Findings show that music expert listeners were more sensitive to microtiming manipulations than non-experts. Across both expertise groups and for both styles, groove ratings were high for microtiming magnitudes equal or smaller than those originally performed and decreased for exaggerated microtiming magnitudes. In particular, both the fully quantized music and the music with the originally performed microtiming pattern were rated equally high on groove. This means that neither the claims of PD theory (that microtiming deviations are necessary for groove) nor the opposing exactitude hypothesis  were supported by the data.This study tested the influence of expert performance microtiming on listeners' experience of groove. Two professional rhythm section performances (bass/drums) in swing and funk style were recorded, and the performances' original microtemporal deviations from a regular metronomic grid were scaled to several levels of magnitude. Music expert ( Groove is a positive experience associated with rhythm and meter in music. Definitions of the concept differ in nuances, but there seems to be a consensus among musicians, music psychologists and music scholars that the groove experience consists in a person's inner urge to synchronize body movement with the beat of the music Doffman, . The groThe use of music in connection with body movement  is ubiquitous. Accordingly, the groove phenomenon has received considerable scholarly attention in recent years, also due to a growing interest in embodiment and musical entrainment  claims that microtemporal patterns arising in music performance—small timing deviations from strict metronomic time, often within a range of ±50 ms—are crucial for the creation of groove. This theory is based on Charles Keil's concept of Participatory Discrepancies or PDs . A surprising result was that the stimuli with loosest timing triggered strong entrainment in music experts.Kilchenmann and Senn  addresseThese results cannot directly be compared to earlier findings given the different methodological approaches: In Kilchenmann and Senn , data onA major concern for the assessment of groove through questionnaires is what dimensions of the experience should be measured and what questions or statements should be used to measure them. Janata et al.  in theirMadison et al.  operatioIn Witek et al.  participEmotional Assessment of Groove (EAG) questionnaire, to measure the intensity of listeners' groove experience. The questionnaire captures three basic dimensions: listeners' felt Entrainment, Enjoyment, and the music's naturalness and flow, assessed by inversely measuring the degree of Irritation experienced by the listeners.In this study we developed a new psychometric tool, the PD theory we hypothesized that the music examples with the original microtiming patterns (as played by the musicians) would receive higher mean groove ratings than music examples with manipulated timing. Specifically, we hypothesized that the groove ratings would be lower the more the timing differed from the originally performed timing.The goal of this study was to clarify the role of real-world performance microtiming in the subjective groove experience of expert and non-expert listeners by systematically manipulating the magnitude of the microtiming deviations in short recorded funk and swing clips. In line with Kilchenmann and Senn  and PD tThe relevance of listeners' musical expertise for the groove effect of microtiming is, as yet, unclear: Davies et al.  and KilcThe experimental stimuli were derived from recorded performances by two professional and internationally renowned musicians, bassist Wolfgang Zwiauer and drummer Dominik Burkhalter. Two recordings were made during a studio jam session for the specific use in this experiment. In one recording, the musicians played an eight-bar funk pattern at 100 bpm on drums and electric bass during several minutes. In another recording, they played a twelve-bar swing pattern at 150 bpm on drums and acoustic bass guitar. The musicians extemporated the music after agreeing on some basic features.During the performance, the musicians heard a metronome click over headphones as a common beat reference; the metronome click was recorded to a separate track. To hear a metronome click while playing is common practice in studio work, and the musicians confirmed being comfortable with it. After the recording session, the musicians indicated segments from each recording that, in their opinion, had the best groove. From these segments, the experimenters chose one iteration of each pattern. As a result, we retained 20 s of music for each style that would serve as basis for the timing manipulations and for creating the experimental stimuli. The musicians agreed with this choice.Participatory Discrepancies (PDs) of the performance with respect to timing. The PDs are given in the transcriptions as a numeric value above each note.Figures Direction and the Δ-Magnitude of the scaling.In a second step, 12 versions were produced for each of the two original recordings (swing and funk) by scaling the microtiming deviations with respect to the metronomic grid. The manipulations were governed by two variables: the Direction variable had two levels, Reduction and Expansion. For Reduction the original deviations were downscaled (in musicians' parlance: the timing gets “tighter”), for Expansion they were upscaled (the timing gets “looser”). The Δ-Magnitude variable determined by which percentage the deviations would be down- or upscaled; it had six levels . Table -Magnitude of 0%, the onset's deviation from the metronomic grid is exactly as in the original recorded performance for both Direction levels (−15 ms). At 100% Reduction, the deviation shrinks to 0 ms, so that the onset is exactly on the metronomic grid. At 100% Expansion, the deviation doubles (−30 ms).The -Magnitude; at the 100% Reduction level, the music was perfectly quantized. In the Expansion series, the timing became looser with higher levels of of Δ-Magnitude. At the 100% Expansion level, deviations were doubled in magnitude.Each individual event onset was down- or upscaled according to this rule, based on its original timing deviation from the metronomic grid. As a result the 0% Reduction and Expansion versions were identical and their timing was exactly as played by the musicians. In the Reduction series, the timing became tighter with higher levels of ΔWith 12 stimuli in either style a total of 24 stimuli was prepared for the experiment. For further details about the preparation of the recordings and the creation of the stimuli, please refer to Kilchenmann and Senn . The stiEmotional Assessment of Groove (EAG) questionnaire was developed by the authors during a workshop in Lucerne in 2012. The aim of the questionnaire was to obtain listeners' feedback on three aspects that have consistently been associated with the groove experience: (1) participants' urge to move their bodies while listening to music; (2) their feeling of enjoyment, and (3) the effortlessness and flow of the music. The last aspect was inversely conceived as a degree of irritation.The twenty-item Answers were collected on five-point Likert scales . Five items loaded on a factor related to their interest or Enjoyment of the music (α = 0.88). And finally 5 items loaded on a factor concerning the listeners' impression of unnaturalness of the stimuli or their feeling of Irritation (α = 0.97).The questionnaire was validated at Justus-Liebig-University Giessen with 90 students who listened to stimuli unrelated to the experiment. Factor analysis revealed a three-factor structure, as intended by the authors: 9 items loaded on a first factor related to listeners' feeling of The actual experiment was carried out in Lucerne, and the experimental data confirmed the three-factor structure. Thirteen items had factor loadings of ≥|0.5| on one of the three factors, and they were sufficient to create reliable scales; the additional 7 items did not augment reliability. Hence, only these 13 items were used for the present analyses. Items and factor loadings are presented in Table Entrainment items, 0.88 for the five Enjoyment items, and 0.94 for the four Irritation items. Overall, the reliability of the scales is good according to the standards defined by Nunnally and Bernstein  questionnaire was used to measure participants' affective reactions to each listening experience , Arousal (quiet/excited), and Dominance (powerful/powerless). It has successfully been used to measure affective reactions to music in the past . The remaining 81 participants were considered to be musical non-experts. They were enrolled in other, non music-related programs and had 5 years of median experience practicing a musical instrument (IQR = 8.25). Overall, there were 82 female and 78 male participants; their mean age was 24.4 years (SD = 4.3). All participants were fluent German speakers.One hundred sixty participants were recruited at the Lucerne University of Applied Sciences and Arts and at Lucerne University. Seventy nine participants were considered to be music experts: they were enrolled in a program to become professional music performers or music teachers (Bachelor/Master of Arts in Music or Music Pedagogy). The experts had 13 years of median experience practicing a musical instrument . No participant was excluded from the experiment because of the gap detection test result. Participants listened to three test stimuli, practiced filling the SAM and EAG questionnaires presented on the screen, and adjusted playback loudness to a comfortable level. Finally, they could ask questions if anything about the procedure was unclear.The experiment was carried out in a quiet university office. Participants took the listening test one person at a time. The technical setup of the experiment is given in detail in Kilchenmann and Senn . During Style . For each Style, 12 stimuli were presented, grouped in two Direction series , each consisting of six stimuli with different Δ-Magnitude. The presentation of the two series and of the stimuli within the two series was randomly ordered for each participant. Participants triggered the stimuli themselves and filled the EAG and SAM questionnaires after each stimulus. This allowed us to capture their affective state immediately after the listening experience. After completing the first Direction series, they filled the PANAS-d questionnaire, and after the second series the NEO-ffi questionnaire. The Ethics Commission of the Canton of Lucerne approved of the design and the procedure of the experiment.After the trial runs, the experimenter left the room, and the participant was guided through the experiment by on-screen instructions. Each participant was randomly assigned to one R (version 3.0.2). There were six dependent variables: Valence, Arousal, and Dominance from the SAM questionnaire, and Entrainment, Enjoyment, and Irritation from the EAG questionnaire. Out of the six dependent variables, the EAG's Irritation scale was positively skewed (γ1 = 0.74), while all others were approximately normally distributed. In spite of the non-normality of the distribution, the Irritation data were kept in the analyses: The limited range of the Likert-type scale prevents extreme outliers; hence the effects of non-normality can be estimated to be mild.Mixed-design analyses of variance were performed using Style  and Expertise . Two further variables were within-subjects: Direction  and Δ-Magnitude  encoded the timing manipulations.In the original design, there were four independent variables, two of them between-subjects: Direction and Δ-Magnitude was problematic. In particular, the Δ-Magnitude variable turned out to be poorly specified: its levels pool responses to stimuli with diverging timing patterns. This led to heteroscedasticity among the levels of Δ-Magnitude; and the main effect of the variable was not interpretable. In order to solve this problem, the timing manipulations were newly encoded using a variable called Signed-Δ-Magnitude. Table -Magnitude and Direction levels were projected onto the levels of Signed-Δ-Magnitude. The combinations from the Reduction series are negatively signed and those from the Expansion series have positive signs. The numeric values from Δ-Magnitude have not changed, except for the sign. The two Δ-Magnitude/Direction combinations referring to the stimuli with the originally performed timing  have both been preserved as separate levels of Signed-Δ-Magnitude  in order to keep group sizes balanced; so, Signed-Δ-Magnitude has twelve levels. In summary, the re-encoding maps the timing manipulations from the two-parameter Δ-Magnitude / Direction setup onto a single Signed-Δ-Magnitude variable that orders the data according to the size of the PDs in the stimuli.During the peer review process for this paper it became clear that the parametrization of the timing manipulations using Style, Expertise (both between subjects), and Signed-Δ-Magnitude (within subjects). The three-way mixed-model ANOVAs tested for effects on all dependent variables: Valence, Arousal, Dominance, Entrainment, Enjoyment, and Irritation. The overall significance level was set to α = 0.05. Šidàk correction , measurements were incomplete due to technical problems; this participant's data were excluded from analysis. Hence, the following results are based on a sample of 159 participants.Signed-Δ-Magnitude had a highly significant effect on Valence, Entrainment, Enjoyment, and Irritation. The main effect of Signed-Δ-Magnitude on Dominance was only near-significant due to the Šidàk correction.The results of the analyses of variance are reported in Table Signed-Δ-Magnitude×Expertise interaction effect was measured on the Irritation scale. The Signed-Δ-Magnitude×Expertise interaction effects on Entrainment and Enjoyment were near-significant. Style and all interactions involving Style did not have a significant effect on any of the dependent variables.A significant Valence, Entrainment, Enjoyment, and Irritation responses for all levels of Signed-Δ-Magnitude. For convenience, the two original Direction series are marked with red (Reduction) and blue lines (Expansion). We observe similar patterns across the mean Valence, Entrainment, and Enjoyment ratings. The ratings were high for negatively signed levels of Signed-Δ-Magnitude; and they decreased for positively Signed-Δ-Magnitude levels. The mean Irritation ratings mirror this pattern: listeners were little irritated while listening to the stimuli of the negatively signed Signed-Δ-Magnitude levels, but Irritation increased for higher positively signed levels of Signed-Δ-Magnitude.Figure Post-hoc pairwise Tukey HSD tests were carried out to pinpoint effects between the twelve Signed-Δ-Magnitude levels  were rated low on groove in comparison to at least one of the other levels of Signed-Δ-Magnitude. Irritation registered these effects most markedly; the largest effect was measured between the −60% and +100% levels of Irritation . The −100% level (quantized stimuli) and the −0% and +0%  levels obtained high groove ratings that were not significantly different from each other on any of the scales.Signed-Δ-Magnitude and the mean ratings. In order to illustrate these relationships, quadratic regression models were fitted to the 1908 data points for each dependent variable. Table Valence, Entrainment, Enjoyment, and Irritation. In each case, the quadratic model fit was significantly better than the best alternative linear model. The quadratic models' lines of best fit are printed in the background of the Figure The plots of Figure Valence ratings for −60.3%, maximum Entrainment for −68.1%, maximum Enjoyment for −56.7%, and minimum Irritation for −53.7% Signed-Δ-Magnitude. All models predict the “point of greatest groove” at approximately −60% Signed-Δ-Magnitude, i.e., roughly halfway between the stimuli with fully quantized timing and the stimuli with original timing.The extrema of the parabolae are indicated by arrows: the quadratic models predict maximum Signed-Δ-Magnitude × Expertise interaction was significant for Irritation  = 20.099, p < 0.001, η2 = 0.205] was considerably larger than the effect on Non-Experts . Figure Irritation responses for the two groups. The plots show a similar general response pattern for Experts and Non-Experts: Irritation ratings are low for all negatively Signed-Δ-Magnitude levels, they increase for the higher positive levels of Signed-Δ-Magnitude. Expert listeners' responses show this pattern more distinctly than the Non-Expert listeners' responses.The on Table . The timIrritation compared to any of the lower levels of Signed-Δ-Magnitude. The timing manipulations in the range from −100% to +60% did not significantly affect the Irritation of Non-Experts. The largest effect in Non-Experts was measured between the −40% and the +100% levels of Signed-Δ-Magnitude .Non-Experts reacted to exaggerated microtiming with seemingly little sensitivity: Tukey HSD pairwise comparisons Table  show thaIrritation ratings were significantly higher than the ratings for several lower levels of Signed-Δ-Magnitude. The difference of the ratings between the neighboring +20% and +40% levels was near-significant (p = 0.073). We can summarize that the timing manipulations did not significantly affect the Irritation of Experts in a range between −100% and +20% but increased strongly above +20%. The largest effect in Experts was measured between the −60% and the +100% levels of Signed-Δ-Magnitude .Expert listeners reacted more sensitively to exaggerated microtiming than Non-Experts: the pairwise comparisons of Table Signed-Δ-Magnitude than Non-Experts: the parabola summarizing the Experts' responses in Figure Signed-Δ-Magnitude (−57.2%), compared to Non-Experts (−47.4%).Quadratic regression models Table  support Valence, Entrainment, Enjoyment, and Irritation) were associated with the timing manipulations. These variables showed a distinct pattern of correlations: Valence, Entrainment, and Enjoyment were mutually positively correlated, the strongest correlation was observed between the EAG's Entrainment and Enjoyment variables  was the dependent variable of Kilchenmann and Senn  = 2.912, p = 0.004, d = 0.462]. With respect to the other personality factors, the two Expertise groups did not differ significantly. The table also presents NEO-ffi scores from a representative German sample (n = 1908) as a reference  reported by Krohne et al.  = 6.535, p < 0.001, see also Table These findings contrast with results from Kilchenmann and Senn  in two p0.148, t106 = 6.53EAG ratings show that expert listeners rated stimuli with exaggerated microtiming to be low on groove, even though they displayed inreased entrainment to these stimuli, as reported in the earlier study. So, the magnitude of entrained body movement is not necessarily associated with groove ratings. It seems that experiencing the enjoyable groove urge is not the only reason why people entrain to music.This means that listeners did not always move along with the music, when they reported an urge to move, and they did not always report an urge, when in fact they did move along with the music. For exaggerated timing, the observations offer some support to an explanation proposed in Kilchenmann and Senn . There, Entrainment ratings is puzzling: listeners experienced the urge to move, but they did not act on it. Why would they hold back? It seems that the expert listeners thought the quantized music inspired them to movement, when in fact it did not. Followers of PD theory might claim that the quantized stimuli lacked PDs and therefore did not have the power “to make us dance, make us want to participate” , while they disagree for the quantized stimuli . This offers some weak evidence against the Findings support our second hypothesis that experts would be more sensitive to microtiming manipulations than non-experts. Both experts and non-experts showed a similar general irritation response pattern to the microtiming manipulations: small PDs were associated with low irritation, and exaggerating the magnitude of the PDs beyond the originally performed magnitude eventually irritated the listeners. But expert listeners reacted more strongly to the manipulations, compared to the non-expert listeners. Also, experts were more sensitive to the magnitude of the timing manipulation: An increase of PD magnitudes by +40% was enough to trigger irritated responses by the experts, whereas non-experts' irritation increased significantly only when PD magnitudes were exaggerated by +80% or more. This resonates with results from Kilchenmann and Senn  who repoPD theory that microtiming generally enhances the groove experience. Non-experts seem to react less to microtiming phenomena than the experts. So, if PDs turn out to have an influence on the groove experience, musical expertise might be an important mediator that determines whether this influence is felt or not. Given that musical experts were strongly involved in the development of PD theory, the claims of this theory might predominantly reflect their expert perspective on music. The PD effect might turn out to be a treat for the musical elite.The expert group's sensitivity to microtiming deviations is not surprising: we expect music experts to have a refined perception of timing nuance, due to their experience and training. However, this dependency on expertise casts a doubt on the claim of PANAS) or personality dimensions (NEO-ffi) were not associated with groove ratings or head movement.Apart from expertise, we did not find any other person-related effects on groove reactions. Affective disposition Δ, as a summary statistic for quantifying the magnitude of microtiming phenomena in performed music. It is calculated as follows:ti is the absolute time of the ith event onset in seconds; n is the overall number of events in the music clip, and bpm is the tempo of the music in beats per minute. The tempo-adjusted standard timing deviation is given as a fraction of the metronomic beat duration.How can we compare the microtiming magnitudes of the swing and funk performances? Previous studies have shown that the just-noticeable difference of timing deviations in isochronous auditory sequences depends on tempo Δ = 0.026 beats; in the swing clip, it amounts to st(B)Δ = 0.068 beats. The large st(B)Δ difference between funk and swing depends on the absolute deviations (in ms), which were larger in swing than in funk, but also on the tempo difference (150 bpm in swing vs. 100 bpm in funk).The timing deviations found in the two originally performed and recorded clips can be inspected in Figures The large timing deviations in swing and the much smaller timing deviations in funk resulted in similar groove ratings when scaled according to the same rules. So, both the performing musicians and the listeners appear to agree on how much microtiming is acceptable in either style. One remarkable aspect of this result consists in the fact that this observation not only concerns the expert group among the participants. The non-experts react similarly to timing manipulations in the two styles, they were simply less sensitive to exaggerated timing deviations. Overall, there seems to be at least an implicit style-dependent notion of adequate vs. exaggerated microtiming in the analyzed population, regardless of expertise.st(B)Δ = 0.020 beats, which is smaller than the st(B)Δ = 0.026 beats measured for the originally performed funk example of the present study Δ = 0.068 of the originally performed swing example). We can conclude that the ratings reported by Frühauf et al.  that no definite conclusions can be drawn. However, we can at least formulate a new hypothesis: we suspect that the patterning of the microtiming deviations makes a relevant difference. Frühauf et al.  created EAG allow to ask very specific questions, and the measured effects seem to be quite strong. On the downside, filling a questionnaire with several scales and several items per scale is time-consuming: the 20-item EAG was rather too long for application in a listening experiment. The laborious task tested the patience of many participants, which in turn may influence the groove experience negatively. Hence, it is certainly beneficial to shorten the questionnaire, preferably without compromising reliability. A first step is reducing the EAG to the 13 items that have been used in the present analysis. In due course, an English version of the questionnaire will be prepared and made available to the scientific community.A final note on methodology: for Kilchenmann and Senn  and for Entrainment scale) rarely agreed with each other. If both methods assess the intensity of the same underlying experience, then the results should confirm one another. This, however, was not always the case. It seems that the groove experience is not that easy to measure. It will take considerable effort to develop reliable methods to assess a listener's groove experience and bodily entrainment.A central methodological problem lies in the disagreement between results derived from the two approaches: participants' bodily entrainment behavior and their subjective groove ratings (particularly on the PD theory) or weakens (exactitude hypothesis) the groove experience in listeners. Both perfectly quantized stimuli and stimuli with PD patterns arising during a competent performance obtained similarly high groove ratings. Hence, if we interpret the theories in their strict mutually exclusive sense, neither is fully supported by the data: PDs are not mandatory for music to groove (as PD theory suggests), and the groove experience is not necessarily damaged when music is not perfectly quantized (as claimed by the exactitude hypothesis).Past research  and the Deutsche Forschungsgemeinschaft (grant BU 2259/1-1 to CB).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.


IDUA gene causing the accumulation of glycosaminoglycans (GAGs). Historically, children with the severe phenotype, MPS-IH (Hurler syndrome) develop progressive neurodegeneration with death in the first decade due to cardio-pulmonary complications. New data suggest that inflammation may play a role in MPS pathophysiology. To date there is almost no information on the pathophysiologic changes within the cerebral spinal fluid (CSF) of these patients. We evaluated the CSF of 25 consecutive patients with MPS-IH. While CSF glucose and total protein were within the normal range, we found a significantly mean elevated CSF opening pressure at 24 cm H2O (range 14–37 cm H2O). We observed a 3-fold elevation in CSF heparan sulfate and a 3–8 fold increase in MPS-IH specific non-reducing ends, I0S0 and I0S6. Cytokine analyses in CSF of children with MPS-IH showed significantly elevated inflammatory markers including: MCP-1 SDF-1a, IL-Ra, MIP-1b, IL-8, and VEGF in comparison to unaffected children. This is the largest report of CSF characteristics in children with MPS-IH. Identification of key biomarkers may provide further insight into the inflammatory-mediated mechanisms related to MPS diseases and perhaps lead to improved targeted therapies.Mucopolysaccharidosis (MPS) type-IH is a lysosomal storage disease that results from mutations in the  IDUA gene resulting in decreased/absent alpha-L-iduronidase activity. The consequent accumulation of the glycosaminoglycans (GAGs), heparan sulfate (HS) and dermatan sulfate (DS), in tissues results in a number of clinical features including hepatosplenomegaly, progressive cognitive impairment, cardiovascular complications, and joint and bone abnormalities (dysostosis multiplex)Severe mucopolysaccharidosis type I, Hurler’s syndrome (MPS-IH), is a lysosomal storage disease due to mutations in the To achieve continuous enzyme delivery as well as provide a cerebral source of cells expressing alpha-L-iduronidase (presumed to be microglia), hematopoietic cell transplant (HCT) is used as standard of care for patients with Hurler syndrome3568910121516As mentioned, prior to the development of HCT, children with MPS-IH were observed to develop progressive, debilitating developmental and cognitive deterioration51819202O (range 14–37 cm H2O). This is higher than what is considered a normal OP in children of this age, which is <20 cm H2OWe performed a lumbar puncture and CSF analysis on 25 consecutive patients with MPS-IH with a median age of 11.2 months. Nearly all CSF was free from erythrocytes or white blood cells. As shown in ® assay, we measured NREs characteristic for MPS-IH, I0S0 and I0S6, and also determined total HS concentrationUsing the Sensi-Pro We also evaluated HCII-T (heparin cofactor II-thrombin) complex, a previously described biomarker of lysosomal storage diseases including MPS-IHWe found six inflammatory markers to be significantly elevated in children with MPS-IH when compared to controls: monocyte chemoattractant protein-1 (MCP-1) , stromal cell-derived factor-1a (SDF-1a) , interleukin-1 receptor antagonist (IL-Ra) , macrophage inflammatory protein 1-beta (MIP-1b) , interleukin-8 (IL-8) , and vascular endothelial growth factor (VEGF)  as shown We report potential CSF biomarkers for patients with MPS-IH. These may be important to consider as further therapies are being developed either through immunomodulation, hematopoietic stem cell transplant, new forms of enzyme therapy and other interventions. These biomarkers may serve as indicators to which we can compare the effectiveness of new interventions. In addition, they may prove useful as a means of identifying future phenotypes in children diagnosed through newborn screening that display novel or poorly characterized genotypes.20et al. analyzed the OP of 197 children and found a mean OP of 19.6 cm H20th/90th percentage of Avery’s data was 11.5 and 28 cm H2O, it has been suggested that an abnormal OP should be consider that of >28 cm H2O232O. While sedation and changes in ventilation can both modulate the OP2 monitored and maintained from 25–40 mm Hg. Classically, elevated OP is associated with intracranial processes such as infection , subarachnoid hemorrhage, pseudotumor cerebri, or any communicating hydrocephalus. Our data suggest that GAG accumulation and perhaps subacute neuroinflammation may contribute to an increase in OP. We should note that none of our patients has evidence of papilledema suggesting that their increased OP was not severe enough to affect the optic nerve head.We found the mean CSF OP was higher in children with MPS-IH than what is considered “typical” for healthy children, which is <20 cm Het al. previously found TNF-alpha to be elevated in MPS VII mice and treatment of MPS VI affected rats with Infliximab, an antibody targeted to TNF-alpha, significantly reduced joint diseaseAn inflammatory process has been implicated as a pathological contributor to MPS diseaseet al. showed significantly high levels of MCP-1 in the brain of MPSI, MPSIIIA, and MPSIIIB miceIn this study, we demonstrate for the first time that markers of inflammation are manifest in the CSF of MPS patients. Our data is consistent with what others have shown in the brain of MPS animal models. For example, Wilkinson Whether the CSF inflammatory mediators are due to GAG accumulation or another process is not known. There are very few reports of CSF GAG evaluation in patients afflicted with a mucopolysaccharidosis diagnosis2829313233342 monitored and maintained from 25–40 mm Hg to ensure opening pressure accuracy.Patients with MPS-IH  had CSF sampling performed 8 weeks prior to hematopoietic stem cell transplant at the University of Minnesota. During the initial evaluation including a sedated MRI, a lateral decubitus lumbar puncture is routinely performed with an opening pressure (OP) measurement and CSF was obtained and analyzed for cell count, protein concentration, glucose concentration, GAG concentration and cytokine analysis. End-tidal COControl patients for biomarker analyses  were those undergoing intrathecal chemotherapy for a prior diagnosis of acute lymphoblastic leukemia, at least 3 months into maintenance therapy, and without a CSF leukemia diagnosis. In controls, CSF was withdrawn just prior to administration of the intrathecal chemotherapy and cytokine concentrations determined by ELISA. Unavailability of “healthy” controls due to the risks inherent to attaining CSF from “healthy” children established these patients as the most appropriate control group available and has been previously published by our group and others3738CSF samples were evaluated using the 22-plex, human panel A,  measured with the Luminex system  and analyzed by Bioplex software . This panel includes ENA-78, bFGF, G-CSF, GM-CSF, IFN-gamma, IL-1alpha, IL-1beta, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17, MCP-1, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, TPO, and VEGF as shown in HCII-T complex was determined by ELISA, following the manufacturer’s instruction .® assay as previously describedThe CSF NREs (I0S0 and I0S6) and total HS  were determined using the Sensi-Pro Cytokine measurements were made in duplicate and the average of the two values was used to determine concentration using standard curves generated with the relevant recombinant human proteins provided with the commercial kits. Means for the MPS-IH and control groups were calculated and subjected to a two-tailed Student’s t-test to compute a p-value.How to cite this article: Raymond, G. V. et al. Elevated cerebral spinal fluid biomarkers in children with mucopolysaccharidosis I-H. Sci. Rep.6, 38305; doi: 10.1038/srep38305 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate.Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs.  Infectious Hantaan virus (HTNV) is a major cause of hemorrhagic fever with renal syndrome (HFRS). In China, it refers to about 100,000 of patients every year . HoweverHTNV viral genome contains three fragments, L, M and S, which encode RNA depended RNA polymerase (RdRp), membrane glycoprotein  and nucleoprotein (NP) respectively . Both GP® and Gardasil® are composed of VLPs which can provide cross protection for different HPV serotypes, also induce ten times higher neutralizing antibodies than natural infection [VLPs usually emerge in natural virus life cycles, which consist of viral structural protein . The fornfection , 16. Altnfection .It has been mentioned above that the expression of M segment in host cells can lead to production of VLPs that were composed of spherical membrane vesicles, with or without nucleocapsid, similar to the morphology of natural virions, but we need VLPs with full components in order to elicit both neutralizing antibodies and virus-specific cytotoxic T lymphocytes. In order to enhance the immunogenicity, the gene expressing VLPs can be altered to display non-structural proteins such as cytokines and specific antigens  that usudhfr− CHO and obtained HTNV VLPs anchoring CD40L or GM-CSF, the protein composition and the structure were verified by Western blot. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. Data of animal research suggest that humoral immune responses and cellular immunity induced by CD40L or GM-CSF decorated HTNV VLP were superior to undecorated VLPs as well as HTNV vaccine, and able to reduce viral load in immunized mice. These results indicated that the recombinant HTNV VLP could be a promising vaccine candidate.In this study we constructed plasmids containing HTNV M segment and CD40L/GM-CSF gene, co-transfected with vector containing S segment into In order to express CD40L/GM-CSF in cis along with HTNV antigens, we developed the VLPs by inserting the membrane bound form of a murine CD40L/GM-CSF gene downstream of the HTNV M gene of our plasmid that expresses HTNV GP. We verified the plasmid by nucleic acid electrophoresis and bands can be seen at 780 bp, and 530 bp , 1.3 kb,dhfr− CHO cells by co-transfected with pCI-S and pCI-M, pCI-S and pCI-M-CD40L, pCI-S and pCI-M-GM-CSF. Purified HTNV VLPs exhibited a consistent size of approximately 100 nm under transmission electron microscopy, in addition, HTNV VLPs were spherical and pleomorphic both in transfected cells  are significantly higher than in the VLP group (68.4%) (p=0.0065)  and M-CSF/GM-CSF as positive controls. As shown in Figure ) Figure , which i) Figure  which ar) Figure . These rPurified recombinant VLPs antigens were tested for the ability to induce specific antibodies against each HTNV NP antigen by ELISA. Sera were obtained from each immunized mice and tested to assess HTNV antigen specific antibodies. NP or GP specific antibody titers were higher after immunization with CD40L-VLP compared to other VLP groups and vaccine group (p=0.0022 for NP and p=0.0015 for GP). However, GM-CSF-VLP group did not have advantage over undecorated VLP groups (p=0.1038 for NP and p=0.0928 for GP), but got advantage over vaccine and saline controls (p=0.0003 for NP and p=0.0004 for GP) Figure . We did Neutralizing antibody responses to HTNV were determined by ELISA analysis of the sera from immunized mice in order to evaluate the effective humoral immune response induced by the VLPs. There is no significant difference amongst VLP group and VLP co-immunized with CD40L/GM-CSF p=0.2567). Compared with vaccine group and saline control, the geometric mean for the neutralizing antibody titer of the CD40L-VLP/GM-CSF-VLP were significantly higher (p=0.0003 for CD40L-VLP and p=0.0002 for GM-CSF-VLP) Figure . The mea567. CompSera from immunized mice were tested for HTNV NP specific antibody subtypes by sandwich ELISA. We detected IgG1, IgG2a, IgG2b, IgA and IgM. The ratio of IgG2a/IgG1 is an indicator of humoral protective effect after viral infection. Sera from mice, which were immunized with CD40L-VLP or GM-CSF-VLP showed higher IgG2a compared to undecorated VLPs (p=0.0031 for CD40L-VLP and p=0.0033 for GM-CSF-VLP), however sera from vaccine group and PBS control showed higher IgG1. Though there were differences in IgG2b, the overall level of this subtype is low Figure . We alsoSpleen cells were isolated on day 34 (7 days after the last immunization) to assess the cellular immune response to the recombinant HTNV VLP. IFN-γ, IL-2, IL-4 and IL-10 cytokine were analyzed by ELISPOT, following stimulation with HTNV GP or NP peptides. There was no significant difference in levels of IFN-γ and IL-2 from undecorated VLP groups compared to inactivated HTNV virion vaccine group (p>0.05). The number of splenocytes secreting IFN-γ and IL-2 were higher in the CD40L or GM-CSF decorated VLP group compared to the vaccine group Figure . The oveMacrophages could be infected by HTNV, which were used as target cells by immune fluorescence assay , HTNV anWe tested the protection ability provided by VLPs by challenging immunized mice with infectious HTNV. Three days after viral challenge, the viral load of heart, liver, spleen, lung, kidney and cerebrum were tested by sandwich ELISA as well as qRT-PCR. We detected small amount of viral antigen Figure  and RNA VLPs have similar structural and physicochemical features with origin virions, however they are non-infectious and possess advantages in terms of safety and manufacturing . One of dhfr gene deficient CHO cell line, which cannot survive in medium without hypoxanthine and thymine (HT) supplement. The pCI-S-neo and pCI-M-CD40L/GM-CSF plasmid contain two parts of dhfr gene, only when the two plasmids are correctly transfected can the deficient cells survive in medium without HT. The dhfr deficient CHO cells are sensitive to G418, but pCI-S-neo can provide G418 resistance, that is to say, correctly transfected cells can survive in medium containing G418. We also applied MTX in culture medium, which can suppress the expression of dhfr gene; it must be amplified to keep cells alive. CD40L/GM-CSF was located downstream of dhfr, the amplification can spur the expression of CD40L/GM-CSF. These measurements can ensure the correct expression of all components of the VLPs we needed. In vitro stimulation with CD40L/GM-CSF decorated VLPs was able to promote bone marrow cells towards DCs and macrophage lineage, which indicate that VLPs had desired construction and basic function.To ensure the expression of VLPs, we used a in vivo [The analysis of humoral immune responses revealed that antigen specific IgG CD40L-VLP and GM-CSF-VLP induced were higher than that of undecorated VLPs, and even higher than HTNV vaccine used in clinical practice. We also found that antibody subtypes changed in sera from immunized mice, CD40L-VLP and GM-CSF-VLP had highest ratio of IgG2a/IgG1, revealing the anti-viral ability of antibodies is high in vivo , 25. Neuin vivo , the lowin vivo . Howeverin vivo –34. ResuThe protective efficacy of the VLPs was tested by viral challenge of immunized mice. The results showed that immunization with either CD40L-VLP or GM-CSF-VLP conferred the most protection, against HTNV challenge, which has the least viral load in important organs. However histopathological analysis show little difference in numbers of lymphocytes except in spleen. This is probably because HTNV can only infect adult mice causing no pathological events and we intend to use suckling mouse to assess the protection effect of the VLPs in our further study.dhfr deficient CHO cells and the immune effect of the VLPs is described. We successfully produced VLPs with desired protein components and construction. The CD40L or GM-CSF decorated VLP induced stronger humoral immune responses against HTNV as well as cellular immunity. Moreover, protective efficacy in mice was better with the CD40L or GM-CSF decorated VLP than with the vaccine. Further study is needed to adjust the productivity of the VLP producing dhfr− CHO cells and to clarify the protective efficacy of that is induced.In conclusion, the construction of a recombinant HNTV VLP vaccine candidate, based on dhfr− cells, purchased from Culture Collection of Chinese Academy of Science, Shanghai) were maintained in 37°C with 5% CO2 in ISCOVE's modified DMEM (IMDM) (Gibco) supplemented with 10% fetal bovine serum (Gibco), 2% HT mix (Sigma), and 100nM methotrexate (MTX) (Sigma). Mouse macrophage cells and bone marrow cells were separated from healthy C57BL/6 mice and similarly cultured in DMEM supplemented with 10% FBS. HTNV strain 76118 was kept in our lab, and all related experiments were performed in a biosafety level 2 (BSL-2) setting.Dihydrofolatereductase deficient CHO cells  and treated with 0.5% Triton-X100 at room temperature for 15 min. After washing with PBS, mouse anti HTNV Gc monoclonal antibody and rabbit anti CD40L or GM-CSF polyclonal antibodies were incubated with fixed cells at 37°C for 1h. Cells were then washed with PBS and incubated with FITC-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit secondary antibody at 37°C for 1h. DAPI was used for nuclear dye. Finally, the cells were washed with PBS and observed using fluorescence microscopy.Indirect fluorescence assays (IFAs) were performed to assess the expression of GP, CD40L and GM-CSF. Dhfr− cells were cotransfected with pCI-S and pCI-M, pCI-S and pCI-M-CD40L, pCI-S and pCI-M-GM-CSF respectively. The cells were cultured for 24h after transfection, and then cells were carefully washed with DPBS before replacing culture medium with serum-free CHO medium (Hyclone). Supernatants were collected 72h after transfection. Culture supernatants containing VLPs were harvested and clarified by low-speed centrifugation at 1000rpm for 10 min at 4°C followed by ultracentrifugation at 30,000rpm for 60 min to pellet. Sedimented particles were suspended in phosphate-buffered saline (PBS) at 4°C.Characterization of HTNV VLPs GP, NP and CD40L or GM-CSF was performed using a sodium dodecyl sulfate (SDS) 10% polyacrylamide gel; Coomassie stain and Western blot analysis. Total VLP protein amounts of 10μg, were loaded into the gel and transferred onto a PVDF membrane using a Mini Trans-Blot . Membranes were blocked using 5% BSA at room temperature for 1h and then incubated with HTNV Gc monoclonal antibody and CD40L or GM-CSF polyclonal antibodies overnight at 4°C. Membranes were washed with PBS containing 0.5% tween-20 (PBST), and then incubated with infrared conjugated antibody and proteins were visualized using an infrared imaging system.Coexpression of HTNV GP and CD40L or GM-CSF was examined by ELISA. We coated a 96-well plate with Gn or Gc monoclonal antibodies at 4°C overnight, the plate was washed with PBST, then samples containing CD40L-VLP and GM-CSF-VLP were addes into the plate and incubated at 37°C for 1 h. Anti CD40L or GM-CSF antibody was added into the plate and incubated at 37°C after washing with PBST. The uncombined antibodies were washed away and HRP conjugated secondary antibodies were added. After incubation at 37°C for 1h, TMB solution was added and 450nm absorbance was detected. The coexpression was further tested by immunoprecipitation. Purified CD40L-VLP and GM-CSF-VLP were incubated with CD40L/GM-CSF antibody or Gn antibody at 4°C overnight. Then we used Protein G beads to capture the antibodies. The samples were boiled with SDS and the beads were discarded before they were subjected to Western blot.Dhfr− CHO cells cotransfected with pCI-S and pCI-M, pCI-S and pCI-M-CD40L, pCI-S and pCI-M-GM-CSF expressing HTNV-VLP, CD40L-VLP and GM-CSF-VLP respectively, were fixed with 1% phosphotungstic acid for 1-2 min to examine the budding of VLPs at 200,000×. Supernatant was dropped onto a piece of bronze net and excess stain was wicked away. The samples were air dried for 1-3 min. VLPs were observed using a H8100 transmission electron microscope at a magnification of 300,000X.6/well nucleated cells were added to 6-well tissue culture plates and cultured in 1 mL of RPMI1640 medium containing 10% FCS. Then the VLPs were added to observe differentiation toward macrophage/DCs lineage that was compared with M-CSF or GM-CSF positive control. After 6 days in culture, contaminating non-adherent cells are eliminated and adherent cells are harvested for BMDM assays. Cells were cultured for 6 d and then harvested for flow cytometry. Population of macrophages is determined by staining with F4/80 and CD11b antibodies, DCs were determined with CD11c and MHC-II antibodies.Bone marrow was flushed from femurs and tibias from healthy C58BL/6 mice. A total of 1× 10FACS analysis were performed with routine protocols using the FACS Calibur flow cytometer (BD Immunocytometry Systems), antibodies used for mice spleen lymphocyte staining are as following: CD3-Percp cy5.5, CD4-FITC, APC, CD8-Percp cy5.5, PE, CD19-FITC, B220-PE, CD69-FITC, CD80-FITC, CD86-APC, CD40-PE (Biolegend) and bone marrow staining: F4/80-PE, CD11b-FITC, MHC-II-PerCP cy5.5 (Biolegend). FACS buffer; 1 × PBS (Gibco) + 2% FBS (Sigma). All data were analyzed using FlowJo .Experiments with mice were conducted in compliance with a protocol approved by the Institutional Animal Care and Use Committee based on the Ethical Principles in Animal Experimentation. Specific pathogen-free female C57BL/6 mice aged four to five weeks, were divided into seven groups according to immunogen, each group contains 12 mice. Animals were inoculated intraperitoneally (i.p.) with CD40L-VLP, GM-CSF-VLP, and undecorated HTNV-VLP respectively (100μg per dose and in 12 mice (n=12)). Same number of mice was inoculated intraperitoneally with HTNV-VLP and mouse CD40L , HTNV-VLP and mouse GM-CSF (VLP+GM-CSF group). HTNV vaccine (β-propiolactone inactive HTNV viron) was subjected to equal number of mice (100μl per dose). Equal amounts of PBS were used as controls Table . Blood sSera from each group of mice were assessed for immunoglobulin by ELISA. Full length HTNV NP or Gc (0.15μg) was coated to each well of 96-well plates and incubated at 4°C overnight. The sera were serially diluted with PBS from 1:20 to 1:1024 and add into NP/Gc coated 96-well plates. After incubation at 37°C for 1h, plates were washed with PBST and then diluted HRP conjugated goat anti mouse IgG antibody was added to each well. Subsequently plates were incubated at 37°C for 1h and then washed with PBST, then TMB solution was added and absorbance at 450 nm was recorded. The titer was determined in samples that had maximum dilution still showing positive result.In order to distinguish the immunoglobulin subtypes, full length HTNV NP (0.15μg) was coated using similar method above. Sera from each group of mice were diluted and HRP conjugated IgG to mouse IgG1, IgG2a, IgG2b, IgA and IgM were applied. Absorbance at 450 nm was assessed to determine the amount of each immunoglobulin subtypes.−5 TCID50) at 37°C for 1h. The virus-serum mixture was aspirated, added along with 0.2 ml overlay medium, and incubated at 37°C with 5% CO2 for 10 days. Plates were subjected to RT/−80°C freeze-thaw cycles 3 times and the supernatant was collected for ELISA. We used HTNV NP monoclonal antibody as capture antibody and HRP-1A8, which is an HRP conjugated HTNV NP monoclonal antibody, as detecting antibody. Neutralizing antibody titers are defined as the maximal serum dilution that 3 out of 4 duplicated wells do not show positive of HTNV NP.Because HTNV do not form plaque in Vero E6 cells, the neutralizing ability of antibodies was measured using a sandwich ELISA method. Briefly, Vero E6 cells were grown to 60% confluence in 96-well plates. Then plates were pre-incubated with serial dilutions of mouse serum  together with HTNV (1×106 cells) from the immunized mice were subsequently transferred to each well and stimulated with NP or Gn/Gc peptide pools at 37°C for 24 h. The cells were then washed away, and a secondary biotinylated anti-cytokine mAb was added to each well, followed by a streptavidin-HRP and AEC substrate solution system. Finally, the spots were counted using an ImmunoSpot® Analyzer (Cellular Technology Ltd. USA)The cytokines, IFN-γ, IL-2, IL-4 and IL-10, from immunized mice were measured using ELISPOT kits (Mabtech), according to the manufacturer's instructions. Briefly, ELISPOT 96-well plates (supplied by the kit) were coated with 100 μl of anti-mouse IFN-γ, anti-mouse IL-2, anti-mouse IL-10, or anti-mouse IL-4 (5μg/ml in coating buffer). The plates were washed twice and blocked with blocking solution for 2h. A volume of 100μl freshly isolated splenocytes  was added to each well. Then a serial of different numbers of splenocytes were applied to the wells . The plates were incubated at 37°C for 6 h, and lactate dehydrogenase (LDH) released was assessed. The percentage of specific lysis was calculated using the following formula.Macrophages separated from healthy mice were infected with 100 TCIDFour mice from all 7 groups were injected intracerebrally with 100μl of infectious HTNV. Three days after injection, the mice were euthanized and tissues from heart, liver, spleen, lung, kidney and cerebrum were collected and divided into two halves. One half was weighted and triturated in PBS. The total protein was measured by BCA assay and all samples were diluted to 1mg/ml, then HTNV antigen was analyzed using the same ELISA method with neutralizing antibody assay.Total RNA of the other half of tissue was extracted following manufacturer's instructions (Axygen), and reverse transcripted into cDNA using a reverse transcription kit (Takara). Copies of HTNV S segments were counted through RT-PCR analysis using a SYBR green RT-PCR kit (Takara), and mouse GAPDH was used as reference.Data were analyzed for statistical significance using GraphPad Prism 5 software. Percentage of flow cytometry was compared using one-way ANOVA, and data were expressed as the mean ± SEM. Statistical analysis of antibody titer was performed using Kruskal-Wallis test, and titers were expressed as the geometric mean. Comparison of antibody subtype level were also using Kruskal-Wallis test. Results of ELISPOT assay were compared by one-way ANOVA, and CTL assay was compared using two-way ANOVA. Protective efficacy was compared using one-way ANOVA. Data was presented as the mean ± SEM.


Rheumatoid meningitis (RM) is a rare disorder that often develops during a remission phase of rheumatoid arthritis (RA). This is the first study to demonstrate differences in regard to immunological disturbance between blood and cerebrospinal fluid (CSF) samples obtained from a patient with RM using flow cytometry.+ Th2 cells and marked decrease in CD8+ cells, suggesting an immunoregulatory disturbance in the CSF. Those findings indicated a CSF-specific activation of humoral immunity, resulting in augmentation of meningeal inflammation, as shown by excess synthesis of intrathecal IgG and markedly elevated interleukin-6 level. Results of the present detailed investigation of lymphocyte subsets revealed a discrepancy regarding the process of inflammation in this RM patient between CSF and blood samples.A 36-year-old woman with RA and generalized myasthenia gravis (MG) developed RM during a remission phase. Although both RA and MG were stable and well controlled, she noticed fever, headache, and transient sensory disturbance. Blood and CSF examination findings suggested aseptic meningitis, while brain magnetic resonance imaging revealed restricted portions of meningitis and associated cortical lesions, compatible with a diagnosis of RM. The dose of oral prednisolone was increased, which ameliorated the symptoms within 1 week along with improvement in CSF findings. This patient exhibited features of RM that were manifested in a manner independent of the activity of RA. An investigation of cellular immunity using CSF specimens with flow cytometry showed differences in regard to the pathogenesis of inflammation in the CSF as compared to outside of the central nervous system. In contrast to results obtained with paired blood samples, CSF cells at the peak stage of RM showed a marked increase in CCR3RM is not a simple reflection of the immune status of RA, as the pathogenesis seems related to, at least in part, CSF-specific immunological dysregulation. Development of rheumatoid meningitis (RM), a rare autoimmune inflammatory disease of the central nervous system (CNS), is often seen in well-controlled rheumatoid arthritis (RA) patients , 2 or evA 36-year-old woman with a 13-year history of generalized MG was diagnosed with RA 8 years prior to the present episode. The patient had undergone a thymectomy, and both diseases were stable with alternate-day administrations of prednisolone (8 mg) and weekly doses of methotrexate (12 mg) until she exhibited new neurological complications. At the time of the initial visit, the patient reported a 10-day history of headache in the left parietal region and also had a moderate fever of 38 °C. On the sixth day after developing the headache, a transient sensory disturbance (dysesthesia) over the right face developed and then spread to the right upper extremity for 30 min, which recurred twice that day; thus, she came to us and also noted headache and fever but no pain in any joints. Neurological examination findings were negative for meningeal irritation signs, and there were no symptoms of MG including ptosis, diplopia, dysarthria, or weakness in the four extremities. Mental state, deep tendon reflexes, and coordination were normal, and there was no sensory disturbance. The patient had no past history of hypertension, diabetes, or smoking.Routine laboratory tests revealed elevated C-reactive protein (CRP)  and erythrocyte sedimentation rate (ESR) , while white blood cells were normal . Serum electrolytes, creatine, liver enzymes, and the coagulation system were also normal, while titers for anti-acetylcholine receptor (AChR)  and anti-ribonucleoprotein (RNP)  antibodies were elevated, though to the same extent as seen in previous examinations. Complements, as well as cytoplasmic and perinuclear anti-neutrophil cytoplasmic antibodies, were normal. The CSF sample obtained at admission was lymphocytic  with elevated levels of protein  and IgG  and a normal glucose level . The oligoclonal IgG band was positive (seven bands), and intrathecal IgG synthesis was elevated , as well as levels of protein (35 mg/dl) and IL-6 (3.4 pg/ml) in the CSF (Fig. /μl, as wWe treated a patient with RM who had a long history of MG and RA. This case showed reported features of RM, which were manifested independently of RA activity , 2, as wIn the present case, it is noteworthy that RM developed during a phase of remission of both RA and generalized MG. If an extrinsic factor is present, such as an infectious event that can induce general T and/or B cell activation before development of RM, symptoms related to RA and MG will likely worsen along with elevation of immunological markers for those disorders. The good response of clinical symptoms, as well as improvement in ESR and CRP following administration of a moderate dose of corticosteroids alone in the present case and others , is simiIn summary, we performed flow cytometric analysis of cellular immunity in a patient with RM, which showed that immunoregulatory disturbance associated with elevated Th2-type response in the CSF may have stimulated intrathecal IgG synthesis. Results obtained in this case indicate that the pathogenesis of RM differs from that of a systemic manifestation of RA and MG.


Advances in neuroscience and pharmacology have led to improvements in the cognitive performance of people with neurological disease and other forms of cognitive decline. These same methods may also afford cognitive enhancement in people of otherwise normal cognitive abilities. “Cosmetic”, or supranormal, cognitive enhancement offers opportunities to enrich our social or financial status, our interactions with others, and the common wealth of our community. It is common to focus on the potential benefits of cognitive enhancement, while being less than clear about the possible drawbacks. Here I examine the harms or side-effects associated with a range of cognitive enhancement interventions. I propose a taxonomy of harms in cognitive enhancement, with harms classified as (neuro)biological, ethical, or societal. Biological harms are those that directly affect the person’s biological functioning, such as when a drug affects a person’s mood or autonomic function. Ethical harms are those that touch on issues such as fairness and cheating, or on erosion of autonomy and coercion. Societal harms are harms that affect whole populations, and which are normally the province of governments, such as the use of enhancement in military contexts. This taxonomy of harms will help to focus the debate around the use and regulation of cognitive enhancement. In particular it will help to clarify the appropriate network of stakeholders who should take an interest in each potential harm, and in minimizing the impact of these harms. Practical ethical decisions about cognitive enhancement require an analysis of the potential benefits and the potential harms of any drug or technology. But “harm” rarely means the same thing in different contexts or when used by different people. So there is a need for greater clarity in debates where potential harms and potential benefits are being balanced. Here I will outline one possible way to organize our thinking around the harms of cognitive enhancement, with the aims of clarifying the types of harms that may be encountered, and of understanding which groups may take an interest in each type of harm.In this review article I propose that the harms that we encounter in cognitive enhancement fall into three categories:I.(Neuro)biological harmsII.Ethical harmsIII.Societal harms.I will examine each of these categories, and show how a framework such as this helps in guiding thinking about the desirability of particular routes to cognitive enhancement. Although not intended as a hierarchy or scale of seriousness, these harms are roughly grouped in order of their directness, or immediacy from application of the enhancer.To focus the discussion, this review article will only consider pharmacological enhancers and device-based enhancements. What does this focus exclude? Primarily it excludes lifestyle-based enhancement techniques such as getting plenty of sleep, avoiding recreational drugs and alcohol, and having a healthy diet and getting plenty of exercise. It excludes paying attention in school and reading for pleasure. These are proven, efficacious techniques that are not usually ethically troubling. It also excludes cognitive training and training via neurofeedback, which are techniques of uncertain efficacy and do not have the quasi-invasive nature of drug- or device-based techniques  works to increase levels of dopamine in the brain by increasing dopamine availability through an effect on its receptors , and particularly the form that uses direct current (tDCS), since the devices are already appearing on the consumer market .People may become dependent on stimuli that activate their reward systems. For example, nicotine is a highly addictive compound that appears to increase dopamine availability in the midbrain biological effect of an enhancer is the responsibility of the manufacturer and of the person delivering the enhancer to another person. So in a university laboratory the researcher may rely on the manufacturer of an enhancing device to have adhered to proper engineering standards, and must take responsibility for correct application of the device to a participant and for following accepted safety guidelines. It is also the researcher’s duty to monitor for adverse effects of the enhancer, and to ensure the comfort and safety of the participant at all times. In a wider sense, it is the researcher’s responsibility to contribute knowledge of these harms to the community through good scientific practice, namely: conducting well-designed and well-controlled experiments; reporting the results of all experiments, including ones with null or “inconvenient” results; and reporting any adverse effect resulting from the experiment.In this section I have discussed some of the possible harms that a person may experience as a direct result of taking an enhancer. Harms could be clustered according to how the enhancer creates the harmful effect: so changes in mood and changes in seizure threshold both arise from the changes in cortical excitability induced by the enhancer. Alternatively, one could divide these harms into short-lived or long-lasting harms: a skin lesion may last for hours or maybe a day or two, while an alteration in emotional processing may last for months or years . However we classify them, these harms might properly be called Ethical harms here are those which do not arise directly from the specific enhancer, but rather from the covert or non-reciprocal use of an enhancer, or from the use of an enhancer in new contexts. Bioethical analysis almost invariably considers interactions among a small number of people, where one person may act unfairly towards another, or may seek to gain an advantage within a group. The recent rise of cognitive enhancement methods has generated much interest among philosophers, as a novel angle on the longer debate around enhancing physical capabilities.Covert use of an enhancer is always likely to be ethically problematic, compared to open use. Many discussions of pharmacological enhancement focus on the use of drugs such as Modafinil on college campuses, where students may gain (perceived) advantages in examinations or coursework through the use of drugs designed to treat neurological conditions. Covert use of enhancement in education is invariably taken to be wrong in such discussions. Analogously, enhancement of sporting performance (“doping”) may be perceived as allowable or not according to one’s view of what is the goal of sport  to change their potential to learn information, we risk a lack of respect for those who do not or cannot make the same changes, and a lack of respect for the notion that the variation among people is exactly what defines our individuality Jordan, . It is cThe defense industry has a long-standing interest in human enhancement, both for offensive and defensive purposes. Amphetamines have a long history of use in the military, for their ability to promote wakefulness and alertness during long operations. The US Air Force has a programme that generates enormously useful declassified information on the limits and the safety of tDCS in particular (see the Air Force Research Laboratory at Wright-Patterson Air Force Base)To this point we have considered the use of an enhancer as a voluntary decision, albeit one which may be more likely to be made if societal norms shift. But could there be situations where a person could be compelled to use an enhancer? For example, could a surgeon be compelled to take a pill that would keep her awake and alert for the duration of a long and complex surgery? Goold and Maslen  suggest,The criminal justice system relies on establishing the true course of events, and on determining a level of punishment for wrongdoing. This process is inevitably imperfect, but there is an interest in methods that increase the reliability of judgements and the appropriateness of punishment. So far the law has been reluctant to use neuroscience as probative evidence, although neuroscience may be introduced in many jurisdictions. One controversial use of neuroscience is the possibility of uncovering concealed knowledge, either through prompting a confession from a suspect or from increasing the reliability of an eyewitness’s memory Moreno, . These aSocietal harms are harms that threaten the stability of societies. That is, they may threaten the stable values of a community, or threaten the stability of interactions between nations. This is not to say that societies are unable to absorb change. But such change usually requires acts of government or debates between nations. For example, the conduct of war is guided by treaties that establish reasonable practice, such as the use of chemical weapons or the treatment of prisoners. These treaties can be changed or added to (such as when nations change their position on the use of nuclear weapons), but this again requires concerted political and diplomatic effort. So the bodies responsible for understanding the effects of cognitive enhancement on society are those that represent the moral character of that society. Depending on the society, that may be the democratic representatives of the population, or the religious leaders, or senior legal experts, or non-governmental organizations that press for change. These bodies need input from researchers and primary users, and take direction from philosophers and legal scholars.In this review article I have attempted to set out a framework for assessing the harms arising from a particular enhancement intervention, and for identifying the interested parties whose expertise may be required in judging the relative acceptability of the enhancer given all the available information. For example, a neuroscientist considering using tDCS to enhance a cohort of undergraduates’ ability to memorise strings of numbers may need to know about the side-effects of tDCS, but may not be overly concerned with the potential use of tDCS in manipulating political beliefs. Conversely, a government’s defense minister will need to understand the societal implications of authorizing the use of tDCS to extract information from a battlefield prisoner, but may be less concerned with the risk of skin lesions in that prisoner.This is not to suggest that a politician need be ignorant of the side-effects, nor that a scientist should close her ears to issues of military uses of enhancement technology. However the level at which a person considers the issues surrounding enhancement technologies is generally limited. The three-level taxonomy of harms that I propose here, of biological, ethical and societal harms, will help in guiding the discussion of cognitive enhancement for a particular enhancer or a particular use.I have not dealt here with the issue of who, precisely, is best placed to make decisions around the use of cognitive enhancement in a given situation. There are many domains, such as professional sports, where there are many layers of regulatory oversight, and many different demands and obligations on the person taking the enhancer. For example, a drug may be banned by criminal law in an athlete’s home country but not the country of competition, or the athlete may have signed a civil contract with a sponsor not to take that drug, or the sport’s governing body may allow a drug in some circumstances but not others. Navigating overlapping systems is always a fraught process Black, . HoweverGood policy decisions in biomedicine can only arise through an interaction among legal experts, ethicists and scientists, with the goals of bringing greater reflective practice to ethically significant science, and of grounding speculative reasoning in scientific possibility Maslen, . I wouldNJD conceived and wrote this article.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies. Neural stem cells (NSCs) in the adult mammalian brain are regarded as a useful tool to regenerate neurons in pathological conditions such as neurodegenerative diseases or acute brain injury. Neurogenic potential has been demonstrated in only two specific areas of the normal mammal brain in adults, namely, the subventricular zone (SVZ) of the lateral ventricle (LV) and the subgranular layer of the hippocampal dentate gyrus (DG). In mammals, both locations show neurogenic activity throughout life , as previously described  to detect cellular replication. Following culture, the samples were fixed in Carnoy’s solution for 1 h, embedded in paraffin and sectioned at 8 μm for immunohistochemistry. We performed a total of five different cultures, generating 30 SVZ cultured brain slices for experimental condition .Organotypic cultures were performed according to current protocols .To assess whether the NSCs population brings about an increase in SVZ neurogenesis, we evaluated the presence of BrdU co-localized with Sox2, a marker for NSCs in an undifferentiated state; Neurod1, a transcription factor widely used as an early stage neuron commitment marker; βIII-Tubulin (Tuj-1) a widespread young neurons marker; Doublecortin, a specific marker for migratory NSCs and Calretinin, a differentiated neuronal marker ; Anti Sox2 (D-17) ; Anti Neurod1 ; Anti β-III-Tubulin (Tuj 1) ; Anti Calretinin ; and Anti Doublecortin (DCX) . Secondary antibody for Anti Sox2 was Antigoat Ig G-Alexa 594 . Secondary antibody for the rest was Antimouse Ig G-Alexa 488 , 1/1000 dilution.Immunolabeling was performed following standard procedures and the antibodies used for immunostaining were as follows: Immunolabeled cells were photographed with a Leica TCS SPE confocal laser microscope.n = 10) for each experimental condition  and immunolabeling type. All images (which had a 0.0269 mm2 area) were carefully obtained in the selected zones: SVZ, striatum (ST) and RMS defined in Figure post hoc Bonferroni test, or alternatively we used the two-tailed Student’s t-test. In both cases the significance threshold was set as p ≤ 0.05 or p ≤ 0.001.To quantify the results, we randomly selected 20 laser confocal brain images taken from 10 different animals  the nuclear co-localization of BrdU and SOX2, which is a molecular marker of undifferentiated NSCs , there were also many cells (BrdU positive and Sox2 negative) in the SVZ (data not shown), which could be those in a more advanced state of differentiation. To assess whether the NSCs amplified population detected in E-CSF treated sections also brings about an increase in SVZ neurogenesis, we evaluated the expression of Neurod1 at an early stage in the neuronal commitment marker and βIII-Tubulin (Tuj-1) as a young neurons marker.As is shown in Figures In order to test whether the increase in neuronal commitment induced by E-CSF leads to an effective and persistent increase in new neurons, we evaluated the number of cells with co-expressed BrdU and βIII-Tubulin in the SVZ. Given the presence of two types of co-labeled cells in the striatum area studied in accordance with the amount of βIII-Tubulin in the cytoplasm and identified as low Figure , in E-CSIn order to evaluate the normal behavior of the new neurons induced by E-CSF, we studied their migratory capacity by the cytoplasmic expression of Doublecortin (DCX), a specific marker for migratory NSCs, co-labeling with nuclear BrdU expression as a cellular lineage marker. For this purpose we determined the co-labeled cells in areas located at the starting point of the RMS Figure , RMS, whThe results are shown in Figure The last part of our study sought to answer another relevant question: the evolution to mature neurons of the newly generated neurons in the SVZ niche under E-CSF influence.Despite not being able to follow the migratory neural precursors to their natural destination, the olfactory bulb, we assume that in our coronal section culture system migratory NSCs stop at the starting point of the RMS, where the maturation of new neurons start. Consequently, here we attempted to locate cells with co-labeling of BrdU  and Calretinin, a mature neurons marker. As can be seen in Figure in vitro”; this previous study only showed that E-CSF is able to induce the initial steps of neuronal differentiation in SVZ NSCs cultured “in vitro” (outside the niche). This is the first report of the influence of E-CSF in organotypic cultures of the SVZ neurogenic niche; moreover, here we show the influence of E-CSF on the main steps of the physiological behavior of SVZ NSCs including expansion of the un-differentiated NSCs population and also the increase of effective neuronal differentiation, migration and final neuronal maturation.We previously published  population. Our results have also shown that, under E-CSF influence, a significant number of proliferative SVZ NSCs initiate a process of neurogenesis (Neurod1 or βIII-Tubulin expression), which shows an “accelerated” neurogenic progress with respect to the controls. The results also suggest that A-CSF could have an inhibitory effect on neurogenesis from both the quantitative (reducing the number of new neurons) and qualitative (delaying the transition from newborn to young neuron) aspects. In our study, we used coronal sections of adult mouse brain; therefore, we were unable to track the final destination of these migratory neuroblasts (olfactory bulb). However, our data relative to Doublecortin positive cells, strongly suggest that E-CSF’s influence on NSC activity involves also an increase in normal migratory behavior. Finally, our study suggests that the final destination of the new neurons generated under the E-CSF influence in the SVZ could be transformation into mature neurons. Taking together the results reported here, E-CSF seems to activate the main steps of SVZ NSCs behavior, increasing neurogenesis.Here we show that the neurogenic instructive signals present in E-CSF are capable of increasing the physiological activity of adult brain NSCs. The CSF is deemed a key factor in contributing to the niche microenvironment diffusible signals in the developing brain  to the adult SVZ niche brings about complete activation of the NSCs including the two main steps in neurogenesis activity: an increase in mitotic activity leading to precursor population expansion and subsequent differentiation to young neurons able to migrate and progress to a mature state. This is in agreement with previous research showing the E-CSF neurogenic properties at the earliest stages of brain development (Zappaterra and Lehtinen, Taking into account these data together with the cellular lineage of the NSCs concept, we propose the theory that CSF is a changing brain component which evolves throughout life and plays a key role in neurogenesis.Further studies are necessary to better understand the molecular basis of embryonic CSF neurogenic properties and the changes induced by age, which probably limit adult neuroregeneration.About how E-CSF influences NSC behavior, E-CSF is a complex fluid involved in neurogenesis (Gato et al., This study supports the idea that the CSF could actually be considered an “inner lake” for brain cellular intercommunication, playing a key role in establishing the niche microenvironment in the developing and adult brain.Here we report new data relating to E-CSF properties. This represents the ability to induce global changes in adult mouse SVZ niche NSCs activity, promoting an expansive increase in the NSCs population, migratory activity and neurogenesis with neuronal maturation.in vivo” neurogenesis induction.Our main conclusion is that E-CSF could be a cocktail of instructive signals, with hidden key information on neurogenesis control from NSCs throughout life. Further studies are necessary to check the usefulness of these properties in adult brain “MIA, AG and FL designed and performed most of the experiments, analyzed data, prepared all figures and wrote the manuscript. EC, JAM contributed to the experiments. AM and JMFF collected the mouse embryonic fluid. MED helped to write the manuscript.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.