerical increase of circulating donor Tregs with a significant higher Treg cell yield in the SC graft, while the number of CD4+ Tregs into effector T cells would be detrimental. Very recently, Miyao et al. could demonstrate that Treg cells constitute a stable cell lineage the committed state of which is ensured by DNA demethylation of the FoxP3 locus (TSDR) also in an inflammatory environment +CD25highCD127- donor Tregs after G-CSF stimulation represent a stable cell lineage robustly maintaining their committed state also in a changing environment. Moreover, donor Tregs remain highly suppressive on the proliferation of effector T cells correlating with a stable expression of relevant molecules like LAG3, CD39, GZMA and IL-10 following G-CSF stimulation in vitro expanded post G-CSF donor Tregs and enhancing their suppressive capacity. With respect to adoptive Treg cell transfer strategies for the prevention and therapy of GvHD, a stable and highly suppressive phenotype of donor Tregs is of pivotal relevance and might compensate the reduced suppressive capacity of Tregs in GvHD patients recently reported by us hiCD62L+CD69− memory Tregs shows a cleavage of CD62L after G-CSF application. However, no signs of activation or induction of the co-stimulatory molecules ICOS or 4-1BB could be observed in donor Tregs. As terminally differentiated activated CD45RA−Foxp3hi Tregs rapidly die after TCR stimulation in vitro and in vivoin vivo.The stability of the suppressive phenotype is of high relevance for the validity and safety of human Treg cell therapy as spontaneous or environmentally induced reprogramming of Foxp3The migration of Tregs to secondary lymphoid organs and target organs of GvHD is critical for effective control of allogeneic immune responses and immune homeostasis following allogeneic SCT. Recently, we have demonstrated that Tregs of GvHD patients show a significant reduction of CCR5 and CXCR3 surface expression likely leading to a diminished migration capacity of Tregs with significantly lower Treg cell infiltration in the inflamed intestinal mucosa in vitro stimulation of Tregs after SC mobilization induces the release of higher levels of IL-12 and TGFß1. However, the secretion of other key cytokines  appears to be independent of G-CSF stimulation and is comparable to data from other investigators in vitro stimulation of Tregs isolated after SC mobilization aligned the expression profile of donor Tregs before and after G-CSF stimulation. Current research focuses on the development of large-scale expansion protocols for Tregs with higher cell yields The comparative analysis of the Treg cytokine profile reveals no differences in the spontaneous secretion whereas In conclusion, our data indicate donor Tregs after G-CSF stimulated SC mobilization retain their potent suppressive and stable phenotype combined with a favorable migration capacity and attractive cell yield. This study outlines that the adoptive transfer of donor Tregs after G-CSF stimulation appears to be feasible and safe. The isolation of donor Tregs from the SC graft will simplify their global clinical application and extend this novel strategy of tolerance induction to the unrelated transplant setting.Figure S1In vitro effects of G-CSF on Tregs. CD4+CD25highCD127−Foxp3+ Tregs were isolated before G-CSF treatment (n = 5) and cultured for 14 days with (black bars) and without (white bars) 100 ng/ml G-CSF stimulated with CD3/CD28 Dynabeads and 500 U/ml IL2. Percentage of CXCR3, CCR5, CD31, CD62L, Ki67 and LAP protein expression of CD4+CD25highCD127− Tregs is shown. For statistical analysis two-tailed Mann Whitney t-test and unpaired t-test was performed, respectively. Not significant p values are indicated as n.s. P values p<0.05 are indicated as *, p<0.01 as **, p<0.001 as *** and p<0.0001 as ****.(TIF)Click here for additional data file.Figure S2Schematic presentation of protocols for adoptive Treg cell transfer. (A) Currently used protocols for clinical trials include isolation of Tregs from the apheresis product befpre G-CSF mobilization of the stem cell donor and injection into the recipient at day −4. A second apheresis is necessary at d0 to isolate the graft. Consequently this protocol is only feasible in a related donor setting. (B) An additional apheresis is not necessary if Tregs are isolated after G-CSF application. This setting is feasible for related and unrelated donors. Moreover, neutropenia is not prolongated.(TIF)Click here for additional data file.


In Hungary, despite the high level of social support, the number of organ recoveries from deceased donors has not changed significantly. The donation activity shows a positive relationship with the level of education of staff in ICUs as well as with their attitude towards transplantation. The aim of this cross-sectional study is to estimate the attitude and knowledge of intensive care specialists and nurses as regard donation and transplantation.n = 179) and nurses (n = 103) were asked about donation activity, participation in an organ donation course, self-reported knowledge of joining Eurotransplant, donor management, legislation, and transplantation. The data were analyzed by SPSS 17.0.The self-completed questionnaire that consisted of 20 items was completed at the Congress of Hungarian Society of Anesthesiology and Intensive Therapy in 2011. Besides the epidemiological data, the intensive care specialists (P 0.01). The average age of those who participated in training was significantly higher among doctors (P 0.01). Fifty-nine percent of doctors and 65.1% of nurses did not even want to participate in such training. Donation activity was higher among staff who joined training (P 0.01). Independently from accepting the presumed consent legislation (91.1%), 66% of physicians agreed with the hospital practice that requests the adult donor's relatives to consent to organ recovery. This standpoint did not depend on donation activity, participation in an organ donation course, opinion about legislation and the nature of staff. A total 95.4% of participants consented to their organ retrieval after death. The staff who participated in an organ donation course had more knowledge regarding the law and ethics of donation (P 0.01), donor management (P 0.01), living and deceased donor transplantation (P 0.01) and joining Eurotransplant (P 0.01). Older professionals had more information about all fields (P 0.01). Nurses had less knowledge concerning donor management (P 0.01), law and ethics (P 0.01) and deceased donor transplantation (P 0.01) than doctors.A total of 53.6% of physicians and 16.7% of nurses attended an earlier organ donation course (Education about organ donation needs to be part of specialist training of intensive care staff, and refresher courses every fifth year as well. The course should include knowledge regarding brain death, donor management and communication with family. This is the first step to improve the number of transplantations.


Melancholic depression is a biologically homogeneous clinical entity in which structural brain alterations have been described. Interestingly, reports of structural alterations in melancholia include volume increases in Cerebro-Spinal Fluid (CSF) spaces. However, there are no previous reports of CSF volume alterations using automated whole-brain voxel-wise approaches, as tissue classification algorithms have been traditionally regarded as less reliable for CSF segmentation. Here we aimed to assess CSF volumetric alterations in melancholic depression and their clinical correlates by means of a novel segmentation algorithm , incorporating specific features that may improve CSF segmentation. A three-dimensional Magnetic Resonance Image (MRI) was obtained from seventy patients with melancholic depression and forty healthy control subjects. Although imaging data were pre-processed with the ‘new segment’ algorithm, in order to obtain a comparison with previous segmentation approaches, tissue segmentation was also performed with the ‘unified segmentation’ approach. Melancholic patients showed a CSF volume increase in the region of the left Sylvian fissure, and a CSF volume decrease in the subarachnoid spaces surrounding medial and lateral parietal cortices. Furthermore, CSF increases in the left Sylvian fissure were negatively correlated with the reduction percentage of depressive symptoms at discharge. None of these results were replicated with the ‘unified segmentation’ approach. By contrast, between-group differences in the left Sylvian fissure were replicated with a non-automated quantification of the CSF content of this region. Left Sylvian fissure alterations reported here are in agreement with previous findings from non-automated CSF assessments, and also with other reports of gray and white matter insular alterations in depressive samples using automated approaches. The reliable characterization of CSF alterations may help in the comprehensive characterization of brain structural abnormalities in psychiatric samples and in the development of etiopathogenic hypotheses relating to the disorders. Melancholic depression is a subtype of major depressive disorder (MDD) that encompasses a constellation of distinctive clinical features such as anhedonia, distinct quality of mood and mood non-reactivity, psychomotor disturbances, feelings of guilt, early awakening, diurnal variation and anorexia In addition to such features, a number of studies have also identified brain structural alterations in melancholia Voxel-wise methods such as voxel based morphometry (VBM) a priori distribution of non-brain tissue. The new algorithm should prevent misclassification of non-brain voxels as CSF Nevertheless, the development of new tissue segmentation algorithms, such as the so-called ‘new segment’ algorithm The study protocol was approved by the ethical committee of clinical research (CEIC) of the Bellvitge University Hospital, and was in compliance with the national legislation and the principles expressed in the Declaration of Helsinki. All participants gave written informed consent after detailed description of the study.A total of 70 inpatients  with melancholic depression were consecutively recruited from the Mood Disorders Unit of the Bellvitge University Hospital, Barcelona. Sample characteristics are shown in A detailed medical history was recorded and a structured interview was administered to detect subjects who fulfilled exclusion criteria. For both groups, exclusion criteria included the presence or past history of any Axis I or Axis II diagnosis (excepting MDD in patients), the presence or past history of neurologic or other serious medical conditions (including dementia), any contraindication to MRI scanning, or abnormal MRI upon visual inspection. Specifically, to ensure there were no cases of cardiovascular etiology, any evidence of ischemic tissue damage in the MRI was regarded as an exclusion criterion. However, subjects with controlled hypertension or diabetes mellitus Type 2 (DM-II) were not excluded . All patients were clinically followed up for at least two years through contact with the Mood Disorders Unit. During this period, the presence of emerging signs of dementia was ruled out by the routine clinical interview, which included the administration of the Mini-Mental State Examination MMSE, .2, and section thickness 2.5 mm). Imaging data were processed on a Microsoft Windows platform using technical computing software  and Statistical Parametric Mapping  Subjects were scanned in a 1.5-T scanner  to obtain a sixty-slice 3-D SPGR sequence in the axial plane  from raw T1 images using a probabilistic framework combining tissue classification, image registration and bias correction. For the purpose of this study, from this step we discarded final output images in normalized space, exclusively keeping files that encoded the segmentation and normalization transformations applied to raw data (the *_seg_sn.mat files). These files were used as an ‘initial import’ in DARTEL tools in order to write out rigidly transformed versions of CSF tissue (rc3*.nii files), which were saved for the subsequent normalization process.On the other hand, the ‘new segment’ algorithm is an extension of the ‘unified segmentation’ algorithm that uses an extended set of tissue probability maps allowing for a different treatment of voxels outside the brain . In addition to gray matter, white matter and CSF tissue probability maps, this algorithm incorporates tissue probability maps of bone, soft tissue and air/background distribution. The ‘new segment’ algorithm also segments, normalizes and corrects bias within the same model, and, also as above, we discarded final output images from this preprocessing and kept the rigidly transformed versions of CSF images that were used for DARTEL normalization (rc3*.nii files).The rigidly transformed images of CSF derived from both the ‘unified segmentation’ and the ‘new segment’ algorithms were normalized using DARTEL Finally, bias corrected, tissue segmented, and DARTEL normalized and modulated CSF images were smoothed with an 8 mm FWHM isotropic Gaussian kernel.Global CSF and total intracranial volume (TIV), obtained from segmented images in native space, were compared between groups by means of independent samples t-tests using SPSS (v. 15).Between-group comparisons of voxel-wise regional volume differences were studied with SPM8 tools. Specifically, two independent sample t-test models  were built, with TIV as a nuisance covariate.Subsequently, in a post-hoc analysis, between-group differences in CSF volume observed in the region of the left Sylvian fissure  with the number of previous depressive episodes, time to remission of the current episode, HAM-D score at admission and percentage of HAM-D reduction between admission and discharge.In all the SPM analyses, the significance threshold was set at p<0.05 (family-wise error [FWE] corrected for multiple comparisons), although, for displaying purposes , we used a threshold of p<0.001 (uncorrected). In SPSS analyses, the significance threshold was set at p<0.05 with the applicable Bonferroni correction when multiple independent analyses were performed .Regarding global CSF volume, no significant between-group differences were observed when images were segmented using the ‘new segment’ approach. Nevertheless, a significant global CSF volume increase in melancholic patients was observed when images were segmented using the ‘unified segmentation’ algorithm. By contrast, significant between-group differences in TIV were not observed with either segmentation approach. These results are presented in FWE = 0.134).Using the ‘new segment’ algorithm, melancholic patients showed a significant CSF volume increase in the region of the left Sylvian fissure surrounding the insular cortex. In addition, we observed a significant volume decrease in the subarachnoid spaces surrounding the medial and the lateral parietal lobe see . ConversTo validate our voxel-wise findings, between-group differences in the region of the Sylvian fissure were studied with a ROI approach. This region constitutes an anatomically defined area that may be reliably traced in non-segmented T1 images in native space. The area selected for this analysis is presented in Within the patient group, we correlated CSF volumes from the regions with significant between-group differences in the above analyses with the clinical data. In these analyses a Bonferroni corrected significance threshold was used. Specifically, the threshold was set at p<0.0042 (0.05/12), since we correlated the volume of three different clusters with four clinical variables. CSF volume at the left Sylvian fissure showed a significant negative correlation with the percentage of HAM-D reduction between admission and discharge . The same region was also correlated with time to symptom remission after treatment initiation, although this finding was not statistically significant at the Bonferroni corrected significance threshold . The number of previous depressive episodes and the HAM-D score at admission were not correlated with CSF volume at the left Sylvian fissure. Likewise, CSF volumes surrounding the medial and lateral parietal regions were not correlated with any of the clinical variables assessed.Finally, in order to assess any medication effects on our results, the number of days of antidepressive treatment at inclusion was correlated against CSF volumes at the regions where between-group differences had been detected in the above analyses, but no significant correlations were found.To our knowledge, this is the first whole-brain voxel-wise study comparing CSF volumes of melancholic patients with healthy controls. Specifically, we observed a significant CSF increase in the region of the left Sylvian fissure and a CSF volume decrease at the level of the medial and lateral parietal cortex. We also observed that such findings were not detected using earlier segmentation algorithms. By contrast, we validated the left Sylvian fissure alterations by means of a non-automated ROI analysis. Finally, we detected that CSF content in the region of the left Sylvian fissure was inversely related to clinical improvement , which provided our findings with clinical significance.CSF volume increases in the left Sylvian fissure have been previously reported in other studies with samples of depressive patients By contrast, significant volume decreases in the CSF spaces surrounding medial and lateral parietal cortices have not been observed in previous studies with melancholic or general depressive samples, in part because this region has not been typically evaluated in region of interest studies. There are, however, some reports of gray matter alterations in medial and lateral parietal areas in depressive patients, both in terms of volume reductions In addition to complementing the description of putative structural alterations in gray and white matter, and thus providing a more comprehensive characterization of the structural abnormalities associated to psychiatric conditions, the identification of CSF abnormalities with reliable whole-brain automated approaches may assist the development of etiopathogenic hypotheses relating to the disorders. For example, CSF alterations located in regions with concomitant gray and/or white matter abnormalities may fit within neurodegenerative disease models According to the above notions, our findings relating to an increased CSF content in the left insular region of melancholic patients, in combination with earlier reports of brain parenchyma reductions in the same region In addition to the above ideas, the CSF alterations reported here seem to be of clinical relevance as, in our sample of melancholic patients, larger CSF volumes in the left Sylvian fissure were related to a smaller improvement of depressive symptomatology at discharge. This observation is in accordance with previous correlations obtained with closely related measures, such as regional CSF insular volume Contrasting the results obtained with the two different image segmentation strategies used here, we observed that the CSF content of the left Sylvian fissure was only significantly increased when data were pre-processed using the ‘new segment’ algorithm. Although there is no ‘gold-standard’ for comparing the results obtained with the two pre-processing approaches, we validated the left Sylvian fissure CSF volume increase detected here under the ‘new segment’ approach with a non-automated ROI analysis. Left, but not right, Sylvian fissure showed a lower mean signal intensity in combination with a larger volume. Also, such a finding is in accordance with earlier studies reporting brain structural alterations in melancholia also using non-automated image analysis techniques Global CSF measurements also differed between the two segmentation methods studied. While the ‘new segment’ algorithm detected no significant between-group differences, the ‘unified segmentation’ approach showed an increased global CSF volume in melancholic patients. Previous reports showed inconsistent results regarding this measurement, with some studies reporting no changes The current study does, however, have certain limitations. Firstly, it is possible that antidepressant treatment may have influenced the reported volumetric measurements, although we did not detect any significant association between our structural measurements and antidepressive treatment time. Secondly, as we did not recruit a sample of non-melancholic MDD patients, our results cannot be considered exclusive to melancholic depression. Such a comparison would be an informative addition to the current findings. Finally, although the ‘new segment’ approach is regarded here as the most valid algorithm to automatically segment CSF content from brain MRIs, further comparisons with other approaches will be required to confirm such an assumption. Nevertheless, the lack of a ‘gold-standard’ protocol for CSF content assessment may perhaps prevent us from obtaining a definite conclusion as to the validity of the different CSF segmentation methods. Be that as it may, voxel-wise alterations located at left Sylvian fissure have been validated with a non-automated ROI analysis, and, moreover, the location of such CSF alterations is not only concordant with earlier reports of regional alterations in the CSF spaces of melancholic samples, but also with previous reports of gray matter alterations in general depressive samples.In conclusion, this is the first study to show regional  CSF volume alterations in melancholic depression using an automated whole-brain segmentation algorithm. Such alterations would seem to have clinical relevance, and may indeed constitute a relevant measurement to be considered in studies seeking imaging predictors of remission or treatment response. Moreover, the description of CSF alterations is necessary for a complete characterization of the neuroanatomical abnormalities of psychiatric conditions, which may help in the development of etiopathogenic hypotheses relating to the disorders. Finally, we have also shown how the characterization of such alterations may depend on the selection of the algorithm used for the automated segmentation of the CSF content from brain images. The results obtained with the ‘new segment’ algorithm, validated here by means of a non-automated ROI assessment, are in agreement with earlier reports also using non-automated methods of CSF segmentation and with findings from similar whole-brain automated methods used for regional gray and white matter volume assessment.


Biomarkers of disease progression in amyotrophic lateral sclerosis (ALS) could support the identification of beneficial drugs in clinical trials. We aimed to test whether soluble fragments of beta-amyloid precursor protein (sAPPα and sAPPß) correlated with clinical subtypes of ALS and were of prognostic value.SMI35) were measured by multiplex assay, Progranulin by ELISA. CSF sAPPα and sAPPß levels were lower in ALS with a rapidly-progressive disease course  and with longer disease duration . CSF NfHSMI35 was elevated in ALS compared to PD and controls, with highest concentrations found in patients with rapid disease progression (p<0.01). High CSF NfHSMI3 was linked to low CSF sAPPα and sAPPß . The ratios CSF NfHSMI35/CSF sAPPα,-ß were elevated in patients with fast progression of disease (p = 0.002 each). CSF Progranulin decreased with ongoing disease (p = 0.04).In a cross-sectional study including patients with ALS (N = 68) with clinical follow-up data over 6 months, Parkinson's disease , and age-matched controls (N = 40), cerebrospinal fluid (CSF) levels of sAPPα a, sAPPß and neurofilaments (NfHSMI35) and to progression of disease.This study provides new CSF candidate markers associated with progression of disease in ALS. The data suggest that a deficiency of cellular neuroprotective mechanisms (decrease of sAPP) is linked to progressive neuro-axonal damage (increase of NfH Purified bovine NfH was obtained from US Biological . Standards ranged from 0 to 2500 pg/ml, and the sensitivity of the assay (background plus three standard deviations) is 2.4 pg/ml, with an intra- and inter-assay coefficient of variation of 4.8% and 8.4%. The standards were stored at −20°C. All samples were measured in duplicates.A highly sensitive immunoassay developed in-house for NfHLevels of Progranulin in CSF and serum were determined using ELISA according to the instructions as supplied by the manufacturer . The accuracy of the ELISA was assessed by “Spike-and-recovery” and “linearity-of-dilution” experiments. The serum samples were diluted 1∶600, the CSF samples 1∶12. The sensitivity of the assay (background plus three standard deviations) was 32 pg/ml. All samples were measured in duplicates.SMI35/CSF sAPPα and CSF NfHSMI35/CSF sAPPßα for identification of ALS patients with a rapid progression of disease. The Youden index was calculated to find the cut-off values which maximize discriminating accuracy of the tests Data analysis was performed using SPSS . Because of non-normal data distribution (Kolmogorov-Smirnov test), the medians and interquartile ranges are shown. Differences between two groups were compared using Mann-Whitney Rank Sum Test. To compare raw data of multiple groups, Kruskal-Wallis analysis of variance on ranks was applied, followed in case of significance by Dunn's Method. All correlations were studied using Spearman's rank correlation coefficient. Multiple correlations were corrected using the Bonferroni method. Receiver operating characteristic (ROC) analysis was used to compare the sensitivity and specificity of the ratios CSF NfHMotor function as measured by the MRCS decreased from a median 56 (range 33–60) points at onset of study to a median 49 (range 7–60) points after 6 months. The median disease progression on the MRCS was 0.92 in this cohort. The ALSFRS-R decreased from a median 40 (range 6–48) points at onset of study to a median of 35 (range 6–46) points after 6 months.alb was observed in 17% of patients with ALS.No significant difference of the CSF cytology was observed between patients with ALS and controls . A mild alb . Furthermore, no correlation of CSF sAPPα or CSF sAPPß with age of patients was detectable .Both CSF sAPPα and CSF sAPPß concentrations were decreased in a subgroup of ALS patients with a rapid-progressive course of disease , disease duration (p = 0.09) or blood-CSF barrier function (p = 0.52). No difference of CSF NfHSMI3 between patients with bulbar and extremity-onset (p = 0.62) or with and without riluzole was detectable (p = 0.42). CSF NfHSMI3 decreased with increasing CSF sAPPα and sAPPß concentrations .We observed a difference of the ratio CSF NfHSMI35/CSF sAPPα of 2.1  and a ratio CSF NfHSMI35/CSF sAPPß of 4.3  as optimal cut-off to differentiate patients with a fast progression of disease from patients with a slower progression of disease.Using ROC analysis, we determined a ratio CSF NfHSMI35, ROC analysis yielded a cut-off of 55.8 pg/ml , for CSF sAPPα a cut-off of 27.5 ng/ml , and for sAPPß a cut-off of 29.5 ng/ml  to differentiate patients with a fast progression of disease from patients with a slower progression of disease.For NfHalb (p = 0.9). We observed an inverse correlation of CSF Progranulin levels with duration of disease . No correlation of serum levels with duration of disease was detectable (p = 0.78).We observed no difference of either CSF or serum Progranulin concentrations between patients with ALS, PD and controls . Similarly, there was no difference of CSF Progranulin between subgroups of ALS patients with fast and slow disease progression (p = 0.07). Likewise there was no difference of CSF Progranulin between patients with bulbar or extremity-onset (p = 0.67) or treatment with Riluzole (p = 0.17). We observed no correlation of CSF Progranulin with age of patients (p = 0.12). We also found no correlation of CSF Progranulin with blood-CSF barrier function as measured by QWe observed CSF levels of sAPPα and sAPPß to be altered in subgroups of ALS: They were a) decreased in patients with a rapid-progressive course of disease over follow-up  and b) iCleavage of APP by the α-and ß-secretase pathway results in the production of the sAPPα or sAPPß fragment SMI35 in the CSF (SMI35 in ALS to mirror extensive axonal damage and indicate a rapid progression of disease SMI35 to be elevated in the CSF of patients with ALS as compared to all other groups (SMI35 which was prominent in patients with a rapid progression of disease suggests that low CSF concentrations of sAPP in ALS are linked to extensive neuro-axonal damage. Our data support the relevance of sAPPα and -ß as neuroprotective agents and provides in vivo evidence that a deficiency of cellular mechanisms protective against the formation of proteinaceous aggregates could be a determinant of disease progression in ALS. As both high CSF NfHSMI35 and low CSF sAPPα and -ß were associated with a rapid deterioration of motor function over follow-up, we determined a combined analysis of those markers. The ratios CSF NfHSMI35/CSF sAPPα and CSF NfHSMI35/CSF sAPPß were superior to NfHSMI35 and sAPPα,ß alone to delineate patients with a fast progression of disease and are therefore promising candidate biomarkers of disease progression for clinical trials of ALS. In analogy to what has been proposed for combining CSF tau and abeta levels in Alzheimer's disease we propose combining CSF NfH and sAPP in ALS. However, there was an overlap between the groups and the number of patients included was comparatively low, so the cut-offs determined here using ROC-analysis will have to be validated on a large cohort of patients with ALS. We propose this to be done in an unbiased, staged multicenter, validation strategy.We found an inverse correlation of both sAPPα and sAPPß levels with NfH the CSF . In a prr groups , with hiThough no significant difference of CSF or serum Progranulin between patients with ALS and controls was detectable, we observed a tendency of CSF Progranulin to decrease with ongoing disease. Progranulin mediates proteolytic cleavage of TDP-43 to generate ∼35 and ∼25 kDa species in vivo evidence that a deficiency of neuroprotective mechanisms involved in protein degradation and cleavage is linked to progressive neuro-axonal damage in ALS. On a clinical level, this study provides new CSF candidate markers associated with progression of disease in ALS.Our data provides indirect biomarker


Embryonic cerebrospinal fluid (e-CSF) has an important role in developmentof embryonic and adult brain. Proteomic analysis suggests that this fluid has many morphogenesand cytokines that alter in time and space throughout embryonic life. The aim ofthis study was to evaluate the developmental effect of embryonic CSF on proliferation anddifferentiation of neuroprogenitor cells in different gestational age.in vitro. Glial differentiation study was carried out by immunocytochemistry.Neurospheres size and percentage of glial fibrialy acidic protein (GFAP) positivecells were measured by image analyzer (image J). The data were analyzed by one-wayANOVA, followed by the Tukey’s post hoc test. Data were expressed as mean ± SEM, anddifferences were considered significant when p<0.05, 0.01 and 0.001.In this In this experimental study, we examined the role of e-CSF on proliferation and differentiation of neuroprogenitor cells using neurosphere culturemethod. Neurospheres size analysis and MTT assay were used to assess cell proliferationafter four days Viability and proliferation of neuro progenitor cells in cultures conditioned withE16 CSF and E18 CSF were significantly increased compare to control group. A dramaticdecrease in percentage of GFAP-positive cells was found following the application of CSFfrom E16 and E18 embryos, but not E20 CSF.Our data suggest that, e-CSF altered proliferation and differentiation of neuro progenitorcells in age dependent manner. E16 and E18 CSF enhanced proliferation and viability ofneuro progenitor cells, and inhibited differentiation to glial fate in comparison with control group. Neural stem cell expansion and cell fate determinationare regulated by two intrinsic and extrinsicfactors. Intrinsic factors include transcription factors,chromatin remodeling and micro RNAs. Theextrinsic factors include extracellular signalingmolecules, such as FGFs, WNTs, SHH and BMPs. The proIn this experimental study,Wistar rats, bred in house,were received from the Research Facility of the Facultyof Biological Sciences, Kharazmi University, followedby obtaining the ethical approval from the AnimalUses Committees of Kharazmi University. Theywere kept in large rat boxes at constant temperatureand a 12/12-hour light/dark cycle with free access tofood and water. Individual male and female rats werepaired in mating cages and checked regularly for thepresence of a vaginal plug, which was taken as an indicationof successful mating, and the day was notedas embryonic day 0 (E0). Embryonic age was calculatedfrom E0. At a particular time point, pregnantdams were euthanized by intra peritoneal injectionof an overdose of sodium pentobarbitone. After rapidremoval of the uterine, they were placed on ice, whilethe fetuses were dissected out. Each pregnant damusually produced between 10-15 fetusesCSF was collected from the cisterna magna of ratfetuses at E16, E18, and E20 using glass micropipettesand capillary action without aspiration. Aspirationinvariably resulted in bleeding and contaminationof the samples. Fetuses were positioned with headsflexed down onto the chest to allow penetration intothe cisternal cavity through the skin and underlyingmuscle. Samples containing undesirable blood contamination,visualized as a pink color in the fluid,caused by damaging a blood vessel within the cisternalcavity, were discarded. All samples were collectedinto sterile microtubes and centrifuged at 14,000 rpmto remove cells or debris from the fluid, and the supernatantwas transferred into another sterile tube. Thesesamples were stored at -80˚C until use. The volumeof CSF collected from each fetus by this method wasbetween 5 and 50 µl, and samples were then pooledfor each experiment.2  supplement, 1% penicillin/streptomycin, and 20 ng/mlepidermal growth factor (EGF) , 20 ng/ml basic fibroblast growth factor (b-FGF) . Cultures wereincubated at 37˚C in a humidified atmosphere and 5%CO2, 92% N2, and 3% 6 2% O2. Fresh medium andgrowth factors were supplemented every two daysNeurosphere were prepared as described previously. Brieflyin vitro, cells obtainedby neurosphere splitting were counted, seeded intopoly-L-lysine coated-surfaces dish , and grown as a monolayer in the samemedium without mitogens in order to allow them todifferentiate. After four days in vitro (DIV) cells wereprocessed by immunocytochemistry in order to performantigen expression and morphological analysis.The cultures were divided into following four studygroups: i.control: No CSF exposure, ii. E16: exposureto CSF of E16, iii. E18: exposure to CSF of E18and iv. E20: exposure to CSF of E20. For differentiationstudies, after four days Cell growth and viability were also studied by theMTT assay , a biochemical approach,which was based on the reduction of MTT into formazan crystals by the action of the mitochondrialde hydrogenase enzymes, were present inviable cells. The crystals formed were then dissolved inacidified isopropanol, giving a spectrophotometricallymeasurable purple solution. Neurospheres were treatedwith a solution of five mg/ml MTT. After two hours at37˚C , the formed formazan crystals were dissolved ina solution consisting of 10% Triton X-100/0.1N HCl/isopropanol, then incubated for one hour at RT in thedark. Absorbance was read at a wavelength of 570 nm.All experiments were carried out in duplicates.Cells were fixed for 20 minutes in 4% paraformaldehydecontaining phosphate-buffered saline (PBS)(pH=7.4), washed in PBS and permeabilized for fiveminutes with PBS/0.5% Triton X-100 .Adherent single cells were incubated overnightat 4˚C in PBS containing 5% bovine serum albumin(BSA) and the appropriate mixture of antibodies. Primaryantibody used was rabbit polyclonal anti-GFAP for astrocytes. After washingin PBS, differentiating cells obtained from spillitedneurosphere were incubated for one hour with Cy3-conjugated secondary antibodies .Nuclei were counterstained with propidiom iodate.After four DIV in proliferation condition, digitalimages of the neurospheres cultures were taken usingan inverted microscope . Themagnification of the image (×10) covered a significantarea of each well from 24 well plates. An imageanalysis program (image J) was used to analyze thesize of neurospheres. After four DIV in differentiationcondition, ten non-overlaping fields were randomlyselected from each well, and images were capturedusing a fluorescence microscopy . Randomly chosen field were counted, andpercentage of GFAP-positive cells was determined.All experiments were carried out in duplicates.Data are presented as the mean ± standard error ofthe mean (SEM). Statistical analysis was performedusing the one-way ANOVA and Tukey’s post hoc test,and significance was accepted for p values of <0.05.In this study, we examined the effect of e-CSFon proliferation and differentiation of neuroprogenitorcells. Increasing in the size of neurospherewas considered as a sign of increasing in proliferationrate of neuroprogenitor cells. As shownin figures 1 and 2, significant increase in the sizeof neurosphere were detected in groups treatedwith CSF from E16  andE18  in comparison withcontrol (109.26 ± 4.26). No obvious differencesbetween culture treated with CSF from E20 andcontrol were observed. Our results showed addingCSF to culture medium caused differential effectson growth characteristics and morphologyof neuroprogenitor cells. The media was immediatelytreated with 10% of CSF after cell seedingestablished sphere forming characteristic. WhenCSF was added to medium in high ratio, the neuroprogenitorcells showed adherent characteristicand began to differentiate .Measurement of neurosphere size in differentcultures condition (in presence and absence of e-CSF) provided interesting results. E16 Figs ,2 and ETo examine the effects of e-CSF on the survival andproliferation of neurospheres, MTT assay was usedto quantify cell proliferation and viability. It is notedthat in this technique, the MTT conversion relies onthe ability of the viable cells to reduce a water solubleyellow dye to a water-insoluble purple formazanproduct. Each absorbance value of MTT reduction activitywas normalized by the results of control group.The MTT conversion of neurospheres in the presenceof e-CSF from E16 and E18 was significantly higherthan control group p<0.05, These results suggest that e-CSF may play animportant role in regulating the differentiation ofneural progenitor cells. We examined whether theaddition of e-CSF, isolated from different embryonicage, could modulate the number of differentiatedneuroprogenitors. Upon differentiation ofneuroprogenitor cells through exposure to serumon a poly-L-lysine coated dish, we found out thataddition of E16 and E18 CSF strongly decreaseddifferentiation of adherent neuroprogenitor cells toglial fate. E16  and E18 CSF-treated media wereshown to cause significant decrease in percentageof GFAP-positive cells in comparison with controlgroup (77.16 ± 2.10). It was not increased overcontrols in CSF from E20 (79.01 ± 1.11, Fig in vitro on neuroprogenitor cells behaviorusing rat embryos of different gestational ages. Itseems that e-CSF neurotrophic component modulatesproliferation and differentiation of neuroprogenitorcells in age dependent manner.We have investigated the regulatory effect ofCSF in vitro studies have showed that thiscomponent enhances proliferation of embryonicneuroprogenitor cells (This study demonstrated e-CSF involvementin the proliferation of neural progenitor cells.e-CSF enhances proliferation of neuroprogenitorsafter placing them in non-adhesive culturedish, also it significantly increases neurospheresize compared to control group. It has beenreported previously that e-CSF has mitogeniceffect on neural progenitor cells isolated fromthe embryonic brain of chicken and rat withdifferent culture method -15. In tor cells , 19. Alsor cells . Since sor cells .in vivostudies that astrocytes are immuno-reactive toGFAP, first detected at E16 (in vivo findingsof Liu et al. (The data suggested that e-CSF modulates differentiationof neuroprogenitor cells to GFAPpositive cells in age dependent manner, whileit decreases during early rat embryonic life atE16 to E20. It has been supported by d at E16 . Recent d at E16 . The evid at E16 . Our resu et al.  about GFu et al.  have demu et al. . The abnu et al. . Also, ru et al. .in vivoand in vitro. Thus, further studies seem necessaryto investigate CSF components and synergistic effectsof them.Our result demonstrated that e-CSF altered proliferationand differentiation of neuroprogenitorcells during embryonic period. E16 and E18 CSFenhanced neurospheres sizes as sign of proliferation,and inhibited differentiation to glial fate incomparison with control groups. Since CSF proteomiccomposition was altered throughout braindevelopmental processes and in neurodegenerativestate, modulation of CSF components couldprovide a complementary therapeutic method toregulate the neuroprogenitor cells behavior