A 2-week intracerebroventricular (i.c.v.) infusion of CSF altered home-cage behavior and induced olfactory dysfunction, excessive immobility in the forced swim test, and perseveration in a learning task. Conversely, sustained administration of purified BRAs produced relatively mild, both inhibitory and stimulatory effects on olfaction, spatial learning/memory, and home-cage behavior. In vitro studies revealed that administration of some CSF samples induces a rapid influx of extracellular Ca2+ into murine neurons, an effect that could be partially mimicked with the commercial anti-NR2A antibody and blocked with selective N-methyl-D-aspartate (NMDA) receptor antagonists. The current findings confirm that the CSF from CNS SLE patients can be neuroactive and support the hypothesis that intrathecal BRAs induce synergistically diverse effects on all domains of behavior. In addition, anti-NMDA receptor antibodies may alter Ca2+ homeostasis of central neurons, thus accounting for excitotoxicity and contributing to the heterogeneity of psychiatric manifestations in CNS SLE and other autoantibody-related brain disorders.Systemic lupus erythematosus (SLE) is a potentially fatal autoimmune disease that is often accompanied by brain atrophy and diverse neuropsychiatric manifestations of unknown origin. More recently, it was observed that cerebrospinal fluid (CSF) from patients and lupus-prone mice can be neurotoxic and that acute administration of specific brain-reactive autoantibodies (BRAs) can induce deficits in isolated behavioral tasks. Given the chronic and complex nature of CNS SLE, the current study examines broad behavioral performance and neuronal CaThe online version of this article (10.1186/s40478-017-0473-1) contains supplementary material, which is available to authorized users. Systemic lupus erythematosus (SLE) is a chronic autoimmune/inflammatory disorder accompanied by damage to multiple organs, including the brain . NeuroloFor many years, autoantibodies reactive to diverse brain antigens  have been attracting considerable attention as terminal factors mediating brain damage and behavioral manifestations in neurological malignancies, seizures, movement disorders, ischemic syndromes, as well as autoimmune diseases including multiple sclerosis and CNS SLE , 25. How2+) from internal stores. Taken together, the results obtained from these studies suggested that antibodies in the CSF bind antigen(s) that are not only shared between immature and differentiated neurons but also conserved amongst mammalian species.More direct evidence supporting a causative role for CSF BRAs stems from experimental studies in murine forms of lupus-like disease . Led in N-methyl-D-aspartate (NMDA) receptor i) transients, induced by diluted CSF samples or commercially available antibodies, are presented as the mean value of normalized fluorescence intensity ± SEM, with n being the number of regions of interests (ROIs). On average, the number of ROIs per frame was 31 ± 3 (mean ± SEM). In pharmacological experiments, amplitudes from the same ROIs were compared before and after drug treatment and analyzed on normalized data using paired t-test. Summary histograms indicate mean of peak amplitudes ± SEM with significant differences of p < .001 shown as *. The peaks of the fast and slow component of calcium transients of two effective CSF samples and two effective autoantibodies were compared using Kruskal-Wallis One-Way ANOVA on Ranks with post-hoc Dunn’s Method.Raw data were analyzed using SigmaPlot , with the criterion for statistical significance set at 14 = 2.529, p < .05; Day 5: t14 = 2.841, p < .05; Day 6: t14 = 2.724, p < .05; Fig. 1,14 = 9.909, p < .01, Fig. 1,13 = 28.044, p < .001, Fig. 1,14 = 28.722, p < .001, Fig. 1,14 = 12.672, p < .01) and increased time in the shelter .As expected, a significant drop in postoperative body weight (taken as a percentage of body weight at surgery) was observed in both groups, likely due to the stress induced by general anesthesia and pain. However, mice receiving CSF from lupus patients exhibited a more pronounced reduction in body weight  or number of beam slips  were noted. Consistent with the results from the beam-walking task, latency to fall from the Rotarod was comparable among mice receiving CNS SLE CSF and those treated with NMO CSF . Taken together, these results suggest post-surgical behavioral performance was not confounded by deficits in balance, motor coordination, or muscle strength. However, mice assigned to receive CNS SLE CSF spent significantly more time investigating cinnamon in their first trial  and similar exploration of a paprika-laced filter paper in the dishabituation trial . When exposed to the same paradigm following mini-pump implantation and CSF infusion, no group differences were noted during cinnamon habituation, although an overall drop in mean sniffing duration was evident. Importantly, mice treated with lupus CSF spent significantly less time investigating the dishabituation odor in comparison to control animals . Similarly, when exposed to the novel object test, groups did not differ in overall locomotion, the topography of movement and exploration of the object (data not shown). They also performed similarly in the open field in terms of distance moved, velocity and time spent in the center and thigmotaxic zones (data not shown). In contrast to dry-land paradigms probing anxiety-like behavior, administration of CNS SLE CSF led to a significant reduction in swimming distance . However, infusion of control CSF from an NMO patient led to a significant reduction in spontaneous alternation rate post-surgery  or the hidden platform over the next 4 days . However, control mice displayed longer swim paths to locate the platform over the same acquisition trials . These results suggest that anti-NMDA receptor treatment alters responsiveness to previously-exposed scents, without necessarily affecting habituation or discriminatory capacity.Behavioral performance between mice receiving anti-NR2A antibodies and controls treated with aCSF was comparable in home-cage setting and in paradigms reflecting neurological function, locomotion, motivated behaviors, and learning/memory (data not shown). Task-specific differences, however, were evident in the olfactory tasks and the MWM. Namely, mice treated with anti-NR2A antibodies spent significantly less time sniffing cinnamon, during the four habituation exposures, compared to mice that were administered aCSF only  that consistently failed to find the platform were excluded from MWM analyses. Performance in the cued and acquisition phases of the test was comparable between groups (data not shown). When the task was made more complex in reversal trials, mice that received the anti-NR2A antibodies exhibited shorter path lengths to locate the newly-positioned hidden platform  and sucrose preference , mice treated with anti-RPLP0 antibodies displayed alterations in water intake , allowing us to evaluate the changes in [Ca2+]I by normalizing the change in fluorescent intensity to the resting level (ΔF/F0) in the dynamic range of the acquisition.Analysis consisted of extracting average fluorescence intensities from ROIs that were drawn around the neuronal bodies. Hippocampal cultures at 7-10 DIV exhibited typical pyramidal neuronal bodies and many interconnected branches forming a network Fig. . Astrocy2+ homeostasis in cultured hippocampal neurons. Two of the four CSF samples from CNS SLE patients induced transient changes in [Ca2+]i. Ten times diluted CSF from patient #4 increased the [Ca2+]i above the dynamical range of the acquisition, so further tests were done with higher dilutions. At a dilution of 1:25, this sample induced two types of responses: a fast [Ca2+]i transient  and a late, slow component that was seen in some neurons , to 1.23 ± 0.08 . Moreover, the effect of CNS SLE CSF #4 was repeatable, as the second stimulation, applied 100 s after the first stimulus (both in 1:50 dilution), induced the [Ca2+]i transient of the same shape and amplitude  and exhibited only one type of response with fast [Ca2+]i transient  at 0 s time, washed around 5 min, and then challenged with K+ solution at the 8-min mark. (WMV 11519 kb)2+]i transients, further experiments were conducted in Ca2+-free external solution. External calcium was proven to be the major source of intracellular transients, as Ca2+-free external solution substantially reduced the effect of 1:25 CNS SLE CSF #4 to 0.32 ± 0.04  reduced the amplitude of the control response from 3.39 ± 0.21 to 2.54 ± 0.15 , suggesting that voltage-gated receptors take part in CNS SLE CSF-induced calcium transients  further reduced the amplitude to 1.97 ± 0.13, and this reduction in amplitude was significant vs. both control response , and VGCCblock  implying a role for ionotropic receptors  and both ionotropic and voltage-gated receptors (VGCCblock + GluRinh). Finally, to distinguish between AMPA/kainate and NMDA receptor involvement, 200 μM DL-AP5 (NMDA receptor blocker in higher concentration) was applied. This blockage completely abolished the control response in every tested ROI  at the end of experiments. The averaged shape of 1:10 CNS SLE CSF #2 calcium response exhibited only a fast transient that peaked 3 s after CSF application, with an amplitude of 1.28 ± 0.18  that peaked at 6 s, showed a slow component (0.46 ± 0.03) 3 min after and remained even after wash  3 min after application, that also could not be washed . On the other hand, all pairwise comparisons of the slow components among treatments  revealed a significant difference only between the two commercial autoantibodies . Neither anti-α-tubulin nor albumin induced any perturbations in [Ca2+]i levels (data not shown). Taken together, the results obtained suggest that the effects of CNS SLE CSF are heterogeneous and complex, and cannot be mimicked by an individual antibody.To narrow down immunoglobulins accounting for neuroactive effects of autoimmune CSF, we assessed Caash Fig. . Anti-RPhed Fig. . Pairwis2+ influx, which in one case seemed to be mediated via the NMDA receptor system. However, anti-NR2A and anti-RPLP0 antibodies could not induce [Ca2+]i responses comparable to the effects of patient CSF. Although the importance of other BRAs is not excluded, the present results suggest that the synergistic effect of multiple BRAs underlies altered neuronal metabolism and behavioral dysfunction in CNS SLE.Given the chronic and complex nature of CNS SLE, the current study examines in vivo and in vitro effects of sustained i.c.v. infusion of human CSF samples and putative autoantibodies on broad behavioral performance and neuronal calcium signaling. Significant differences were found in several behavioral domains, but the sustained administration of putative BRAs could not recapitulate the broad spectrum of behavioral manifestations detectable in the spontaneous murine MRL model of lupus, or in healthy mice treated with undiluted CNS SLE CSF. Moreover, we showed that CSF from two out of four lupus patients induced neuronal activation through CaThe results obtained with CNS SLE CSF are largely consistent with previous reports detailing CSF neurotoxicity in human and murine forms of lupus , 78, 92.2+ store channels. By employing a more specific Ca2+-sensitive probe, the results herein confirm the induction of intracellular ionic misbalance by CNS SLE CSF with a clear role of Ca2+ signaling in this process. Contrary to our previous study, the CSF effect in the present study was fully dependent on extracellular Ca2+. This discrepancy may be attributable to the different neuronal cell types employed , as well as to the application of CSF from different patients. Other groups have also tested the effects of serum BRA on [Ca2+]i homeostasis, including ARPA in primary cortical neurons i rise, which was not observed in previous studies i could be induced with a 1:50 dilution. However, these in vitro experiments also demonstrate significant heterogeneity, which is typical of the overall CNS SLE symptomatology . The effect was shown to be predominantly attributed to postsynaptic NMDA receptors, as it could be completely abolished by DL-AP5. Nevertheless, the effect of VGCC cannot be completely excluded via Ca2+-dependent presynaptic release or by removing voltage-dependent Mg block from NMDA receptor. One may hypothesize that the effect of CNS SLE CSF is mediated directly by IgG Fab’2 fragments, unrelated to complement activation, as shown for DNA/NMDA receptor-reactive antibodies i transient amplitude. Using only the NMDA receptor inhibitor DL-AP5 could completely abolish the response to CNS SLE CSF, albeit at a rather high concentration . Our cell cultures also contained astrocytes, which may interact with neurons and their Ca2+ signaling via gliotransmission i. However, the concentration used (100 μg/ml) was above the maximal IgG titer in the tested undiluted CNS SLE CSF sample  reaction, while the response of neurons to diluted CSF may be less specific, yet well defined. On the other hand, the IgG fraction isolated from sera of CNS SLE patients could also induce a similar, well-defined [Ca2+]i responses in rat neuronal cultures (ms in preparation), reminiscent of the effect of whole CSF herein. In addition, the effect of BRA could become apparent through synergy with glutamate release and action as in the case of NMDA receptor-reactive antibodies in lupus [2+]i response phase remains to be elucidated.It is noteworthy that the in vitro tested commercially available antibodies against NR2A or RPLP0 had a slight  effect on neuronal I, but seemingly impaired performance in spatial learning and memory tasks. NMO is an inflammatory demyelinating disorder of the CNS that is primarily characterized by the presence of autoantibodies to AQP4 in serum [2+. This would support the hypothesis that NMO-specific IgG acts through a mechanism distinct from a [Ca2+]i signaling pathway [The interpretation of the present findings is complicated by the observation that CSF from an NMO patient had no effect on [Cain serum  and CSF in serum . The tarin serum . Passivein serum , 11, 88.in serum . One mayin serum , a more in serum . Neverth pathway , 109 tha pathway , 89. In  pathway  and targ pathway , autoimm pathway , autism  pathway , 17, and pathway .2+ into murine neurons via the glutamatergic system.The sustained infusion of autoantibody-rich cerebrospinal fluid from CNS SLE patients into the brains of healthy animals induces alterations in home-cage behavior, olfactory dysfunction, depressive-like behavior and perseveration in a learning task. The administration of putative BRAs in a similar manner produces relatively mild, both inhibitory and stimulatory effects on olfaction, spatial learning/memory, and home-cage behavior. In vitro studies reveal that some CSF samples induce a rapid influx of extracellular Ca


Sc). In the studies conducted to date in CJD, cerebrospinal fluid (CSF) RT-QuIC showed good diagnostic sensitivity (82–96%) and virtually full specificity. In the present study, we investigated the diagnostic value of both prion RT-QuIC and surrogate protein markers in a large patient population with suspected CJD and then evaluated the influence on CSF findings of the CJD type, and the associated amyloid-β (Aβ) and tau neuropathology. RT-QuIC showed an overall diagnostic sensitivity of 82.1% and a specificity of 99.4%. However, sensitivity was lower in CJD types linked to abnormal prion protein (PrPSc) type 2  than in typical CJD (MM1). Among surrogate proteins markers -tau, and t-tau/phosphorylated (p)-tau ratio) t-tau performed best in terms of both specificity and sensitivity for all sCJD types. Sporadic CJD VV2 and MV2K types demonstrated higher CSF levels of p-tau when compared to other sCJD types and this positively correlated with the amount of tiny tau deposits in brain areas showing spongiform change. CJD patients showed moderately reduced median Aβ42 CSF levels, with 38% of cases having significantly decreased protein levels in the absence of Aβ brain deposits. Our results: (1) support the use of both RT-QuIC and t-tau assays as first line laboratory investigations for the clinical diagnosis of CJD; (2) demonstrate a secondary tauopathy in CJD subtypes VV2 and MV2K, correlating with increased p-tau levels in the CSF and (3) provide novel insight into the issue of the accuracy of CSF p-tau and Aβ42 as markers of brain tauopathy and β-amyloidosis.The differential diagnosis of Creutzfeldt-Jakob disease (CJD) from other, sometimes treatable, neurological disorders is challenging, owing to the wide phenotypic heterogeneity of the disease. Real-time quaking-induced prion conversion (RT-QuIC) is a novel ultrasensitive in vitro assay, which, at variance with surrogate neurodegenerative biomarker assays, specifically targets the pathological prion protein (PrPThe online version of this article (doi:10.1007/s00401-017-1683-0) contains supplementary material, which is available to authorized users. PRNP)  are also consistent with this finding. Thus, as with CSF protein surrogate markers, the in vitro conversion reaction exploited by RT-QuiC is significantly affected by the neurobiological heterogeneity of human prions.Our results confirm the very high specificity of RT-QuIC for the diagnosis of sCJD in a large series of CJD and non-CJD CSF samples. However, they also provide, for the first time, convincing evidence for a lower sensitivity of the assay in atypical subtypes of the disease characterised by PrPRecent studies using a different recombinant PrP substrate or olfactory mucosa brushings instead of CSF as an alternative seed reported a 96–97% sensitivity of the RT-QuIC assay for the diagnosis of sCJD , 46. HowSc levels. Nevertheless, our observation of a higher conversion activity of the samples from gCJD patients carrying the E200K-129M haplotype (and PrPSc type 1), in comparison to those of the sCJD MM(V)1 group, which is confirmatory of the results of a previous study [Sc in seeding rec-PrP conversion and aggregation.The reasons behind the variability in seeding activity among CSF samples from CJD patients remain, unfortunately, partially unclear. Indeed, besides the above-mentioned correlation with the disease subtype, we failed to reveal other common features among the CSF samples giving a negative RT-QuIC result. In particular, there was no significant correlation with age at onset, disease duration at the time of lumbar puncture, or with the results of both conventional protein assays and brain MRI. Future studies should further address this issue and establish, in the first instance, whether seeding activity correlates or not with PrPus study , supportRegarding the more conventional CSF surrogate diagnostic markers, such as proteins t-tau and 14-3-3, our data demonstrate that t-tau is (moderately) superior to 14-3-3 in the diagnosis of sCJD. More specifically, t-tau yields a lower number of false positive results, especially in cases suffering from inflammatory-related conditions and subacute dementias; moreover, it has a higher sensitivity than 14-3-3 for the sCJD MV2K type. These results, which are in line with those reported by Hamlin et al. , togethePRNP codon 129 genotype, one of the two major molecular determinants of the disease type, can be obtained from a blood sample, it is crucial to include codon 129 genotyping in the diagnostic work-up of patients with suspected CJD. This information, combined with those obtained from the patient’s clinical history and neurological examination, which is usually able to discriminate between a typical (MM(V)1) or atypical  disease subtype are crucial for the correct interpretation of the results of CSF assays. The most striking example of the importance of codon 129 genotyping for the clinical diagnosis of CJD concerns the VV2 subtype, the second most common sCJD type [Our results further underline the profound implications that the phenotypic heterogeneity of the disease has on diagnostic criteria and the overall clinical approach to a patient suspected of having CJD. Indeed, as also clearly revealed by our study, the sensitivity of both surrogate protein markers and RT-QuIC varies significantly according to the CJD type. Given that knowledge about the patient’s CJD type . Given tSc deposition also develops in classic CJD, especially in the VV2 or MV2K subtypes. Initial studies were either negative [Sc burden in the neocortex, is consistently found in sCJD brains, irrespective of the molecular subtypes and the AD-related associated pathology. Consistently, in a recent study by Kovacs et al. [Sc burden than those of the MM(V)1 type [One of the major issues in the field of diagnostic biomarkers concerns their correlation with the underlying brain pathology. Besides the analyses of the effect of CJD subtypes on all biomarkers analysed, in this study we have correlated neuropathological and CSF findings for both p-tau and Aβ. Our findings of increased CSF p-tau concentrations in sCJD types VV2 and MV2K, (both caused by CJD strain V2), is of particular significance in this respect. Indeed, although the CSF tau profile in CJD patients usually combines excessively high t-tau with relatively low p-tau concentrations , 67, 68,negative . More renegative  demonstrs et al.  these sms et al. . OverallV)1 type  somehow V)1 type  and ReinV)1 type . MoreoveV)1 type .APOE genotype or the number of Aβ-positive plaques in the brain [APOE-4 on the decrease in Aβ42 in the CSF of sCJD patients. Furthermore, other preliminary data suggest that Aβ42 levels in the CSF are significantly higher in sCJD than in AD. In the present study we definitely show, in a large group of sCJD cases, that (1) the CSF levels of Aβ42 are significantly higher in sCJD than in AD and that (2) about 60% of sCJD cases have a concentration of Aβ42 in the CSF within the range of controls. Nevertheless, our analyses of Aβ load in neuropathologically verified patients clearly shows that the decrease in CSF Aβ42 in CJD patients, although influenced by the co-morbid AD pathology, may occur irrespective of the plaque load. Thus, the more likely explanation for the reduced Aβ42 levels in a subgroup of sCJD patients seems to be a combined effect of age-related co-morbidity, influenced by the APOE genotype, and the effect of neuronal loss which can be very significant in CJD even a few months after clinical onset. An alternative explanation, which has been put forward in previous studies [Aβ42 concentration in the CSF of sCJD patients has been measured in a number of studies, but with inconsistent results , 75, 77.he brain , 47, 71.he brain  demonstrOur findings have also implications for the issue of CSF biomarkers reliability in AD. Indeed, at variance with most neurodegenerative disorders including AD, brains affected by CJD and other rapidly progressive dementias provide the unique opportunity to correlate the CSF findings with the post-mortem neuropathology within a short time interval. Although our data confirm an overall positive correlation between p-tau levels in the CSF and the extent of brain tauopathy and an inverse correlation between Aβ42 levels and β-amyloid load, it must be emphasized that the correlation for Aβ42 and β-amyloid is rather gross. Of most significance in this respect, also given the “negative” effect of CJD on Aβ42 concentration in the CSF, is our finding of Aβ42 levels within the control range in 68% of CJD brains with a co-occurring significant Aβ deposition in the brain . If confirmed in brains with other pathologies, this finding would suggest that the Aβ42 level in the CSF is far from being an optimal marker of the early phases of Aβ deposition in the brain in pre-clinical AD. Given that our data are limited to Aβ42, it will be important to expand the correlative analysis to other Aβ peptides such as Aβ40. Indeed, evidence suggests a better diagnostic performance of the Aβ42/40 CSF concentration ratio compared to the Aβ42 concentration alone , 76. FurAt variance with Aβ42, according to our data, CSF p-tau level represents a more reliable marker of brain AD-related tau pathology since it fails to consistently discriminate between patients with or without neurofibrillary pathology only when p-tau deposition is relatively mild and limited to the transentorhinal/entorhinal cortices (Braak stages I–II).In summary, our study provides confirmatory and novel evidence for a significantly improved value of CSF biomarkers for the clinical diagnosis of CJD. Although RT-QuIC is the most promising assay, given its high specificity, t-tau remains the most important surrogate marker. Our data support a change in the current diagnostic criteria for CJD, indicating both RT-QuIC and t-tau as primary laboratory investigations to be implemented in suspected cases of CJD, in combination, when possible, with codon 129 genotyping. In cases of a negative RT-QuIC result and elevated t-tau in a patient with rapidly progressive cognitive impairment, the differential diagnosis between CJD and atypical AD is also supported by the combined analyses of CSF p-tau, Aβ42 and possibly total-PrP . FinallyDespite the significant advances, the diagnostic value of both CSF biomarkers and cerebral MRI remain very low in some rare atypical variants of sporadic prion disease, such as sporadic fatal insomnia (MM2T) and VPSPr. Efforts should continue in the attempt to recognise such atypical forms through disease surveillance and the neuropathological examination of suspected cases. Future studies should also aim to develop a type-specific RT-QuIC or other assay to discriminate the molecular subtypes of sCJD cases in vitam to bring the epidemiological surveillance of sCJD to the next level.Supplementary material 1 (DOCX 42 kb)Below is the link to the electronic supplementary material.


Optical coherence tomography (OCT) has been the imaging technique for detection of scaffold malapposition during implantation, the method of choice at follow up for assessing strut resorption and measurement of cap thickness overlying atherosclerotic plaques . BioresoCoronary artery calcification is a risk factor for coronary adverse events . DespiteBioresorbable scaffolds may be a treatment option for calcified plaques by inducing a neointimal cap at follow up. The advantage of biodegradable device compared to the metallic stent is temporary vessel scaffolding which reduces the inflammatory response in treated segments at follow up. Reduced inflammation with re-capping may decrease the vulnerability risk of the plaques.


Macrophages play a central role in the pathogenesis of inflammatory and fibrotic lung diseases. However, alveolar macrophages (AM) are poorly available in humans to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL). In this study, to identify the best alternative in vitro model for human AM, we compared the phenotype of AM obtained from BAL of patients suffering from three lung diseases -associated interstitial lung disease) to human blood monocyte-derived macrophages (MDMs) differentiated with M-CSF or GM-CSF. The expression of eight membrane markers was evaluated by flow cytometry. Globally, AM phenotype was closer to GM-CSF MDMs. However, the expression levels of CD163, CD169, CD204, CD64 and CD36 were significantly higher in SSc-ILD than in lung cancers. Considering the expression of CD204 and CD36, the phenotype of SSc-AM was closer to MDMs, from healthy donors or SSc patients, differentiated by M-CSF rather than GM-CSF. The comparative secretion of IL-6 by SSc-MDMs and SSc-AM is concordant with these phenotypic considerations. Altogether, these results support the M-CSF MDM model as a relevant in vitro alternative to simulate AM in fibrotic disorders such as SSc. Macrophages constitute a heterogeneous population of mononuclear cells. They can indeed adopt various degrees of polarization with distinct functional and phenotypic properties in vivo and in vitro . DependiPulmonary macrophages are poorly available in human to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL) and to an even more limited access to interstitial macrophages . The curThis study aims: a/to further characterize the phenotypic properties of GM-CSF/M-CSF MDMs and b/to confront these characteristics to those of AM obtained from BAL of patients suffering from various inflammatory and/or fibrotic disorders with pulmonary involvement, i.e., lung cancers, sarcoidosis and SSc.n = 16 patients, with lung neoplasia (n = 6), sarcoidosis (n = 5) and SSc-ILD (n = 5)), plated for 24 h after BAL fluid recovery, was compared to MDMs from healthy blood donors, differentiated in the presence of GM-CSF (GM-MDMs) or M-CSF (M-MDMs) for 6 days. Two panels of four membrane markers were used to characterize these cells. Flow cytometry graphs are shown in The phenotype of all AM, regardless of their origin  lung cancers (Neo group), (b) sarcoidosis (Sarco group) or (c) SSc-ILD (SSc group). Flow cytometry graphs are shown in neg and CD169neg. By contrast, M-MDM CD206+ cells co-expressed both CD163 and CD169  or of M-CSF (M-SSc). The In order to support our data on the phenotype in SSc-ILD, we quantified the secretion levels of IL-6, a pro-inflammatory cytokine, and CCL18, a chemokine found in pro-fibrotic lung tissue. IL-6 secretion was significantly higher in GM-SSc than M-SSc and than GM-MDMs. By contrast, IL-6 levels of AM-SSc were similar to those of M-MDM or M-SSc . There wPulmonary macrophages are poorly available in human to perform in vitro studies due to a limited access to broncho-alveolar lavage (BAL) and to an even more limited access to IM. This statement is especially true considering rare systemic inflammatory and fibrotic lung disorders in which macrophages could play a key pathogenic role ,7,9. MorWe did not obtain BAL samples from healthy subjects in this study, however, AM of patients suffering from lung cancers were obtained from the alveolar lumen and were thus not specifically in contact or inside the tumor tissues, as the collection of macrophages inside the tumor would imply obtaining direct histopathological samples of the cancer. Therefore, AM from these patients could be considered closer to healthy AM rather than authentic tumor associated macrophages (TAMs).Recent insights concerning macrophages in inflammatory and fibrotic conditions have shed light on the necessity of considering macrophage polarization in a given micro-environment, influenced both by the considered organ but also by the disorder affecting this organ ,17. MartIn our work, the proportion of AM expressing CD204 of patients suffering from SSc-ILD is significantly enhanced, in comparison with AM of patients suffering from lung cancer. The expression of CD204 (scavenger receptor I Class A), in AM from SSc-ILD and M-CSF MDMs is an important feature since CD204 seems to play an important role in the pathogenesis of lung fibrosis . CD204, In our study, AM of patients suffering from SSc-ILD, in comparison with AM from lung cancers, also had a significantly enhanced expression of the M2 polarization marker CD206 and a higher percentage of positive cells for CD169 and CD163. In the literature, skin macrophages of SSc patients with skin fibrosis also have a higher expression of CD169 and CD163 . CirculaTo support these phenotypic considerations on AM in SSc-ILD, we compared the secretion of the pro-inflammatory cytokine IL-6 and the pro-fibrotic chemokine CCL18 by MDMs and SSc-AM. We have chosen these secreted markers as they are both respectively considered highly representative of the inflammatory and fibrotic processes involved in the pathogenesis of SSc ,32,39,40By illustrating that M-CSF MDMs share some phenotypic features with human lung AM from fibrotic disorders such as SSc, our work reinforces the vision of a wide and heterogeneous functional range of activated macrophages, not only depending on the organ of interest but also on the pathological disorder at stake ,2,13,33.v/v) of heat-inactivated fetal bovine serum  and antibiotics. At day 6, culture media were replaced with a fresh medium with 5% of FBS. At day 7, conditioned media were removed and stocked at −20 °C for ELISA analysis whereas the cells were washed and harvested for flow cytometry analysis.Peripheral blood mononuclear cells were obtained from blood buffy coats from healthy donors  through Ficoll gradient centrifugation. Monocytes, selected after 1-h adhesion step, were differentiated into macrophages for 6 days using GM-CSF (400 IU/mL)  or M-CSF (50 ng/mL) from Miltenyi Biotec SAS  in RPMI 1640 medium GlutaMAX  supplemented with 10%  Good Clinical Practice (GCP) guideline (1997) and approved by the Ethic Committees of Rennes University Hospital  and by the “Commission nationale de l’informatique et des libertés (CNIL)” N° 1998854 (17 October 2016). Patients were recruited by the Pulmonology department of Rennes University Hospital and all of them gave written informed consent. The clinical features of patients are reported in 6 cells/cm2. After an adhesion step of 3 h, the medium was replaced with 5% of FBS. The next day, conditioned media were removed and stocked at −20 °C for ELISA analysis whereas the cells were washed and harvested for flow cytometry analysis.BALs were performed under local anesthesia with 2% lidocaine. BALs were obtained from 6 patients suffering from lung cancers (Neo), from 5 sarcoidosis (Sarco) patients and from 5 patients with systemic sclerosis (SSc). BAL collecting was performed in accordance with the standard procedures using a fiberoptic bronchoscope. About 100 mL of sterile 0.9% saline was instilled in the bronchial and alveolar lumen, and then, aliquoted and recovered into sterile propylene tubes. The average volume obtained for this study by our lab was about 10 mL. BALs were then centrifugated at 450 rcf for 5 min, washed once with 5 mL of PBS 1× and suspended in RPMI 1640 Glutamax containing 10% FCS and antibiotics and plated at 0.14 × 10TM , in order to maintain expression of M2 membrane marker [Phenotypic analysis of monocytes-derived macrophages and of macrophages from BALs was performed using flow cytometric direct immunofluorescence. Cell detachment was performed using Accutasee marker . A firstLevels of IL-6 and CCL18 secreted in culture media were quantified by ELISA using specific Duoset ELISA development system kits .t-test or One-way analysis of variance (ANOVA) followed by Newman–Keuls’ post-hoc test were applied for statistical analysis and p-values < 0.05 were considered significant.Data were analyzed using GraphPad prism software . Student’s


Crustal components may be incorporated into continental basalts by either shallow contamination or deep mixing. While the former proceeds at crustal depths with common preservation of refractory minerals, the latter occurs at mantle depths with rare survival of relict minerals. Discrimination between the two mechanisms has great bearing to subcontinental mantle geochemistry. Here we report the occurrence of relict zircons in Cenozoic continental basalts from eastern China. A combined study of zircon U-Pb ages and geochemistry indicates that detrital zircons were carried by terrigenous sediments into a subcontinental subduction zone, where the zircon were transferred by fluids into the magma sources of continental basalts. The basalts were sampled from three petrotectonic units with distinct differences in their magmatic and metamorphic ages, making the crustal contamination discernible. The terrigenous sediments were carried by the subducting oceanic crust into the asthenospheric mantle, producing both soluble and insoluble materials at the slab-mantle interface. These materials were served as metasomatic agents to react with the overlying mantle wedge peridotite, generating a kind of ultramafic metasomatites that contain the relict zircons. Therefore, the occurrence of relict zircons in continental basalts indicates that this refractory mineral can survive extreme temperature-pressure conditions in the asthenospheric mantle. The primary zircon of magmatic and peritectic origins is common not only in magmatic rocks of felsic composition but also in some mafic intrusives such as gabbro and pyroxenite. However, zircon rarely occurs in mafic volcanics such as basalts because the crystallization of zircon from silicate melts requires the oversaturation of both ZrO2 and SiO24. This is not justified in basaltic melts that were derived from partial melting of the depleted MORB mantle, the ordinary asthenospheric mantle. Nevertheless, zircon megacrysts with U-Pb ages consistent with those of host rocks do occur in some alkali basalts. There are different interpretations for their origin, including xenocrysts from metasomatized lithospheric mantle5, and crystallized from the primitive magma of host basalts6, fractionated alkali basaltic magma8, or felsic melts derived from incipient melting of amphibole-rich mantle domains9. In addition, some basalts contain zircons with variably older U-Pb ages, which were interpreted as xenocrysts captured from metasomatic lithospheric mantle10 or felsic wallrocks11 during ascent of basaltic magmas.Zircon is a refractory mineral in crustal rocks. Because of its chemically resistant property, zircon can survive from high-grade metamorphism and even partial melting13. Such basalts were derived from partial melting of ultramafic metasomatites in the ordinary asthenospheric mantle. This involves both chemical and physical transports of crustal materials from the subducting slab to the mantle, which is illustrated by the occurrence of relict zircons in Cenozoic continental basalts from east-central China. As such, survival of the detrital zircons becomes possible not only during partial melting of the subducting crustal rocks at the slab-mantle interface in the subcontinental subduction channel but also during partial melting of the metasomatic mantle domains above the subducted oceanic slab.On the other hand, old zircons in basalts can be produced through recycling of crustal components during the metasomatic formation of mantle sources at different depths14. Nevertheless, there is no considerable difference in basalt composition, exhibiting not only variable depletions in radiogenic Sr-Nd isotopes relative to bulk silicate Earth but also oceanic island basalts-like trace element distribution patterns on the primitive mantle-normalized spidergram17.The target basalts were sampled from three petrotectonic units with significant differences in crustal composition, making the shallow contamination discernible. The three petrotectonic units are tectonically located in the southeastern edge of the North China Craton, the northeastern edge of the South China Block, and the Dabie-Sulu orogenic belt. The Dabie-Sulu orogenic belt was built by the Triassic subduction of the South China Block beneath the North China Craton18.Thirty basalt samples were selected for this study. They were collected from east-central China, close to the Tan-Lu Fault Fig. . Geograp17.All these basalts are generally porphyritic in texture. Phenocryst are olivine ± clinopyroxene ± plagioclase, matrix contains all these three minerals in fine grains. Olivine phenocrysts are subhedral to anhedral and usually show fractures. Plagioclase exhibits as tabular with small olivine and pyroxene grains filling between them. They usually have polysynthetic twinning. Clinopyroxene phenocrysts are subhedral. Compositional zoning is present in some olivine and clinopyroxene phenocrysts. Zircon is rare in the basalts and hardly observed in thin sections under a microscope. Nevertheless, mineral separation yields considerable amounts of zircon grains for U-Pb dating and geochemical analyses. The all basalt samples from the Sulu orogen and part of samples from the North China Craton and the South China Block show slight low-temperature alteration after eruption, resulting in transformation of some olivines to iddingsites. Petrography and geochemistry of the target basalts have been investigated in detailMost zircon grains from the basalts are subhedral or rounded, without euhedral ones. The internal structural of these zircons are complicated in the CL images Fig. . Many ziHf(t) values of −18.9 to 9.9 (Table Hf(t) values from −18.9 to 9.4  values, with a positive correlation between εHf(t) values and U-Pb ages. On the other hand, zircons with U-Pb ages younger than 1000 Ma show a large variation in 176Lu/177Hf ratios relative to those of zircons older than 1000 Ma (0.000301 to 0.004743 vs. 0.000060 to 0.003006). Moreover, Th/U ratios are correlated neither with 176Lu/177Hf nor with 176Hf/177Hf ratios. Zircons exhibit variable HREE enrichments with highly variable (Yb/La)n ratios from 1.6 to 19750 and (Lu/Gd)n ratios from 2.4 to 107 and highly variable Eu anomalies with Eu/Eu* ratios from 0.01 to 0.64 and Ce anomalies with Ce/Ce* ratios from 0.29 to 83.3  but absent in the North China Craton22. This precludes the possibility that these zircons would be of xenocrystic origin through the crustal contamination during the ascent of basaltic magmas through the continental crust of the North China Craton. Furthermore, the Triassic U-Pb ages of 200 to 245 Ma are consistent with those for zircons from UHP metamorphic rocks in the Dabie-Sulu orogenic belt but absent in crustal rocks from either the North China Craton or the South China Block outside the Dabie-Sulu orogenic belt21. Therefore, the old zircons in the target basalts are not the xenocrysts from the local continental crust.The concordant zircon U-Pb ages for the Cenozoic continental basalts are mainly clustered in the middle Neoproterozoic, Triassic and Early Cretaceous ages Fig. , respect2 and LILE , which are melt-mobile during assimilation. However, these features are absent in our basalts, arguing against the effect of crustal contamination17.It is known that zircon only occurs as a tiny accessory mineral in very small amounts in crustal rocks; even silica-saturated granitic rocks contain very little zircons. If the crustal contamination could be responsible for the origin of old zircons in basalts, there would be concurrent changes in the whole-rock major and trace element compositions of the resulted basalts. In this regard, the effect of crustal contamination on zircon addition to continental basalts can be tested by using a simple calculation to see whether there is a problem in mass balance. This is primarily gauged with the amount of crustal rocks in basaltic magmas in view of melt-mobile incompatible elements relative to the amount of xenocrystic zircons in erupted basalts. Supposing that 1 gram zircon is captured by an ascending basaltic magma, several hundreds to thousands grams of the crustal rocks are necessarily incorporated into the basaltic magma at the same time. Consequently, the finally erupted basalt must show a considerable increase in typical crustal components such as SiO12 and in volcanic arc rocks above the Caribbean subduction zone in Cuba13. If these zircons are inherited from crustal components, they must survive during metamorphic dehydration and partial melting at UHP conditions24. The survival of detrital zircons during subduction zone metamorphism is also indicated by the occurrence of inherited zircons with Precambrian relict cores and Triassic metamorphic rims in UHP metamorphic rocks from the Dabie-Sulu orogenic belt in east-central China25, where the crustal rocks were subducted to subarc depths for the Triassic UHP metamorphism21.An alternative interpretation for the origin of old zircons in the Cenozoic continental basalts is the source inheritance from the metasomatic agents of terrigenous origin. This mechanism has been used to explain the occurrence of old zircons in metasomatic pyroxenites veins within peridotite xenoliths carried by continental basalts in North China28. Despite small amounts, relict magmatic zircons do not show significant disturbance of not only U-Pb and Lu-Hf isotope systems but also trace element composition in UHT metamorphic rocks from the Bohemian Massif in central Europe28 and the Napier Complex in Antarctica27. In fact, a subset of detrital zircons were identified in the enriched MORB from the Macquarie Island ophiolite29. In addition, old zircons have been recovered from mafic igneous rocks from Indian mid-ocean ridges31. These observations indicate that zircons do have survived at temperatures for the generation of enriched MORB, which are derived from decompressional melting of the asthenospheric mantle at temperatures of 1300–1400 °C. In this case, the old zircons may be inherited from the mantle sources of their host rocks rather than captured from an unknown continent crust fragment.Furthermore, detrital zircons may survive during partial melting at ultrahigh-temperature (UHT) conditions of 900 to 1100 °C33. Both newly grown and relict zircon domains occur in orogenic peridotites from the Dabie-Sulu orogenic belt37. Such observations indicate that both the chemical transport of dissolved Zr and the physical transport of crustal zircon by metasomatic agents  have taken place at the slab-mantle interface in subcontinental subduction channels14. During partial melting of the metasomatic mantle domains, these tinny zircon grains may not be completely dissolved into basaltic melts, and some of them may survive and be physically transported by the basaltic melts to the surface.It is known from the study of zircons in metamorphic veins inside UHP metamorphic rocks that tinny zircon grains can be physically transported from subducting crustal rocks by metamorphic fluids12. The second stage is analogue to the occurrence of inherited zircons in intra-arc mafic igneous rocks above the Caribbean subduction zone13. In order to document this possibility, we have made a detailed discussion in Supplementary Information on the occurrence of magmatic zircon in mafic igneous rocks and the survival of detrital zircons at deep subduction zones. The results indicate that the old zircons were capable of survival at the lithosphere-asthenosphere boundary. As such, they were inherited from their mantle sources that contain the tinny zircon grains of crustal origin from the recycling of subducted crustal rocks in the subcontinental subduction channel.The occurrence of relict zircons in the Cenozoic continental basalts indicates that subduction zone fluids would have served as metasomatic agents to transport the detrital zircons from the orogenic crust via the mantle wedge to the basaltic melts. This involves two-stage processes in mass transfer at the slab-mantle interface in the subcontinental subduction channel. The first stage is analogue to the occurrence of Precambrian zircons in metasomatic pyroxenite veins inside peridotite xenoliths entrained by Cenozoic continental basalts44. This is consistent with the concordant U-Pb age peaks at 2.5 Ga and 2.7 Ga for Paleoproterozoic to Archean zircons from the Cenozoic continental basalts  values  values for zircons from the Jiaobei terrane44. Thus the ancient crustal rocks from the Jiaobei terrane could be the source of the Paleoproterozoic to Archean zircons in the Cenozoic continental basalts. In other words, the Paleoproterozoic to Archean zircons in the basalts would be sourced from weathering of the continental crust in the Jiaobei terrane.It is known that zircon U-Pb ages for high-grade gneisses from the Jiaobei terrane in the southeastern margin of the North China Craton exhibit three episodes of magmatic events around 1.8 Ga, 2.5 Ga and 2.7 Gas Tables  and S2. ts Table . Thus, tues Fig. , which a49. These relict zircons show Th/U ratios of 0.15 to 1.56, which resemble Th/U ratios of 0.19 to 4.49 for the relict zircon cores of Neoproterozoic U-Pb ages in the UHP metaigneous rocks from the Sulu orogen. Furthermore, the 176Hf/177Hf ratios of Neoproterozoic relict zircons in the Cenozoic continental basalts are similar to those of most relict cores in the UHP metaigneous rocks51, giving similar εHf(t) values (Fig. The Neoproterozoic relict zircons show concordant U-Pb ages of 705 to 983 Ma Tables  and S2. 55. Because the South China Block was subducted subsequent to the subduction of the Paleotethyan oceanic crust in the Late Paleozoic, the Early Cretaceous mafic igneous rocks should contain the geochemical signature from the interaction between the cratonic mantle and the subducting Paleotethyan oceanic crust. However, no such a signature has been found so far in these rocks. In this regard, the influence of the subducted Paleotethyan oceanic crust on the mantle sources of Cenozoic continental basalts is insignificant. Therefore, the Neoproterozoic relict zircons in the Cenozoic basalts would be not originated from the seafloor sediment overlying the subducting Paleotethyan oceanic crust in east-central China.On the other hand, some of the studied Cenozoic continental basalts are spatially distributed along the southeastern edge of the North China Craton. It is likely that the subducting Paleotethyan oceanic slab could have influenced the overlying cratonic mantle wedge. In this case, zircons could come from the terrigenous sediments that were weathered from the continental crust in the northeastern margin of the South China Block and they would be carried by the subducting Paleotethyan oceanic crust into the mantle sources of Cenozoic continental basalts. As a consequence, the Neoproterozoic magmatic zircons in the Cenozoic basalts could be inherited from the seafloor sediment overlying the subducting Paleotethyan oceanic crust. However, the mantle source of Early Cretaceous mafic igneous rocks in the southeastern margin of the North China Craton has been documented as the metasomatic product through reaction of the ancient cratonic mantle with felsic melts derived from partial melting of the subducting continental crust of the South China Block56. They are the product of metamorphic growth during the subduction of the South China Block beneath the North China Craton. These zircons exhibit similar 176Hf/177Hf ratios to the Triassic zircons in the Cenozoic continental basalts. The majority of these metamorphic zircons show very low Th/U ratios of <0.12,51, consistent with the low Th/U ratios for the majority of Triassic zircons in the basalts (Tables The metamorphic zircons of Triassic U-Pb ages are common in crustal rocks from the Dabie-Sulu orogenic belts Tables  and S2. 65. Zircons in Mesozoic gabbros with U-Pb ages around 130 Ma always show Th/U ratios of >1.0, with Th and U contents mainly between 50 to 300 ppm64. These are consistent with those for zircons with U-Pb ages around 130 Ma in the Cenozoic continental basalts, which exhibit Th/U ratios generally higher than 1.0 with Th and U contents mainly between 100 to 300 ppm (Tables A few zircons from the Cenozoic continental basalts show concordant U-Pb ages of 101 to 131 Ma Tables  and S2. m Tables  and S2. 65. As such, the crustal rocks would be eroded from the continental margin in the Sulu orogen, delivering the terrigenous sediments into the trench adjacent to the continent margin (Fig. As discussed above, the crustal rocks exposed in the Dabie-Sulu orogenic belt may be the source of detrital zircons in the Cenozoic continental basalts. This is consistent with Mesozoic subduction of the Paleo-Pacific slab beneath the continental edge of eastern Chinagin Fig. . After hgin Fig. . It is p17. In this regard, these youngest relict zircons were not provided by the terrigenous sediments. Instead, they were generated during the melt-peridotite reaction at the slab-mantle interface in the subcontinental subduction channel (Fig. 2-undersaturated magmas and eventually occur in the Cenozoic continental basalts.On the other hand, some basalt zircons show U-Pb ages of younger than Triassic Tables  and S2. nel Fig. . In thisnel Fig. . Rapid aSupplementary informationTable S1 to S7


To evaluate Bacterial Meningitis Score (BMS) on its own and in association with Cerebrospinal Fluid (CSF) lactate dosage in order to distinguish bacterial from aseptic meningitis.3 (1 point); CSF protein ≥80 mg/dL (1 point); peripheral blood neutrophil count ≥10,000 cells/mm3 (1 point) and seizures upon/before arrival (1 point). Cutoff value for CSF lactate was ≥30 mg/dL. Sensitivity, specificity and negative predictive value of several BMS cutoffs and BMS associated with high CSF lactate were evaluated for prediction of bacterial meningitis. Children diagnosed with meningitis at a tertiary hospital between January/2011 and December/2014 were selected. All data were obtained upon admission. BMS was applied and included: CSF Gram staining (2 points); CSF neutrophil count ≥1,000 cells/mm Among 439 eligible patients, 94 did not have all data available to complete the score, and 345 patients were included: 7 in bacterial meningitis group and 338 in aseptic meningitis group. As predictive factors of bacterial meningitis, BMS ≥1 had 100% sensitivity (95%CI 47.3-100), 64.2% specificity (58.8-100) and 100% negative predictive value (97.5-100); BMS ≥2 or BMS ≥1 associated with high CSF lactate also showed 100% sensitivity (47.3-100); but 98.5% specificity (96.6-99.5) and 100% negative predictive value (98.3-100). 2 point BMS in association with CSF lactate dosage had the same sensitivity and negative predictive value, with increased specificity for diagnosis of bacterial meningitis when compared with 1-point BMS. Therefore, studies comprising more healthcare centers are necessary so that results can be validated. Thus, our primary goal was to evaluate the discriminatory power of BMS to differentiate MB and MA, in the context of a Brazilian tertiary, private hospital. Secondary purpose was to verify CSF lactate also for the distinction between bacterial and aseptic meningitis.All children aged 1 month and 14 years who were assisted at the emergency service of Hospital Israelita Albert Einstein  between January 1, 2011 and December 31, 2014, with meningitis-related International Classification of Diseases (ICD)  were initially selected for the study.This is a retrospective cohort study, carried out by medical records data analysis and search for laboratory tests in the institution’s website. All data were collected at admission. Two authors (FRP and ACBFF) were responsible for collecting and compiling data.Inclusion criteria were: age between 1 month and 14 incomplete years on the day of medical assistance; Diagnosis of meningitis with CSF leukocyte >9 cells/µL (considering leukocytes-erythrocytes ratio of 1:500 for puncture accident). Exclusion criteria were: hemodynamic instability, evidence of cerebral herniation, presence of ventricular shunt or recent neurosurgery; previous epilepsy occurrence, immunosuppression; other bacterial infection requiring parenteral antibiotics or having received antibiotics 72 hours prior to lumbar puncture.®).The variables analyzed were gender, age, occurrence of seizures, absolute neutrophil and C-reactive protein (CRP), assessment of CSF glucose, lactate, neutrophils, protein and Gram, as well as blood and CSF cultures. All exams were collected at admission. Differential cytology for CSF was performed with microscopic counting after cytospin concentration and panchromatic staining. Biochemical features, lactate included, were processed by a dry-chemistry automated method (Fusion3) associated with positive blood culture for bacterial pathogen were considered. The aseptic meningitis group comprised all other children, who did not fit the standards described.The outcomes analyzed were presence of BM or AM. In the bacterial meningitis group, children who had positive CSF culture or pleocytosis (>9/mmThe study sample (345 patients) was obtained for convenience in a 4-year period, which is sufficient for the estimation of a 95% confidence interval (95%CI) and 65% accuracy measure, with 5% sampling error. As both primary and secondary objectives of the study did not involve testing hypotheses, sample statistical power estimation was not applied.In statistical analysis, the patients’ gender was described in groups as absolute frequencies and percentages and compared with by Fisher’s exact test. Numerical variables were described as medians and quartiles and compared with by the Mann-Whitney test. BMS power to distinguish children with BM and AM was assessed by measuring accuracy, sensitivity, specificity, positive and negative predictive values; kappa coefficient was also used to compare agreement between score and cultures in BM scenario, all measures being accompanied by respective 95% intervals. The calculations were made in compliance with recommendations by AltmanThe project was approved by the Ethics Committee of HIAE and Platforma Brasil, under CAAE consubstantiate opinion: 46335015.2.0000.0071.A total of 503 patients admitted to HIAE (SP) between 2011 and 2014 were identified and met the aforementioned inclusion criteria. Among these, 64 were excluded due to the following conditions: previous use of antibiotic (n=26); no diagnosis of meningitis (n=29); two previous hospital admissions (n=8); previous occurrence of epilepsy (n=1). Among 439 children eligible for examination, 94 did not perform all tests required for BMS analysis, 90 were not submitted to blood count, and four did not have CSF Gram staining, so they were excluded afterwards. Thus, 345 patients fulfilled all prerequisites and were selected for the study : seven wStreptococcus pneumoniae (five cases), Neisseria meningitidis (one case), and Enterococcus faecalis (one case). In AM group, 292 had CSF investigated for polymerase chain reaction enterovirus and 237 tested positive (81%).The microorganisms isolated in cultures of patients in BM group were: versus 15.5 mg/dL in AM), CSF neutrophils count , blood PCR (23.3 versus 1.53 mg/L in AM) and CSF protein (83 versus 30 mg% in AM), as shown in When comparing groups, no evidence of difference in age, gender, or blood neutrophils count was found . The BM In BMS analysis, 1 point was the cutoff for differential diagnosis of MB and MA; that is, if the patient scored 1 or more in BMS, it should be treated as probable MB. Results were: 100% sensitivity (95%CI 47.3-100), 64.2% specificity (95%CI 58.8-69.3), 100% negative predictive value (95%CI 97.5-100), and kappa coefficient for score and CSF culture of 0.07 (95%CI -0.07 -0.2).When using the 2 as cutoff point in BMS and considering patients who scored 2 or more as BM, the same 100% sensitivity (95%CI 47.3-100) and negative predictive value were obtained (95%CI 98.3-100), but specificity was increased to 98.8% (95%CI 97.0-99.7), and kappa coefficient for CSF score and culture was 0.77 (95%CI 0.55-0.99) .When using CSF lactate dosage with 30 mg/dL as cutoff value for BM or AM diagnosis, results were: 85.7% sensitivity (95%CI 42.1-99.6), 99.1% specificity (95%CI 97.4-99.8), 99.7% negative predictive value (95%CI 98.4-100.0), and kappa coefficient for CSF score and culture of 0.74 (95%CI 0.49-0.99).Combining 2-point and 1-point BMS results and CSF lactate ≥30 mg/dL, findings were: 100% sensitivity (95%CI 47.3-100.0), 98.5% specificity (95%CI 96.6-99.5), 100% negative predictive value (95%CI 98.3-100.0), and kappa coefficient for CSF score and culture of 0.73 (95%CI 0.49 -0.96).,,,,When analyzing 1-point BMS predictive value for BM, results were 100% sensitivity and negative predictive value, but specificity was low (64.2%), which is in accordance with what has already been reported by other authors.In the analysis of 2-point BMS, we found 100% sensitivity and a negative predictive value, besides specificity being raised to 98.8%, which would generate low hospital admission rate, but still counting BM hospitalization cases. However, because of the small number of BM cases compared to AM, care should be taken when interpreting such data, since Nigrovic et al.For CSF lactate with ≥30 mg/dL cutoff, we found 85.7% sensitivity, 99.1% specificity, and 99.7% negative predictive value. Our results are in agreement with the literature, that is, this is a very specific but not very sensitive criterion.Since 2-point BMS did not individually demonstrate 100% sensitivity in another studyIn our study, the frequency of BM was 2% (95%CI 0.8-4.1). This data differs a lot from those obtained by Mekitarian Filho et al., who reported 9% of BM cases when analyzing the clinical picture of 547 children between 2001 and 2012 in a public hospital of São Paulo.Our study had, though, a few limitations. Being a retrospective survey may cause important information not to be obtained, especially regarding subjects’ general state and toxemia, or their vaccination history. Also, as the study was performed in one institution alone, it reflects the reality of that place solely. In addition, the number of BM cases found was small, and this may have impaired the reproducibility of the modified score. The number of BM cases also explains the broad values in confidence intervals for sensitivity in all scores evaluated.Bearing such limitations in mind, this is the first Brazilian study to analyze accuracy of BMS in a private tertiary service which showed promising results like those reported in the literature and also confirmed previous findings of a Brazilian study conducted in a public hospital.


In the present study, we validated a commercially available ECL-based a-syn ELISA platform as a diagnostic test for correct classification of sCJD cases. CSF a-syn was analysed in 203 sCJD cases with definite diagnosis and in 445 non-CJD cases. We investigated reproducibility and stability of CSF a-syn and made recommendations for its analysis in the sCJD diagnostic workup. A sensitivity of 98% and a specificity of 97% were achieved when using an optimal cut-off of 820 pg/mL a-syn. Moreover, we were able to show a negative correlation between a-syn levels and disease duration suggesting that CSF a-syn may be a good prognostic marker for sCJD patients. The present study validates the use of a-syn as a CSF biomarker of sCJD and establishes the clinical and pre-analytical parameters for its use in differential diagnosis in clinical routine. Additionally, the current test presents some advantages compared to other diagnostic approaches: it is fast, economic, requires minimal amount of CSF and a-syn levels are stable along disease progression.The analysis of cerebrospinal fluid (CSF) biomarkers gains importance in the differential diagnosis of prion diseases. However, no single diagnostic tool or combination of them can unequivocally confirm prion disease diagnosis. Electrochemiluminescence (ECL)-based immunoassays have demonstrated to achieve high diagnostic accuracy in a variety of sample types due to their high sensitivity and dynamic range. Quantification of CSF α-synuclein (a-syn) by an The online version of this article (doi:10.1007/s12035-017-0479-5) contains supplementary material, which is available to authorized users. Creutzfeldt-Jakob disease (CJD) is the most common human prion disease characterised by the accumulation of pathological misfolded prion protein (PrPSc) in the central nervous tissue. Most patients present an unknown aetiology being classified as sporadic Creutzfeldt-Jakob disease (sCJD), which is clinically characterised by the presence of dementia and ataxia , 2. sCJDIn this context, electrochemiluminescence-based systems have demonstrated high accuracy, reproducibility, recovery rates and broad dynamic range in the detection of several biomarkers in biological fluids , 19. Besin-house a-syn electrochemiluminescence-based enzyme-linked immunosorbent assay (ELISA) platform presented high accuracy in the discrimination of sCJD cases [The detection of elevated α-synuclein (a-syn) in the CSF of sCJD cases using colorimetric assays was previously reported , 21. HowJD cases , 22. HowIn the present study, we analysed a commercial electrochemiluminescence-based human a-syn ELISA kit as a novel test method in sCJD diagnosis. The present test allowed discriminating non-CJD from sCJD cases with high diagnostic accuracy. Importantly, data were collected from three independent cohorts of sCJD autopsy-confirmed cases. Since standard protocols and sample handling have become more important in routine diagnostic, recommendations for the CSF a-syn diagnostic of sCJD are reported based on the study of the influence of pre-analytical and analytical parameters.n = 41) and patients suffering from neurological diseases (n = 149) or neurodegenerative conditions and cognitive impairment/dementia (n = 255). Neurological diseases included psychiatric disorders , ischemic stroke, epilepsy, autoimmune diseases, meningitis, alcohol abuse, headache, vertigo, pain syndromes, acute hypoxia, encephalopathy, cerebral vasculitis, normal pressure hydrocephalus and alternative neurologic conditions. Neurodegenerative disease included Parkinson’s disease, dementia with Lewy bodies, multiple system atrophy, Alzheimer’s disease, frontotemporal dementia, Huntington’s disease, vascular dementia, corticobasal degeneration, progressive supranuclear palsy and olivopontocerebellar atrophy.The study included a total of 648 patients recruited at the National Reference Center for Transmissible Spongiform Encephalopathies of the Department of Neurology and Neuropathology at the University Medical Center of Gottingen, Germany (cohort 1), at Polish neurologic and psychiatric hospital departments (cohort 2) and at the Unit of Bio-diagnostic of CJD and other prion diseases at the Hospital Clinical of Barcelona, Spain (cohort 3). All patients with sCJD were classified as definite cases by neuropathological examination according to diagnostic consensus criteria . Non-CJD3 and/or haemoglobin contamination were intentionally excluded from this study in order to avoid possible false positive outcomes. Information about demographics and CSF biomarkers tau and 14-3-3 of the study population are supplied in Table CSF samples were tested for blood contamination using Hemastix strips (Siemens), and cases containing more than 25 erythrocytes/mmU tests were used to compare two groups of samples. For analysis of a-syn stability, Wilcoxon signed rank sum tests were applied. In order to assess the diagnostic accuracy of a-syn, receiver operating characteristic (ROC) curve analyses were carried out and areas under the curve (AUC) with 95% confidence intervals were calculated using GraphPad Prism 6.01. The best cut-off value was estimated based on the Youden index [Mann-Whitney en index  derived Quantification of a-syn was performed using the electrochemiluminescence-based ELISA-based human a-syn kit from Meso Scale Discovery (MSD)  following manufacturer’s instructions. Briefly, assay plates were blocked by adding 150 μl of Diluent 35 to each well. Plates were sealed and incubated at room temperature with shaking at 300 rpm for 1 h. Then, plates were washed three times with 150 μl per well of PBS-T (PBS supplemented with 0.05% Tween-20). Detection antibody (25 μl) and CSF (diluted 1:8) or calibrator (25 μl) was added to each well. Plates were sealed and incubated at room temperature with shaking at 700 rpm for 2 h. After washing as indicated above, 150 μl of 2× read buffer T was added to each well. Measurements were performed on a MSD Sector Imager 6000. Calibrators were run in duplicate to generate calibration curve.Tau was quantified using the INNOTESThTAU-Ag  ELISA test. 14-3-3 was semi-quantitatively tested by using Western blot as previously described .To evaluate the effects of CSF storage conditions (temperature and freezing/thawing cycles) on a-syn concentration, CSF samples from 8 sCJD patients were stored in polypropylene tubes at room temperature and 4 °C for 1, 3 and 6 days. In addition, CSF samples were subjected to 2, 4 and 8 repeated freezing and thawing cycles. a-syn concentrations at each time point or cycle were calculated as percent of control (time point zero), which was defined as 100%.The study was conducted according to the revised Declaration of Helsinki and Good Clinical Practice guidelines and approved by local Ethics committees Goettingen . Informed consent was given by all study participants or their legal next of kin.CSF a-syn levels were measured using the electrochemiluminescence-based human a-syn kit Fig. . The dynp < 0.001 and AUC = 0.997, 95% CI: 0.995 to 0.999)  presented sensitivities of 91 and 92%, respectively, in detecting sCJD cases, which was significantly lower than those obtained for CSF a-syn (98%). Neither a specific diagnostic group nor a correlation between a-syn and tau/14-3-3 profile was detected in the non-CJD cases tested positive for a-syn. Comparison of sensitivities in cohort 1 between the three biomarkers revealed statistical differences between a-syn vs tau (p = 0.035) and a-syn vs 14-3-3 (p = 0.021) comparisons, but not in tau vs 14-3-3 (p = 0.763). Additionally, we found a positive correlation between CSF a-syn and tau levels (p < 0.001) in control subjects and in sCJD cases, in agreement with the observations from our previous study [PRNP codon 129 polymorphism on CSF a-syn levels. Information about codon polymorphism was available for 182 cases . As we previously reported [p < 0.05). Mean values in MV cases were also lower than in VV (9941 ± 9733 pg/mL a-syn). However, these differences were not statistically significant.Our study cohort included a total of 445 non-CJD and 203 sCJD cases. CSF a-syn showed an excellent accuracy in discriminating non-CJD from sCJD cases (99) Fig. , b. Mean99) Fig. . Using a99) Fig. . Since hus study . Next, wreported , a-syn lNext, we assessed the influence of time of CSF sampling on a-syn concentration in the diagnosis of sCJD. For this, samples from 10 sCJD patients for which CSF at different stages of disease was available were tested. Since disease duration in sCJD is highly variable, depending on demographic and genetic characteristics , the timp < 0.001) in the 185 cases where disease duration was known –0.57*((a-syn (pg/mL)/10,000)–1.02).CSF a-syn levels were inversely associated with disease duration  was detected after 8 cycles  , RT-QuIChttp://www.eurocjd.ed.ac.uk). Here, we also demonstrated that CSF a-syn levels are stable along disease duration as reported in a group of sCJD cases ranging from the lowest to the highest a-syn levels for which serial LPs were available. Altogether, our study supports the use of CSF a-syn quantification in diagnostic routine as a first robust, cheap and fast discriminatory assay to detect sCJD cases, which could be further validated by means of RT-QuIC test to achieve a defined pre-mortem diagnosis.sCJD disease duration varies depending on demographic and genetic factors from several weeks to 3 years [We summarised the outcome of our study and prepared a recommendation table for pre-analytical sample handling and storage to improve standardisation of CSF a-syn quantification for the diagnosis of sCJD patients in clinical routine Table . See alsA potential further application of CSF a-syn quantification is its use as prognostic marker, since disease duration showed a negative correlation with a-syn levels, comparable to CSF tau . In thisa-syn, α-synuclein; AUC, area under the curve; CJD, Creutzfeldt-Jakob disease; ECL, Electrochemiluminescence; ELISA, enzyme-linked immunosorbent assay; LP, lumbar puncture; CSF, cerebrospinal fluid; PrPSc, pathological misfolded prion protein; ROC, receiver operating characteristic; RT-QuIC, real-time quaking-induced conversion; sCJD, sporadic Creutzfeldt-Jakob disease; SD, standard deviation.Supplementary Table 1Number of cases and a-syn values stratified for the three different cohorts analysed in the present study (A) and stratification of controls according to differential diagnosis (B). (PPTX 57 kb)Supplementary Table 2Comparison of CSF sCJD biomarkers according to literature. Biomarker outcome, methodology and clinical accuracy values (sensitivity and specificity) expressed in ranges, when more than one study is cited, are reported. (PPTX 69 kb)


In contrast to virtually all organ systems of the body, the central nervous system was until recently believed to be devoid of a lymphatic system. The demonstration of a complex system of paravascular channels formed by the endfeet of astroglial cells ultimately draining into the venous sinuses has radically changed this idea. The system is subsidized by the recirculation of cerebrospinal fluid (CSF) through the brain parenchyma along paravascular spaces (PVSs) and by exchanges with the interstitial fluid (IF). Aquaporin-4 channels are the chief transporters of water through these compartments. This article hypothesizes that glymphatic dysfunction is a major pathogenetic mechanism underpinning idiopathic intracranial hypertension (IIH). The rationale for the hypothesis springs from MRI studies, which have shown many signs related to IIH without evidence of overproduction of CSF. We propose that diffuse retention of IF is a direct consequence of an imbalance of glymphatic flow. This imbalance, in turn, may result from an augmented flow from the arterial PVS into the IF, by impaired outflow of the IF into the paravenous spaces, or both. Our hypothesis is supported by the facts that (i) visual loss, one of the main complications of IIH, is secondary to the impaired drainage of the optic nerve, a nerve richly surrounded by water channels and with a long extracranial course in its meningeal sheath; (ii) there is a high association between IIH and obesity, a condition related to paravascular inflammation and lymphatic disturbance, and (iii) glymphatic dysfunction has been related to the deposition of β-amyloid in Alzheimer’s disease. We conclude that the concept of glymphatic dysfunction provides a new perspective for understanding the pathophysiology of IIH; it may likewise entice the development of novel therapeutic approaches aiming at enhancing the flow between the CSF, the glymphatic system, and the dural sinuses. Hints on the existence of this system came from the identification of endothelial lymphatic cells by intraventricular injections of immunohistochemical markers in meningeal vessels. These vessels drain the CSF and contain T and MHCII+ cells . Additio+ cells . Because+ cells , 4.The discovery of the glymphatic system has challenged classical views on the “third circulation” and rapidly galvanized the interest of basic researchers. Over the past few years, the recognition that the glymphatic system may be involved in the pathophysiology of common neurologic diseases, such as idiopathic intracranial hypertension (IIH), has also attracted the interest of clinicians. These advances require a review of some critical aspects of some of the classical views on CSF circulation.According to classical knowledge, the CSF is formed as an ultrafiltrate of the blood in the choroid plexus at a daily rate of 500 mL, but the CSF volume in the human central nervous system (CNS) is 150 mL, indicating the CSF is replaced three to four times a day . From thvia the cisternal CSF. In this sense, the paravascular pathways within the CNS parenchyma enable the clearance of solutes from the brain to the CSF, whereas the extra-axial meningeal lymphatic vessels propel the solute-laden CSF on to the systemic vascular system  with direct communication with the interstitial fluid (IF) all over the way. The CSF also drains into the lymphatic system through the meningeal sheaths of the spinal and cranial nerves, especially the optic, trigeminal, facial, and vestibulocochlear , 3. The r system , 4, 8–10The role of the PVS has been documented in the optic nerves. Denniston et al.  argue thInjections of paramagnetic agents and fluorescent tracers in the SAS of mice have shown that the CSF moves swiftly from the SAS into the Virchow–Robin PVSs , 8. Linivia the Virchow–Robin spaces is enhanced by arterial pulsations and sleep  administration of acetazolamide, a carbonic anhydrase inhibitor that reduces CSF production, (ii) cisterna magna puncture, which eliminates the small pressure gradients that allow the convective exchange of CSF with the IF, (iii) AQP4 deletion, and (iv) body position adjustment . In all The glymphatic system has also been implicated in the removal of heat excess from the brain. Narebski stipulated a brain–blood temperature gradient in which the Circle of Willis is approximately 0.5°C cooler than the brain tissue, which is under metabolic heat. The cooler blood flow was defined as the main mechanism for the removal of heat excess by convection . RefreshIn the following section, we review the clinical features of IIH, a common neurological disorder that may be caused by glymphatic dysfunction.2O, and the CSF composition is normal  and specificity (90%) in differentiating IIH from normal controls. The area of the pituitary has been reported to be very sensitive (100%) for detecting improvement following pharmacological treatment of IIH. Furthermore, an empty sella is a non-specific finding, which is often incidentally found in healthy subjects as well as in obesity, meningioma, pediatric nevoid basal cell carcinoma, and following therapy for growth hormone deficiency. Regardless of its limited specificity, the finding of an empty sella in conjunction with other radiological signs support the diagnosis of IIH provided tumor, venous thrombosis, and infection are ruled out .Chronic increased ICP leads to bone erosion and remodeling, resulting in widening of the bone canals of the cranial base. Enlargement of the foramen ovale has high specificity (81%), but low sensitivity (50%) for a diagnosis of IIH. The jugular foramen and hypoglossal canal were also reported to be enlarged in IIH. Enlargement of CSF spaces or spontaneous CSF-filled outpouchings of the dura (meningoceles) resulting from chronic increased ICP may also support the diagnosis of IIH. These enlargements have been reported in Meckel’s cave, the oculomotor nerve sheath, and in the CSF spaces around the abducens nerve .Neuroimaging is decisive for ruling out causes of raised ICP in space-occupying lesions. Venous sinus thrombosis can present with severe headache from intracranial hypertension secondary to impairment of CSF reabsorption, which can mimic IIH symptoms . MRI of 2O with 88–100% sensitivity and 93–100% specificity. Optic disc elevation can also be detected by ultrasound. MRI is another reliable imaging modality for the detection of increased ONSD with a sensitivity of 72–80% and specificity of 96%. ONSD decreases 30 min after lumbar puncture (LP), while normalization of optic disc protrusion requires longer treatment times. Flattening of the posterior globe in MRI is highly specific (close to 100%), but not sensitive, to IIH. Although these imaging features reliably differentiate IIH patients from individuals without intracranial hypertension, these signs can also be seen in increased ICP from other causes, as in cerebral venous thrombosis  with good sensitivity (90%) and specificity (84%) at a cutoff value of 5.8 mm in detection of IIH. The ONSD of 5.5 mm predicted a CSF pressure of 20 cm Hrombosis .Stenosis at some point of the dural sinus system has been reported in 30–93% of IIH patients. Stenosis of the transverse and sigmoid sinuses detected by magnetic resonance venography is reliable markers of IIH with high sensitivity and specificity (93% for both). It is still debated whether venous sinus stenosis plays a causal role or results from increased ICP. Acute CSF volume reduction by CSF diversion or a single LP can ameliorate the symptoms of venous sinus stenosis. Stenting of transverse sinus stenosis, in turn, can improve intracranial hypertension. These findings suggest that venous sinus stenosis and intracranial hypertension share a circular loop relationship .in vivo is an urgent need so that the role of glymphatic dysfunction in the development of symptoms in several common pathological states can be better understood.In contrast to the blood, which runs through a network of dedicated vessels, the CSF flows through paravascular channels and brain parenchyma at a much slower pace . The dev3D-FLAIR (hT2-FL) sequences are sensitive to very low concentrations of gadolinium (IV-GBCA), which appears as a slight enhancement of CSF 4 h after a single dose of IV-GBCA in healthy subjects. Following this lead, Naganawa et al.  investig1-weighted MRI in a recent investigation with the intrathecal contrast agent gadobutrol injected via the lumbar route (in vivo.The human glymphatic system was successfully imaged with Tar route . There wGlymphatic dysfunction compromises the clearance of IF and leads to diffuse stagnation of flow and solutes in (a) the interstitium, (b) in the PVSs, or (c) in both, resulting in IIH; andGlymphatic dysfunction results from (a) augmented flow from arterial PVS to the IF, (b) impaired outflow of IF into the paravenous space, or (c) both.Hypotheses (1) and (2) are not mutually exclusive and may assume different weights in individual patients.The main hypotheses of this article may be summarized as follows:Kress et al.  observedIn vitro study showed that inducing microglia to express macrophage like surface proteins was especially effective at lowering lysosomal pH and activating the degradation of fAβ sheet structures . Projection fibers follow a head-to-foot direction (z-axis) and the intra-hemispheric fascicles run in the anterior–posterior direction (y-axis). This configuration—in which vessels and white matter are perpendicular to each other—allowed an independent analysis of diffusivity in PVS, which was taken as a surrogate of glymphatic function. The authors found a significant positive correlation between PVS diffusivity and Mini-Mental State Exam scores, indicating impaired water diffusivity in the PVS of projection and association fibers, and this impairment was related to AD severity. The authors concluded that glymphatic dysfunction is the rule in patients with severe AD. The results were consistent with the impaired glymphatic activity observed in experimental animals  may exhibit remarkable improvements, especially if the symptom presented is gait impairment . iNPH isThe impairment in dynamic of CSF flow in iNPH is illustrated by the study of Jeppsson et al.  where itWostyn et al.  discuss There is growing evidence of lower ICP in patients with primary open angle glaucoma (POAG) when compared with non-glaucomatous control subjects and additionally, is lower in the normal-tension versus the high-tension form of POAG. The authors sustain that the lower ICP reported in POAG patients could be an indicator of decreased CSF production and turnover, what may demonstrate that changes in CSF circulatory physiology may be even more pronounced in the normal-tension compared with the high-tension form of POA .Although acute exposure to microgravity has immediate physiologic effects, it is the chronic exposure to microgravity that poses a more insidious consequence, as experimented during space flight. The longer the mission lasts the higher is the evidence of visual acuity changes in astronauts associated with structural abnormalities of the retina and optic nerve. The etiology of the ocular changes remains speculative; however, MRI abnormalities identified in postflight astronauts have implicated ICP as the underpinning cause. Intracranial hypertension can hypothetically affect the visual pathway through propagation of elevated CSF pressure from the intracranial compartment to the orbit with resultant expansion of the optic nerve sheath and structural modification of the posterior globe. Kramer et al.  verifiedMRI studies provide indirect evidence of cerebral edema, associated with clinical data on the swollen brain  . This seGiven the high sensibility of the empty sella for a diagnosis of IIH and the fact that the pituitary recess is one of the most proximal glymphatic inflow conduits, the sellar glymphatic compartment is probably affected early in the course of IIH and represents an area of interest for study . MoreoveThe AQP4 has also been in the focus of HII research. Kerty et al.  searchedConsidering the existence of a possible glymphatic dysfunction associated with slower CSF circulation and reduced CSF production, it would be interesting to verify whether IIH patients present higher levels of CSF protein and other cumulative metabolites  than controls. The LP aiming to relieve ICP might unblock stagnated CSF flow: if the restraint or resistance is in the interstitium → venous paravascular direction, as in NPH, significant rises of waste products might be noted comparing CSF composition before and after this procedure. However, if there is an overload in arterial paravascular → interstitium direction, the removal of CSF might not interfere with the interstitium drainage. In the experiment by Lundgaard et al.  using miThe concentration of Aβ is another candidate of cumulative metabolite to be checked. Current medicines used to treat IIH, such as acetazolamide, while hampering the production of CSF enhances the acidification in brain, which might lead to the formation of fibrillary Aβ degradation. However, the analysis of this product may have a low sensibility since patients with IIH tend to be young, and the glymphatic flow is disturbed. In this case, a prospective study of IIH might elucidate whether it contributes any risk to the development of AD.The discovery of the glymphatic system may bear important implications for the diagnosis and management of several common neurological diseases in the near future. For instance, it has been proposed that a failure in the clearance of soluble β-amyloid from the brain interstitium contributes to amyloid plaque deposition and AD progression , 8.Idiopathic intracranial hypertension is responsible for severe headache, papilledema, and visual loss that may evolve into irreversible brain damage. The incidence of IIH is expected to rise given the worldwide obesity epidemic and the well-known predisposing role of obesity for the development of IIH . The rolThis article discusses the role of IF accumulation as a consequence of glymphatic dysfunction leading to increased ICP in IIH. MRI signs indicative of IIH have not included direct evidence of overproduction of CSF. Indeed, there seems to occur the clearance saturation of the glymphatic system, whether by a primary disturbance  or, for example, by an increase in volume of the PVSs, which could take place in inflammatory conditions such as obesity.Regardless of the exact mechanism at play in individual cases, we argue that glymphatic dysfunction is a final common pathway for abnormal increases of ICP. Currently, increases of ICP are treated with (a) measures to reduce the total volume of CSF by direct removal either from the SAS through cisternal or LP, (b) by attempts to inhibit CSF secretion at the choroid plexus, or a combination of (a) and (b). The second strategy is provided by drugs, such as acetazolamide and topiramate, which inhibit carbonic anhydrase. It is known that one of the main determinants of CSF secretion is the production of al cells . These mal cells . If glymThe discovery of the glymphatic system may bring novel insights into the pathogenesis of IIH. Confirmatory evidence that IIH is associated with CSF circulatory dysfunction and stagnation in the cerebral microcirculation should redirect the attention of clinicians and researchers to new therapeutic measures aiming at enhancing the rate of cerebral clearance. The present hypothesis affords a direct clinical application of the glymphatic concept. As such, it may open the way for new studies on the role of the glymphatic system in several common neurological diseases, such as AD and other degenerative diseases.MB is the mentor of the central idea explored in the article. He also contributed to the writing of the manuscript. AF contributed in the writing of the manuscript and researched for more arguments to defend the presented hypothesis. RO-S searched for more papers to illustrate the cited arguments. He contributed a great deal to the edition and the improvement of the text.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.


Data matters. Senator Cory Booker's mantra is ‘in God we trust … Everybody else, bring me data’,Gamete Donor Anonymity and Limits on Numbers of Offspring: The Views of Three Stakeholders acknowledges existing data collection on assisted reproduction, studies that tend to offer patchwork pictures of different populations at different times and places, answering different questions. Their article represents a marked departure in its attempt to tell a broader story.The literature review in Nelson, Hertz, and Kramer's First, the data set is expansive. It compiles the views and experiences of sizeable population: 325 donors, 2134 parents, and 419 children. The researchers posed a range of questions to these donors, parents, and donor-conceived children, all at essentially the same time. Moreover, some of their respondents are in the same family, providing a detailed comparison of the three main stakeholders in assisted reproduction. As a whole, the data set should enable researchers to map similarities and differences within each stakeholder group, between stakeholder groups, and to some extent even within a particular family.A piece of this large study appears in this volume, exploring two issues: donor anonymity and the number of offspring born of each donor. The authors provide a metric to evaluate scholarly proposals, professional practices, and legal rules regarding these two issues. The leading proponent of increased state involvement on both of these issues is Naomi Cahn, who proposes that donor registries replace anonymity and suggests stamping birth certificates ‘by donor’ for children conceived via assisted reproduction.Lurking behind these proposals for increased state regulation is the question of whether assisted reproduction is more like coital conception or like adoption. If it is like coital conception, then constitutional doctrines as well as privacy norms stand in the way of bans on anonymity or limits on numbers of children conceived unless the state is prepared  to take actions such as genetically testing all babies to make sure that the approximately 3% of babies conceived in infidelity know their genetic provenance, or mandate sterilization or birth control for particularly fecund people like the Duggans of reality television fame.Space constraints preclude a review of what Nelson, Hertz, and Kramer's data tell us about the advisability of these actions. Instead, this comment focuses on three surprising findings that should interest law makers and policy shapers who seek to conform reproductive technology rules to the lived realities of donor-conceived families. Two core principles make these three surprises stand out: family law's oft-repeated insistence on protecting children's best interests and the dangers of interfering in adults’ freedom of action and decision-making in most matters related to family creation and child raising.As a whole, donor-conceived children in this study were not determined to abolish anonymity. Instead, just a third (33%) were ‘neutral’ about the issue of anonymity rather than for or against it.But just under a third (31%) of the offspring strongly opposed anonymity. At first glance, this datum seems to support calls for national registries and a ban on anonymous donation. Nelson, Hertz, and Kramer look closely at this anti-anonymity group, however, and find that older donor-conceived people are most likely to express hostility to anonymity. They were born decades ago, when assisted reproduction was less acceptable legally and socially, and available to married heterosexual couples more than single and gay people. Because of those norms and sometimes professional advice to not tell children about their origins, these offspring were also more likely to discover their origins relatively late in life.Younger respondents—in their teens and 20s—came into a world more friendly to reproductive technologies and family variety. These younger subjects were more likely to be raised by single or gay parents, and, as the authors put it, tended to emphasize that ‘families can take all different forms and that love is not based on genes’.Nelson, Hertz, and Kramer largely allowed the data to speak for itself. They acknowledge that the response to anonymity expressed by older donor-conceived children may be an artifact of the more homogenous social and legal conditions that shaped those offsprings’ growing up, and also the possibility that older people may be more thoughtful about the complex interplay of genetics and social relationships, especially once they have children themselves.The second surprising bit of data is that offspring who have had contact with half-siblings through online networks like the DSR were more supportive of anonymity than those who have not had contact with half-siblings of the same donor (sometimes known as ‘diblings’).Third and finally, I was surprised to read that the authors predict that future proposals for reform will focus on limiting the number of children conceived by any particular donor. This issue has been debated less in scholarly or policy circles than anonymity. The authors reason that parents and offspring report strong support for preventing one donor from siring scores of children. Notably, their data also indicate that gay and lesbian parents express less concern about this issue. They—unlike heterosexual parents—are less able to credibly lie to their children about their origins, and accidental incest is a bigger risk for those who do not know that they were donor conceived. The study also documents that gay and lesbian parents are generally more cautious about regulation, an unsurprising data point given the state's very recent transition from ignoring or punishing gay people to marriage equality and other gay rights.I am less confident of this prediction than others put forward by Nelson, Hertz, and Kramer. Even if the issue of the number of children conceived with any particular donor matters a lot to donor-conceived families, I do not expect pushes for regulation unless partnered, heterosexual parents greatly outnumber gay parents in the overall population of donor-conceived families, and these heterosexual parents organize for lobbying efforts. Yet, the greater likelihood of this population remaining closeted about using donor gametes presents a formidable barrier to that organizing. Finally, since the fertility industry is the most organized and well-funded stakeholder in donor-conceived families, I see no reason to expect it to cede its prominence in policy and legal debates. Accordingly, while Nelson, Hertz, and Kramer provide compelling evidence that the state may increase its participation in reproductive technology arrangements, my money is on continued deregulation.