Biomarkers of Huntington’s disease (HD) in cerebrospinal fluid (CSF) could be of value in elucidating the biology of this genetic neurodegenerative disease, as well as in the development of novel therapeutics. Deranged synaptic and immune function have been reported in HD, and concentrations of the synaptic protein neurogranin and the microglial protein TREM2 are increased in other neurodegenerative diseases. We therefore used ELISAs to quantify neurogranin and TREM2 in CSF samples from HD mutation carriers and controls. CSF neurogranin concentration was not significantly altered in HD compared to controls, nor was it significantly associated with disease burden score, total functional capacity or motor score. An apparent increase in CSF TREM2 in manifest HD was determined to be due to increasing TREM2 with age. After age adjustment, there was no significant alteration of TREM2 in either HD group, nor any association with motor, functional or cognitive score, or brain volume quantified by MRI. Both analyses were well-powered, and sample size calculations indicated that several thousand samples per group would be needed to prove that disease-associated alterations do in fact exist. We conclude that neither neurogranin nor TREM2 is a useful biofluid biomarker for disease processes in Huntington’s disease. HTT encoding mutant huntingtin protein1. The pathogenesis of HD is multifactorial and includes synaptic dysfunction2 and activation of the innate immune system, most likely due to a direct effect of mutant huntingtin in myeloid cells5. We previously showed that cytokines4 and chemokines6 are increased in plasma in HD mutation carriers and that CSF in HD contains increased levels of the microglia-associated proteins chitotriosidase and YKL40, with the latter independently associated with the severity of motor symptoms7. Modulating the immune system has the potential to offer therapeutic benefit in HD8 and one trial of a putative microglial-modulating agent (laquinimod) is currently underway10.Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansions in 11 that has been proposed as a synaptic function biomarker12. Neurogranin has been shown to be increased in CSF in Alzheimer’s disease (AD)13 but not in other neurodegenerative conditions such as frontotemporal dementia (FTD), Lewy body disease, Parkinson’s disease (PD), progressive supranuclear palsy and multiple system atrophy14. There is evidence that synaptic dysfunction contributes to HD pathology16, and a whole-brain gene expression study in post-mortem HD patient brains identified that NRGN, encoding neurogranin, was among the most robustly downregulated genes in HD caudate compared to controls18. However, it has not previously been quantified in CSF in HD.Neurogranin is a postsynaptic protein that regulates the availability of calmodulin19. Missense mutations in TREM2 are associated with CNS disease20 and single-nucleotide TREM2 polymorphisms have been reported as genetic modifiers of AD21, amyotrophic lateral sclerosis22, PD and FTD23. Soluble TREM2 is quantifiable in CSF and has been reported as elevated in AD25, and in multiple sclerosis, where it normalised upon immunomodulatory treatment26. While TREM2 has not specifically been linked to the pathobiology of HD, dysfunction of myeloid cells due to cell-autonomous expression of mutant huntingtin is a well-described feature of the disease5, and other microglial-associated proteins have shown disease-related alterations in HD patient CSF7. Our previous work demonstrates the principle that biomarker studies in human biofluids can provide novel pathogenic insights by highlighting links with substances previously reported to be linked to HD29.Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor expressed by myeloid cells including monocytes, macrophages and microglia whose activation is inhibitory to the immune responseOn the basis of these findings in other neurological conditions and the potential to show alteration in CSF in HD, we therefore set out to quantify neurogranin and soluble TREM2 in CSF samples from HD mutation carriers and matched controls.The neurogranin cohort consisted of 32 participants: 12 healthy controls and 20 HD gene expansion carriers. The HD group contained 17 manifest and 3 premanifest HD participants pooled together. Details are given in Table Medians and interquartile ranges (IQR) of CSF neurogranin are shown in Table A post-hoc power calculation showed that this dataset had 98% power to determine that CSF neurogranin levels are equivalent between healthy controls and HD gene expansion carriers. A sample size calculation indicated that 14,661 samples per group would be needed to establish with 80% power at p = 0.05 that neurogranin levels are in fact higher in HD than in controls. Therefore the analysis was not repeated in the larger TREM2 cohort.HTT mutation carriers, and 40 patients with manifest HD, stages 1–3. Demographics and clinical characteristics are given in Table The TREM2 cohort consisted of 80 independent CSF samples collected from 20 healthy controls, 20 premanifest CSF TREM2 concentrations were strongly associated with age overall Fig. . HoweverThis sample had 97% power to demonstrate that CSF TREM2 levels are equivalent between healthy controls and HD mutation carriers after age-adjustment. A sample size calculation indicated that 3,288 samples per group would be needed to establish with 80% power at p = 0.05 that a significant CSF TREM2 increase does in fact exist in HD versus controls, after adjustment for age.In two adequately-powered sample sets, we have shown that neither neurogranin, a putative marker of postsynaptic damage, nor TREM2, a putative marker of microglial function, has an altered concentration in CSF in Huntington’s disease, despite each protein having shown alterations in CSF in other neurodegenerative diseases, and each having potential links to HD pathogenesis. An apparent increase in CSF TREM2 concentration in manifest HD was determined to be an artefact of its tendency to increase with age, rather than a disease effect. We show that the sample sizes necessary to demonstrate alterations of these substances in CSF in HD are likely prohibitively large.26. Similarly, they do not indicate that these proteins have no role in the pathogenesis of HD, or diminish the importance of synaptic or immune dysfunction as pathogenic pathways; merely that detectable alterations were not found in CSF, a biofluid enriched for brain-derived proteins. Finally, there is still a potential role for either substance as a pharmacodynamic biomarker of target engagement by a compound intended to ameliorate synaptic or microglial dysfunction, since for such markers, a baseline difference from controls is not a prerequisite.Our results do not negate previously-reported positive findings in regard to neurogranin and TREM2 in other diseases, where both mechanistic links to pathobiology and consistent CSF changes have been shownNonetheless we conclude that neither neurogranin nor TREM2 is likely to be of value as a CSF biomarker for disease processes in Huntington’s disease.30. In brief, samples were collected after an overnight fast at the same time of day and centrifuged and aliquoted rapidly on ice using a standardised protocol and polypropylene plasticware30. Healthy controls were contemporaneously recruited, drawn from a population with a similar age to patients, and clinically well, so the risk of incidental neurodegenerative diseases was very low. Relevant aspects of clinical phenotype were quantified using the total functional capacity and total motor score components of the Unified Huntington’s Disease Rating Scale (UHDRS)31. Manifest HD as opposed to premanifest HD was defined as UHDRS diagnostic confidence level of 4. Disease burden score, a function of age and CAG repeat length that predicts many features of HD onset and progression, was calculated33. The TREM2 cohort was drawn from the baseline of a larger, more recent CSF collection initiative entitled HD-CSF. This is a single-site study aligned to the HDClarity study (NCT02855476) with added optional MR imaging and a longitudinal design. Inclusion and exclusion criteria, and methods of CSF collection were virtually identical to the neurogranin cohort; clinical assessment included the measures above plus a cognitive battery consisting of Symbol Digit Modality Test, Categorical Verbal Fluency Test, Stroop Color Naming and Stroop Word Reading (full protocol available at http://hdclarity.net).Ethical approval was given by the joint University College London/University College London Hospitals ethics committee (neurogranin cohort) and the London - Camberwell St Giles Research Ethics Committee (TREM2 cohort). All patients gave informed written consent before enrolment. All experiments were performed in accordance with relevant guidelines and regulations. For the neurogranin cohort, patient consent, inclusion and exclusion criteria, clinical assessment, CSF collection and storage were as previously publishedFor the TREM2 cohort, T1-weighted MRI data were acquired on a 3T Siemens Prisma scanner using a protocol optimised for this study. Images were acquired using a 3D MPRAGE sequence with a TR = 2000ms and TE = 2.05 ms. The protocol had an inversion time of 850 ms, flip angle of 8 degrees, matrix size 256 × 240 mm. 256 coronal slices were collected to cover the entire brain with a slice thickness of 1.0 mm with no gap. Parallel imaging acceleration was used (GRAPPA) and 3D distortion correction was applied to all images.34. Volumetric regions of the whole-brain, ventricles and total intracanial volume (TIV) were generated via MIDAS using semi-automated segmentation procedures as previously described37. SPM12 segment (MATLAB version 2012b) was used to measure the volumes of grey and white matter38. Finally, to measure caudate volume the images were processed using MALP-EM39, a fully automated software that was recently validated in an HD cohort40. All segmentations underwent visual quality control to ensure accurate delineation of the regions. No scans failed processing. Brain volumes are expressed as a percentage of total intracranial volume, to account for overall head size.Scans underwent visual quality control prior to processing. No scans were excluded due to the presence of significant motion or other artefacts. Bias correction was performed using the N3 procedure41. All samples but 6 were above the limit of detection LOD (i.e. 10.0 pg/mL) – 4 in the HD group and 2 samples in the control. Samples below the LOD were assigned the LOD concentration (i.e. 10.0 pg/mL). Concentrations of TREM2 were quantified with an in-house Meso Scale Discovery based ELISA, using an adapted protocol from Kleinberger et al.42. All samples were above the LOD (65.3 pg/mL). Haemoglobin concentration was measured using a commercial ELISA  to determine CSF contamination by blood. Quantification for each analyte was run on the same day for all samples using the same batch of reagents. Laboratory operators were blinded to clinical data.CSF neurogranin was measured using an in-house ELISA and anti-Ng antibodies NG22 and NG2, essentially as describedStatistical analysis was performed with Stata 14 software . Significance level was defined as p < 0.05. Both neurogranin and TREM2 concentrations were non-normally distributed; square-root transformation produced an acceptable normal distribution for TREM2, while non-parametric tests were used for neurogranin because of the smaller sample size.Potentially confounding demographic variables  were examined in preliminary analyses and those found significant were included as covariates for subsequent analyses.For neurogranin, we used two-sample Wilcoxon rank-sum (Mann-Whitney) test or the exact Fisher test to assess intergroup differences of the cohort’s characteristics. Two-group comparisons were tested using Wilcoxon rank-sum (Mann-Whitney) test, and associations were tested using Spearman’s rank-order correlation.For TREM2, we used unpaired two-samples t-test/ANOVA or the Pearson’s chi-squared test to assess intergroup differences of the cohort's characteristics. Group comparisons were tested using unpaired two-samples t-test/ANOVA for unadjusted comparisons or multiple linear regression for adjusted comparisons. Correlations were tested using Pearson’s correlation and partial correlations for covariate adjustment. Where necessary, multiple comparisons were corrected for using the Bonferroni method.43 to provide an estimation of the statistical power of the data set to prove robustly that analyte levels in healthy controls and HD mutation carriers are not different. We also performed sample size calculations for theoretical experiments to demonstrate that the level of each analyte was higher in HD mutation carriers than in controls, assuming 5% type I errors and 20% type II errors.For both analyses, a post-hoc power calculation for equivalence was computed assuming 1% type I errorsEJW has participated in scientific advisory boards with Hoffmann-La Roche Ltd, Ionis, Shire, GSK and Wave Life Sciences. All honoraria for these advisory boards were paid through UCL Consultants Ltd, a wholly owned subsidiary of UCL. His host clinical institution, University College London Hospitals NHS Foundation Trust, receives funds as compensation for conducting clinical trials for Ionis Pharmaceuticals, Pfizer and Teva Pharmaceuticals. KB and HZ are co-founders of Brain Biomarker Solutions in Gothenburg AB, a GU Ventures-based platform company at the University of Gothenburg. HZ has served at advisory boards of Eli Lilly, Roche Diagnostics and Pharmasum Therapeutics and has received travel support from TEVA.


B cell immunity, including the chemokine CXCL13, has an established role in Lyme neuroborreliosis, and also, T helper (Th) 17 immunity, including IL-17A, has recently been implicated.n = 49), defined by both cerebrospinal fluid pleocytosis and Borrelia-specific antibodies in cerebrospinal fluid and from two groups with possible Lyme neuroborreliosis, showing either pleocytosis  or Borrelia-specific antibodies in cerebrospinal fluid . A non-Lyme neuroborreliosis reference group consisted of 88 patients lacking pleocytosis and Borrelia-specific antibodies in serum and cerebrospinal fluid.We analysed a set of cytokines and chemokines associated with B cell and Th17 immunity in cerebrospinal fluid and serum from clinically well-characterized patients with definite Lyme neuroborreliosis  were significantly elevated in groups 1, 2 and 3 compared with the reference group, except for BAFF, which was not elevated in group 3. Regarding Th17-associated markers , CCL20 in cerebrospinal fluid was significantly elevated in groups 1, 2 and 3 compared with the reference group, while IL-17A and CXCL1 were elevated in group 1. Patients with time of recovery <3 months had lower cerebrospinal fluid levels of IL-17A, APRIL and BAFF compared to patients with recovery >3 months.By using a set of markers in addition to CXCL13 and IL-17A, we confirm that B cell- and Th17-associated immune responses are involved in Lyme neuroborreliosis pathogenesis with different patterns in subgroups. Furthermore, IL-17A, APRIL and BAFF may be associated with time to recovery after treatment. Borrelia burgdorferi (B.b.) interacting with recognition receptors such as Toll-like receptor 2, subsequently resulting in activation and recruitment of B and T cells to the central nervous system (CNS). The chemokine C-X-C motif ligand (CXCL)13 is a key molecule in B cell recruitment to the CNS [B.b.-specific antibodies can be detected; however, different cutoff levels have been discussed, e.g. 142 and 250 pg/mL, respectively [Lyme neuroborreliosis (LNB) is the dominating disseminated form of Lyme borreliosis in Sweden  as well  the CNS –6. CXCL1ectively . The cytectively , and raiectively , 11. Altectively , the relRecent studies have indicated involvement of T helper (Th)17 cells in the intrathecal immune response in patients with LNB –15. IL-1A basic understanding of the molecules involved in the pathogenesis is a prerequisite for the identification of prognostic biomarkers and in the long run for finding potential therapeutic targets. The aims of this study were to evaluate the putative involvement of Th17- and B cell-associated immune response and to assess associations with disease course in LNB by analysing IL-17A and its downstream chemokines CCL20 and CXCL1, as well as B cell-associated factors APRIL, BAFF and CXCL13.n = 49) had both CSF pleocytosis and Borrelia-specific antibodies in CSF. Group 2  had symptoms strongly suggestive of LNB, short duration of symptoms and CSF pleocytosis but not (yet) Borrelia-specific antibodies in CSF. Group 3  had Borrelia-specific antibodies in CSF, but no pleocytosis and symptoms were less suggestive of LNB. As a non-LNB reference group, we selected 88 gender- and age-matched patients from the same cohort investigated for suspected LNB 2007–2009, in whom LNB was excluded based on no Borrelia-specific serum or CSF antibodies, no CSF pleocytosis and normal CSF-albumin. The reference group consisted of patients where the LP was part of a neurological investigation and in whom no neurological diagnosis was verified (n = 56) or they later received other neurological diagnoses such as Bell’s palsy (n = 18) or Alzheimer’s disease, Parkinson’s disease and stroke (n = 14).We included retrospectively 165 patients in Jönköping County, Sweden, who had been investigated by lumbar puncture (LP) and blood sampling during 2007–2009 to verify or exclude suspected LNB. Medical records were scrutinized, and the patients were divided into four groups based on the CSF findings  between 2007 and 2008. Intrathecal antibody index (AI) was calculated using total IgG as a reference molecule [Borrelia-specific IgG in CSF (OD)/Borrelia-specific IgG in serum (OD))//total IgG in serum (g/L)) [Borrelia-specific AI >2 was indicative of intrathecal anti-Borrelia antibody production. From 2009, the laboratory used the IDEIA ). Both antibody assays use purified, native B. afzelii strain DK1 flagellum as test antigen, and results were interpreted according to the manufacturer’s instructions.All tests were performed at the clinical laboratory of microbiology in Jönköping. molecule  accordinAPRIL, BAFF and CXCL13 were analysed by ELISA  Systems, Inc., USA, respectively). IL-17A, CXCL1 and CCL20 were analysed by Luminex multiple bead technology . All analyses were conducted according to the manufacturers’ instructions. The lowest detection limits were as follows: APRIL: 0.02 pg/mL, BAFF: 0.05 pg/mL in serum, 0.04 pg/mL in CSF; CXCL13: 0.04 pg/mL in serum, 0.03 pg/mL in CSF; IL-17A: 0.38 pg/mL in serum, 0.06 pg/mL in CSF; CXCL1: 3.2 pg/mL in serum, 0.14 pg/mL in CSF; CCL20: 0.29 pg/mL in serum, 0.84 pg/mL in CSF. Values under the detection limit were given half the value of the lowest point of the standard curve.p < 0.05 followed by Mann-Whitney U test as a post hoc test. For children (age < 15 years), a covariance analysis has been performed with pleocytosis as a covariate. Data are given as medians and interquartile (i.q. range). For categorical variables, the chi-square test was used. Correlations were determined by Spearman’s rank order correlation. p values below 0.05 were considered significant.For statistical analyses, SPSS version 20 was used. Inter-group comparisons were performed by using the non-parametrical Kruskal-Wallis test and when Table CSF levels of APRIL and CXCL13 Table . No corrp < 0.0001) in groups 1, 2 and 3. No correlations were seen between cytokine and chemokine levels in serum and CSF.CSF levels of IL-17A, CXCL1 and CCL20 (Table n = 34) had significantly higher levels of BAFF  in serum and CXCL13 in serum and CSF  compared to adults. BAFF in serum also showed a strong negative correlation with age in groups 1 and 2 . APRIL, BAFF and CXCL13 in CSF were all positively correlated with pleocytosis . When a covariate analysis was performed with pleocytosis as a covariate, BAFF in serum was still significantly higher in children (p < 0.0001), but CXCL13 in serum and CSF was not. Children in groups 1, 2 and 3 had significantly higher levels of CCL20 in serum and CSF . CCL20 was however not significantly higher when performing a covariate analysis with pleocytosis as a covariate. IL-17A levels in CSF correlated with pleocytosis .There were no significant differences in cytokine/chemokine levels in serum or in CSF between men and women. Regarding differences in relation to age (data not shown), we found that children <15 years of age in groups 1, 2 and 3 (p < 0.01). When stratifying patient in groups 1, 2 and 3 according to duration of symptoms before LP, those with a shorter duration  had higher levels of BAFF in serum  compared to patients with longer symptom duration (n = 28) .Symptom duration before LP did not correlate with levels of cytokines/chemokines in serum or CSF in groups 1, 2 and 3 together. However, within group 2, duration of symptoms before LP correlated negatively with BAFF and CXCL13 in serum  had lower levels of APRIL (p = 0.003), BAFF (p = 0.04) and IL-17A (p = 0.02) in CSF compared to patients with longer time of recovery, group B , (Fig. Regarding relation to disease course, patients in groups 1 and 2 were stratified according to time to recovery after treatment. Patients with shorter duration, group A (<3 months, In this study, we showed that levels of several cytokines and chemokines related to Th17 and B cell immunity are raised in CSF from patients with LNB, strengthening the involvement of both Th17 and B cell immunity in LNB. Furthermore, we noted several relations to demographic and clinical parameters.The lack of correlations between cytokine/chemokine levels in serum versus CSF indicates an intrathecal source of the cytokines and chemokines present in CSF, thus reflecting the pathological process in the CNS.CXCL13 was significantly elevated in CSF of all LNB groups, in particular the pleocytosis groups 1 and 2, as compared to the non-LNB group. CXCL13 has been suggested as a diagnostic marker for acute LNB, and a majority of patients in groups 1 and 2, those with most probable acute LNB, showed raised CSF levels above 142 and 250 pg/mL, respectively, while no patients in group 3 had levels over 142 pg/mL, supporting CXCL13 as a diagnostic tool and corroborating several studies –8, 21. PThe involvement of B cell-related cytokines and chemokines in the pathogenesis of LNB is also supported by the raised CSF levels of both APRIL and BAFF in groups 1 and 2, the groups with most probable LNB. This is, to our knowledge, the first time elevated levels of APRIL has been reported in LNB patients, while BAFF has been studied previously . The raiB. burgdorferi [We found elevated CSF levels of IL-17A in LNB patients, which corroborates previous studies –15. We hgdorferi . Interesgdorferi . Furthergdorferi  and psorgdorferi . In CNS,gdorferi . CCL20 cgdorferi . Our finThere are some limitations of the current study. The retrospective design hampers clinical assessments. Another potential limitation is the lack of truly healthy controls, although the chosen group represents a clinically relevant reference group. Group 2, with mostly children, had CSF pleocytosis and characteristic symptoms of LNB, but other, foremost viral, infections cannot be completely ruled out, especially in cases with low CSF levels of CXCL13, since presence of neurotropic viruses was mostly not investigated. Regarding the EFNS guidelines, we note some limitations in the classification of possible LNB cases. According to the guidelines, patients corresponding to our groups 2 and 3 are classified as possible LNB, while we find important differences between the two groups in terms of clinical presentation and CSF findings, including cytokine and chemokine levels.We here demonstrate additional support for Th17 involvement in the intrathecal immune response in LNB as well as indications that high levels of IL-17A in CSF in the acute phase of the disease may be associated with slower recovery, hence proposing that IL-17A should be further evaluated as a possible biomarker for prognosis. Besides CXCL13, the B cell-related cytokines APRIL and BAFF are elevated in CSF from patients with LNB, and the levels could be associated with time to recovery after treatment.


Cardioinhibitory vasovagal response is uncommon during the tilt test (TT).Heart rate variability (HRV) by use of spectral analysis can distinguishpatients with that response.To compare the HRV in patients with cardioinhibitory vasovagal syncope (casegroup - G1) with that in patients without syncope and with negative responseto TT (control group - G2).64 patients were evaluated  and submitted to TTat 70 degrees, under digital Holter monitoring. The groups were paired forage and sex .2, p=0.001) and a lower low/high frequencyratio in the supine position  in G1,with no difference during TT between the groups. Applying the receiveroperating characteristic curve for cardioinhibitory response, the area underthe curve was 0.74 for the LF component in the supine position (p = 0.001).The following were observed for the cutoff point of 0.35 ms(2)for the LF component: sensitivity, 97.4%; specificity, 83.3%; positivepredictive value, 85.3%; negative predictive value, 96.9%; and positivelikelihood ratio, 5.8.In G1, 21 patients had a type 2A response and 19 had type 2B, with mean TTduration of 20.4 minutes. There was a greater low frequency (LF) component(11,6 versus 4,5 msHRV in the supine position allowed identifying patients with syncope andcardioinhibitory response with a high negative predictive value andlikelihood ratio of 5.8. The latter manifests with orwithout asystole .6 Theincidence of that response varies from 1 to 4.4% of the positive tests, reaching 21%in protocols sensitized with nitroglycerin and 13% in protocols sensitized withisoproterenol, being more frequent in young individuals.9Tilt test (TT) is used to diagnose vasovagal syncope, being safe, of low cost and ofgood reproducibility.11 There are only fourstudies15 including spectral analysis of adult patients withcardioinhibitory response and distinct behavior, evidencing an increase in the LFcomponent at rest, a greater reduction in the HF component after TT, or an increasein the LF/HF ratio before the syncopal event during TT, using only univariateanalysis and no receiver operating characteristic curve for the analysis of the HRVpredictive value regarding that specific response. Thus, the present study aimed atassessing HRV by using spectral analysis before and during TT in patients withvasovagal syncope and cardioinhibitory response, and at comparing it with the HRV ofpatients with negative response to TT and no history of syncope, assessing itspredictive value.Previous studies have shown changes in HR variability (HRV) during TT with gradualinclination in healthy patients, providing a non-invasive quantitative analysis ofthe vagal sympathetic balance, via its components of low frequency (LF), highfrequency (HF) and LF/HF ratio.This is a case-control, observational, prospective study, whose population samplecomprised 64 patients consecutively selected to undergo TT at the Department ofGraphic Methods of the Hospital Madre Teresa, from January 2013 to February 2014,from a total of 435 patients. They were divided into two groups: case group, 40patients with history of syncope and cardioinhibitory response on TT; and controlgroup, 24 patients with other symptoms not related to loss of consciousness, such asdizziness or fall, and neither syncope nor presyncope, with a negative TT . Both groups had sinus rhythmand were paired for sex and age. Patients aged at least 14 years , of both sexes and able to undergo TT were consecutivelyincluded. The following were excluded: pregnant women, patients refusing toparticipate in the study, patients with coexisting conditions that could affect theHRV analysis, such as atrial fibrillation, pacemaker rhythm and use ofantiarrhythmic drugs, and patients undergoing heart transplantation.2, minimum difference to be detected of 100ms2, test power of 90%, significance level of 5%, and one-tailedtest. In addition, the number of participants included in similar studies wasconsidered.The population size was calculated as 52 patients, and such calculation was based onthe 1:1 ratio between the two groups, standard deviation of the spectral analysiscomponents of 200 msThis study project was approved by the Ethics Committee in Research, and theparticipants provided written informed consent.Trendelenburg position) to 70 degrees(upright), support for the patient's feet, in a quiet room under mild and constanttemperature. The fasting patients were allowed to rest in the supine position for 10minutes, and then tilted (70 degree) during the first 20-minute step. When there wasno event (syncope or presyncope), the drug-sensitization step was initiated with thesublingual administration of 1.25 mg of isosorbide for up to 15 minutes. In thepresence of events, such as cardioinhibitory response with symptoms, the test wasconsidered positive. Simultaneously, continuous electrocardiographic monitoring wasperformed, as well as intermittent recording of blood pressure every 3 minutes byuse of the Hewlett Packard Omnicare 24C monitor. Continuous electrocardiographicrecording was performed by use of the Holter system with a digital recorder (DMS300-8) of three channels  to analyze HRV in the supineposition for 10 minutes and in the tilt position. Recording was performed on thefifth minute  in the supine andtilt positions for all patients. In addition, recording was performed on the fifthminute at the end of inclination, in the control group, and on the fifth minuteafter an event, in the case group in the supine position.All TTs were performed in the morning period, using a tilt table with an angleranging from -20 degrees (2).The software Holter DMS, version 76, was used for the HRV spectral analysis,assessing the LF, HF and LF/HF ratio components via the Fourier mathematical model,after processing the data obtained, with correction of extrasystoles and artifacts.The results of that analysis were expressed in absolute units  software, version 14.0, wasused for data analysis. The results were expressed as numbers and proportions forcategorical variables, and as measures of central trend and dispersion forcontinuous variables. Mann-Whitney and chi-square or Fisher tests, when appropriate,were used to compare the differences between continuous and categorical variables, respectively. Wilcoxon test was used to compare HRV valuesbetween the periods of supine position, during inclination and after the event orend of inclination. Logarithmic transformation of HRV values was performed. Receiveroperating characteristic curve was used to assess sensitivity and specificity of HRspectral analysis in the supine position, considering the positive response to thetest. The significance level adopted was 5%.The patients' mean age was 36.2 ± 17.9 years , 35 (54.7%)being males. Regarding the case group, the median time of symptom presence was20 months. The mean time since the last syncope episode was 60.1 months. Themean number of syncope episodes was 4.17 ± 2.6 , and theCalgary score ranged from -8 to +4 points . The hemodynamicvariables of the entire case series are shown in In the case group, 38 patients (95%) reported prodromes, while 8 patients (20%)reported trauma resulting from the syncope episode. The triggers related tosyncope were as follows: body posture type (upright or sitting), 31 patients(77.5%); emotional stress, 8 (20%); and sight of blood, 1 patient. To avoid biasin HRV interpretation, the case and control groups were paired for sex and age:17 women in the case group (42.5% of the group) and 12 women in the controlgroup (50%), p=0.56. In the case group, mean age was 32.9 ± 14.8 years,and, in the control group, 41.7 ± 21.2, p=0.13.During TT, the control and case groups did not significantly differ regarding thehemodynamic variables (HR and blood pressure) in the supine position and duringinclination .In the case group, 21 patients (52.5%) had type 2A response, and 19 (47.5%), type2B response, constituting the subgroups. In the type 2A response subgroup, meanage was 35.9 ± 14.5 years, and, in the type 2B response subgroup, 29.5± 14.9 years . The case group had 17 women (42.5%), with 9 womenand 12 men in the type 2A response subgroup, while the type 2B response subgrouphad 8 women and 11 men. In the type 2A response subgroup, the mean HR achievedwas 28.4 ± 5.2 bpm . In the type 2B responsesubgroup, the mean value of pause was 14.2 ± 16.5 seconds  , while tComparing the HRV components during the supine position and on the fifth minuteof TT by using Wilcoxon test in the entire case series, the mean values of thosecomponents and p values were obtained, being shown in When comparing HRV during only 1 minute (on the fifth minute) between case andcontrol groups, by using Mann-Whitney test, there was a significant differencein the supine position regarding the LF and LF/HF ratio components, but nodifference between the groups during inclination. 2, p=0.004), but no difference regarding the HF (p=0.48) andLF/HF ratio components (p=0.77). During the first five minutes at the beginningof TT (inclination phase), a difference occurred only regarding the LF component. During the five minutes after TT, statistically significantdifference was observed only for the LF component . Withlogarithmic transformation of those HRV component values, the same p values wereobtained. The HR spectral analysis of 1 patient in the case group and of anotherpatient in the control group is plotted in The HRV analysis was also performed during the five minutes accumulated in thesupine position between the case and control groups. That analysis showed asignificant difference regarding the LF component  was 85.3% and the negative predictivevalue (NPV), 96.9%. The positive likelihood ratio was 5.8. For the LF/HF ratioin the supine position, sensitivity was 89.7% and specificity, 66.7%, with PPVof 72.9% and NPV of 86.6%. Considering type 2B response as the stable variable,the receiver operating characteristic curve obtained for the case group showedno statistical significance for any of the HRV components.Applying the receiver operating characteristic curve for the entire case series,considering cardioinhibitory response as the stable variable, that is the casegroup, the areas under the curve obtained were 0.74 and 0.70 for the LF andLF/HF ratio components, both in the supine position, respectively, withstatistical significance according to the Mann-Whitney test. The curves and datawith p values and 95% confidence intervals are shown in 11 being useful to identify autonomicchanges before and during TT in patients with syncope.15 Thepresent study showed that the HRV components could predict the cardioinhibitoryresponse before beginning the tilting, and some of them, when compared in thedifferent phases of TT, differed from those in the control group. The LF componentin the supine position, with better discriminatory power than the LF/HF ratio,showed sensitivity of 97.4%, with excellent diagnostic screening, correctlydetecting patients with history of syncope, and specificity of 83.3%, also detectingthose truly negative, who had no history of syncope, enabling diagnosticconfirmation. The power of that diagnostic test resulted in a high NPV, due to itshigher sensitivity, with a positive likelihood ratio, that is, TT's likelihood of apositive cardioinhibitory response of 5.8.The HRV spectral analysis parameters during TT have been shown to help to understandthe mechanism of syncope,14 Toassess the influence of age, Ruiz et al.16 have compared HRV data between patients with positive andnegative response on TT, divided into two groups according to age , and have shown a significant change in theLF components and an increase in the LF/HF ratio between the supine and tiltedpositions, less evident among the elderly. However, that study has not assessed thecardioinhibitory response. Regarding sex, a study paired for age,17 comparing healthy men and womenwith a mean age of 50 years, has shown in the female sex a lower LF component(p<0.001), a higher HF component (p<0.001) and a lower LF/HF ratio(p<0.001) as compared to the values obtained in male patients. Anotherstudy18 on HRV has shownthat young women have a lower LF component and LF/HF ratio than young men during thepostural maneuver. Barantke et al.,19 in a study with healthy volunteers, have reported a higher LFcomponent in men as compared to women, both in the supine and tilted positions, aswell as no difference in the HF component between sexes, which could justify thedifference between sexes regarding orthostatic tolerance.The HRV components in the frequency domain undergo changes during the tilting phases,being influenced by some factors. Comparing HRV in the supine position and after thetilting postural maneuver, healthy individuals showed an increase in the LF andLF/HF ratio components, and a reduction in the HF component regarding the values atrest in the supine position.Because of the influence of age and sex on HRV, and to prevent interpretation bias,the present study compared the group of patients with history of syncope and allwith cardioinhibitory response on TT with the control group, paired for age andsex.20 and Kochiadakis et al.10 have demonstrated a reduction in the HF component inpatients with positive response on TT, in the time period between the beginning oftilting and immediately after the end of TT, which did not significantly occur inpatients with negative response. However, Furlan et al.21 have shown that different models of spectral HRVcan be detected preceding syncope: one characterized by a progressive increase incardiac autonomic modulation until the sudden occurrence of bradycardia, and anotherwith gradual inhibition of the sympathetic component and concomitant increase invagal modulation. In accordance with the results of the present study, Kouakan etal.,22 assessing 69patients submitted to TT and with history of unexplained syncope, have concludedthat the LF/HF ratio persisted reduced during the entire period of inclination,being the only variable that discriminated the groups with positive and negativeresponses on TT (p=0.005), with 89% sensitivity, 89% specificity, 92% PPV and 86%NPV.Regarding the ability of the HRV components to predict and differentiate patients whowould have syncope during TT, Duplyakov et al.This subject has distinct results regarding the behavior of HRV spectral analysis.This can be explained by the case series of those studies including patients withhistory either suggesting vasovagal syncope or unexplained, with different responseson TT and distinct periods of time to assess HRV, in addition to other variables,such as age and sex.23 comparing a group of patients with syncope ofvasodepressor response with the control group, have demonstrated that, duringtilting, there was a progressive increase in the LF component and a decrease in theHF component. However, during the presyncope period, in the group with syncope, theLF component decreased. In addition, the HF and HF/LF ratio components did notsignificantly differ between groups.24 However, in that study, no patient with cardioinhibitoryresponse was included.The response on TT, whether vasodepressor, mixed or cardioinhibitory, could reflectdifferent behaviors of HRV; however, those responses on TT have been reported instudies only as subgroups of positive response on TT. Only the vasodepressorresponse has been emphasized. Prinz-Zaiss et al.,12 with a small caseseries, have shown a higher LF component at rest in the first minute after syncopeduring TT in patients with cardioinhibitory response (9 patients), as compared tothat value of patients with vasodepressor response (7 patients). Kochiadakis etal.,14 assessing 24 youngindividuals  submitted to TT and with cardioinhibitory responsein 71% of them (17 patients), have reported a decrease in the HF component after TT,as compared to 31 patients with mean age of 56 years, 68% of whom had vasodepressorresponse on TT. Another study,15in which only 8 patients had cardioinhibitory response, has reported no differencein HRV at rest between those with different responses on TT; however, there wassympathetic activation during TT in those with cardioinhibitory response. Thosestudies had a small case series and a small number of patients with cardioinhibitoryresponse as compared to the present study, which compared 40 patients withcardioinhibitory response with those in the control group, paired for sex and age,with a more robust design and avoiding interpretation bias.Considering the HRV behavior in patients with cardioinhibitory response on TT, Guzmanet al.,25 its pathophysiology is stillcontroversial. Differently from patients with vasodilation in the presyncope period,34% of those with vasovagal syncope had a decrease in cardiac output, mainly due toa drop in HR, with no change in total peripheral resistance.26 In addition, there is evidence ofsympathetic innervation activity persistence in patients with vasovagalsyncope.27 That couldexplain the findings of the present study, with an increase in the LF and LF/HFratio components in the case group before the occurrence of syncope or presyncope onTT.Although the vasovagal syncope is attributed to the Bezold-Jarish reflex, withparadoxical bradycardia and hypotension due to sympathetic inhibition and subsequentparasympathetic hyperactivity,28 or up to 21% insensitized protocols.9 Trauma secondary to syncope occurredin 20% of the case group with exclusively vasovagal syncope, while in the literaturethat ranged from 27.5% to 29% in a heterogeneous group . Prodromes, which are important in clinical history, occurred in 95% of thecase group in the present study, being frequently reported in the literature, mainlyamong young indivduals.31Regarding the clinical profile of the case series of the present study, 9.2% of thepatients submitted to TT had cardioinhibitory response, an incidence in accordanceto those in the literature: up to 6.6%, considering only the passivephase,32 and that itsinvestigation has better cost-benefit ratio if initiated during TT than by themonitor of implantable events,33further studies on cardioinhibitory response, especially on type 2B response, arenecessary.Considering that the asystole of the TT is a rare response during spontaneoussyncopeThe results of the present study should not be extrapolated to different and lessselected groups (with other types of response on TT) of patients with vasovagalsyncope. The acquisition of continuous hemodynamic parameters of blood pressurewas limited, because digital pletismography was not used. However, this did notinfluence the HRV results. The number of patients with cardioinhibitory responsewith and without asystole was small for appropriate comparison between bothsubgroups.The spectral analysis in supine position, before TT, by use of the LF and LF/HF ratiocomponents, allowed the identification of patients with history of syncope who hadcardioinhibitory response, and the comparison to patients with no history of syncopeand with negative TT. The case group patients had greater sympathetic activation inthe supine position. Thus, the HRV analysis can be used as a non-invasive tool topredict response to TT, with high NPV and likelihood ratio of 5,8.


Galectin-3  is involved in cardiovascular fibrosis and aortic valve (AV) calcification. We hypothesized that Gal-3 pharmacological inhibition with modified citrus pectin (MCP) could reduce aortic and AV remodeling in normotensive rats with pressure overload (PO). Six weeks after aortic constriction, vascular Gal-3 expression was up-regulated in male Wistar rats. Gal-3 overexpression was accompanied by an increase in the aortic media layer thickness, enhanced total collagen, and augmented expression of fibrotic mediators. Further, vascular inflammatory markers as well as inflammatory cells content were greater in aorta from PO rats. MCP treatment (100 mg/kg/day) prevented the increase in Gal-3, media thickness, fibrosis, and inflammation in the aorta of PO rats. Gal-3 levels were higher in AVs from PO rats. This paralleled enhanced AV fibrosis, inflammation, as well as greater expression of calcification markers. MCP treatment prevented the increase in Gal-3 as well as fibrosis, inflammation, and calcification in AVs. Overall, Gal-3 is overexpressed in aorta and AVs from PO rats. Gal-3 pharmacological inhibition blocks aortic and AV remodeling in experimental PO. Gal-3 could be a new therapeutic approach to delay the progression and the development of aortic remodeling and AV calcification in PO. Chronic pressure overload (PO) results in pathological morphological changes in the cardiovascular system. These changes result in an initially compensatory phase, but if they persist, could produce an important impact on cardiovascular function. PO could be due to aortic stenosis (AS) and may promote heart failure (HF) . Of notePO also induces a modification in the aortic valves (AVs). The valve cusps become progressively thickened, fibrosed, and calcified . A combiGalectin-3  is a 29 to 35 kDa protein and member of a β-galactoside binding lectin family, which interacts with cell surface receptors and extracellular matrix (ECM) proteins . Gal-3 lAnimals subjected to partial occlusion of ascending aorta presented similar body weight (BW) and blood pressure as compared to control rats  after siGal-3 mRNA expression was increased in aorta from PO rats as compared to that of controls, and was normalized by the treatment with the Gal-3 inhibitor modified citrus pectin (MCP) A. This rp < 0.05), fibronectin (p < 0.05), α-smooth muscle actin (α-SMA) (p < 0.05), TGF-β1 (p < 0.05), and connective tissue growth factor (CTGF) (p < 0.05) mRNA levels as compared to that of controls (The PO group presented an increase in aortic collagen type I (Col1a1)  as compared to that of controls (p < 0.05) was normalized by MCP treatment  in PO group (Vascular expression of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein-1 (CCL-2) was higher , and was normalized by MCP treatment (p < 0.05). Moreover, PO animals presented higher staining for total collagen as well as for TGF-β as compared to that of controls and PO rats receiving MCP , BMP-4 (p < 0.05), and SRY-homeobOX-like (Sox)-9  (The immunostaining for leucocytes (cd45) and macrophages (cd68) was higher in AVs from PO rats as compared to that of controls and PO + MCP rats A. The qu Y gene) B. Runx2 The aim of this study was to evaluate the effects of Gal-3 inhibition on vascular and AV alterations in an experimental normotensive model PO. In this model, Gal-3 inhibition was shown to exhibit anti-fibrotic and anti-inflammatory properties in the myocardial tissue . Our resPartial occlusion of the ascending aorta is a common experimental method to induce PO, cardiac hypertrophy, and HF. Although previous studies have shown thickening of the aortic wall, adventitial hyperplasia, collagen deposition, and inflammatory changes, the molecular signaling pathways responsible for this remodeling have not been identified ,21,22,23Gal-3 expression has been recently reported in VICs from AVs in patients undergoing AV replacement . MoreoveIn conclusion, our findings demonstrate that Gal-3 pharmacological blockade delays alterations in aorta and AVs in PO rats. These results indicate that Gal-3 may be a new potential biotarget for delaying aortic remodeling and AV inflammation and calcification in PO conditions.n = 7), PO rats , and PO rats receiving the Gal-3 inhibitor modified citrus pectin  in the drinking water. Treatment was initiated one day prior to the occlusion of the aorta and continued for another six weeks afterwards until the animals were finally sacrificed. The Animal Care and Use Committee of Universidad Complutense de Madrid and Dirección General de Medio Ambiente, Comunidad de Madrid (PROEX 242/15) approved all experimental procedures (12 April 2012) according to the Spanish Policy for Animal Protection RD53/2013, which meets the European Union Directive 2010/63/UE .Adult male Wistar rats were obtained from Harlan Ibérica . Rats were subjected to PO by placing a clip in the ascending aorta as previously described ,22,23,31The ascending aortic section containing the AV and a section of thoracic aorta were dissected. Tissue staining was performed on transversal sections of AVs and aorta. Samples were dehydrated, embedded in paraffin and cut in 5 μm-thick sections.2O2 for 10 min to block peroxidase activity. All sections were blocked with 5% normal goat serum in phosphate buffered saline (PBS) for 1 h and incubated overnight with Gal-3 , fibronectin , TGF-β , CCL-2 , osteopontin , MMP-2 , MMP-9 , cd68 , cd45 , BMP-2 , BMP-4 , Runx2 , Sox-9 , α-SMA , and CTGF , washed three times, and then incubated for 30 min with the horseradish peroxidase-labeled polymer conjugated to secondary antibodies . The signal was revealed by using a 3,3′-Diaminobenzidine (DAB) Substrate Kit .Slides were treated with HFor the staining, the slides were hydrated and incubated for 2 h with haematoxylin and eosin staining and 1 h with picrosirius red. After incubations, samples were washed three times and dehydrated.All measurements and quantifications were performed blind in an automated image analysis system .N′,N′-dimethyl-N-[4-[(E)-methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine (SYBR) green PCR technology   and purified using the RNeasy kit, according to the manufacturer’s instructions . First strand cDNA was synthesized according to the manufacturer’s instructions . Quantitative polymerase chain reaction analysis was then performed with NH, USA) . RelativIL-6, IL-1β, TNF-α, CCL-2, MMP-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-2 concentrations were measured in aortic extracts by ELISA according to the manufacturer’s instructions .2 and 5000 mmol/L NaCl to promote degradation of gelatin. Gels were fixed in 40% methanol and 10% acetic acid and then stained for 30 min in 0.25% Coomassie blue R-250 to identify proteolytic activity of MMPs.Aliquots of aortic tissue containing 15 μg of proteins were resolved on a 10% SDS polyacrylamide gel containing 0.3% gelatin. The gel was rinsed three times for 15 min with a solution of 2.5% Triton × 100 to remove SDS and renature the proteins, followed by incubation for 48 h at 37 °C in 1000 mmol/L Tris-HCl, pH 7.5 with 1000 mmol/L CaClp-value of <0.05 was considered statistically significant.Data are expressed as mean ± SEM. Normality of distributions was verified by means of the Kolmogorov–Smirnov test. Data were analyzed using a one-way analysis of variance, followed by a Newman-Keuls to assess specific differences among groups or conditions. All analyses was done with SPSS version 20.0 (IBM SPSS Statistics) and amtwo-tailed Gal-3 pharmacological inhibition blocks aortic and AV remodeling in experimental PO. Gal-3 emerges a new therapeutic approach to delay the progression and the development of aortic remodeling and AV calcification in PO.


Hydrocephalus is a complex neurological disorder with a pervasive impact on the central nervous system. Previous work has demonstrated derangements in the biochemical profile of cerebrospinal fluid (CSF) in hydrocephalus, particularly in infants and children, in whom neurodevelopment is progressing in parallel with concomitant neurological injury. The objective of this study was to examine the CSF of children with congenital hydrocephalus (CHC) to gain insight into the pathophysiology of hydrocephalus and identify candidate biomarkers of CHC with potential diagnostic and therapeutic value.42), tau, phosphorylated tau (pTau), L1CAM, NCAM-1, aquaporin 4 (AQP4), and total protein (TP) were measured by ELISA in 20 children with CHC. Two comparative groups were included: age-matched controls and children with other neurological diseases. Demographic parameters, ventricular frontal-occipital horn ratio, associated brain malformations, genetic alterations, and surgical treatments were recorded. Logistic regression analysis and receiver operating characteristic curves were used to examine the association of each CSF protein with CHC.CSF levels of amyloid precursor protein (APP) and derivative isoforms , followed by normalized sAPPβ , tau, APP, and L1CAM. Among subjects ≤12 months, a normalized CSF sAPPα cut-point of 0.41 provided the best prediction of CHC ; these infants were 32 times more likely to have CHC.CSF levels of APP, sAPPα, sAPPβ, AβCSF proteins such as sAPPα and related proteins hold promise as biomarkers of CHC in infants and young children, and provide insight into the pathophysiology of CHC during this critical period in neurodevelopment. Hydrocephalus is a debilitating neurological condition affecting approximately 1 in every 1000 children born in the United States , 3. HydrOur previous work in post-hemorrhagic hydrocephalus (PHH) of prematurity has shown alterations in CSF levels of amyloid precursor protein (APP), L1 cell adhesion molecule (L1CAM), neural cell adhesion molecule 1 (NCAM-1), and other protein mediators of neurodevelopment which normalize after initiation of PHH treatment . FurtherApproval from the Washington University (WU) Human Research Protection Office  was acquired prior to initiation of this study. Informed Consent (IC) procedures were in accordance with the approved WU HRPO parameters. Written IC was obtained where possible; however, verbal consent was permitted in cases where parents/guardians were unable to travel to Washington University/St. Louis Children’s Hospital. In all cases, a log was kept with subject ID and date and individual providing IC.th percentile for corrected age; bulging fontanel or splaying of the cranial sutures; papilledema; refractory headache, vomiting, or lethargy without other identifiable cause; or upgaze paresis/palsy. Exclusion criteria included previous surgical treatment for hydrocephalus; history of central nervous system infection or neoplasm; history of open spina bifida; hydranencephaly; and PHH of prematurity. Subjects meeting inclusion/exclusion criteria underwent surgical management of hydrocephalus following routine clinical care pathways at St. Louis Children’s Hospital. For a detailed record of clinical, radiographic, and neurosurgical parameters from the patients recruited for CHC, please refer to All patients ≤18 years of age presenting to St. Louis Children’s Hospital/WU School of Medicine for evaluation and/or surgical management of untreated, non-hemorrhagic CHC from 2010–2014 were considered for recruitment. For inclusion, CHC subjects were required to have ventriculomegaly on cranial imaging  ≥0.4)  and at lA Neonatal CSF Repository was established at WU in 2008 (WU Human Research Protection Office #201101887 and #201203126). For the purposes of this study, CSF samples were acquired at the time of initial CHC treatment. This study was initiated before the introduction of endoscopic third ventriculostomy (ETV) at our institution; thus, 18 subjects underwent placement of ventriculoperitoneal shunts while one patient had an ETV. One other CSF sample was acquired from a patient born at 31 weeks estimated gestational age who underwent initial ventriculosubgaleal shunting. In all cases, once CSF was acquired, the sample was immediately transported on ice from the operating room to the WU Neonatal CSF Repository, where it was centrifuged at 2500 rpm for 6 minutes, and the supernatant was aliquoted into microcentrifuge tubes (500μl each) and stored at -80°C until experimental analysis. For comparative analysis, age matched control CSF samples were obtained from patients ≤18 years of age without known neurological disease (corroborated on neuro-imaging) undergoing lumbar puncture for evaluation for infection, where cytological and microbiological (and in some cases PCR) evaluation of the CSF was negative. Age matched lumbar CSF from infants and children with seizures or stroke, termed other neurological diseases (OND), was also analyzed for an additional comparator group.42 (Aβ42), tau, and pTau ; NCAM-1 ; L1CAM ; AQP4 . Each CSF sample was run in duplicate for each ELISA, and the 96-well plates were read at 450nm on a Versamax microplate reader . The Pierce Bicinchoninic Acid protein assay kit  was used to estimate the concentration of total protein (TP) in each CSF sample as previously described [42, sAPPα, sAPPβ, L1CAM, NCAM-1, tau, pTau, and AQP4) were normalized by total CSF protein to account for any non-specific changes in total CSF protein.As described previously, enzyme-linked immunosorbent assays (ELISAs) were used to measure the CSF levels of each protein , 8. The ® 9.3.Data were expressed using mean ± standard deviation. Associations between continuous factors were assessed using the Pearson correlation coefficient. Simple logistic regression was used to estimate predictive models of CHC with biomarkers. ANOVA with paired contrasts was used to compare means across groups. Odds ratio (OR), sensitivity, and specificity were calculated for candidate cut-points of normalized CSF proteins for predicting CHC. All data analyses were performed using SASth ventricular outlet obstruction. Seven subjects had radiologic evidence of associated developmental brain malformations  and in controls  (p = 0.2847) but was significant in controls  but higher among subjects with OND than among control and CHC subjects (p < 0.0001)  . While C 0.0001) .42, tau, pTau, L1CAM, and NCAM-1 but not AQP4 or TP were elevated in untreated CHC compared with age-matched controls or individuals with OND  are reported throughout the remainder of this study as normalized by TP. With respect to ventricular size, normalized CSF tau levels were associated with FOR in control subjects only  while normalized CSF L1CAM demonstrated association with FOR in CHC subjects only   .42, sAPPα, sAPPβ, L1CAM, NCAM-1, tau, and pTau across the entire cohort of CHC subjects, ranging in age between 0 and 215 months, demonstrated significant increases in CSF APP, Aβ42, sAPPα, sAPPβ, L1CAM, tau, and pTau in CHC compared with control and OND subjects (p = 0.009), while no other biomarkers were associated with age in control or CHC subjects  = 0.99). When data from OND subjects were included, sAPPα remained effective in predicting CHC. Though not as strong, normalized sAPPβ, tau, APP, and L1CAM were also predictive for CHC over control and OND subjects , the presence of developmental brain anomalies, or a known genetic alteration. No differences were seen among the three groups with respect to FOR or TP, and none of the CSF biomarkers demonstrated significant differences among groups .42, tau, pTau, L1CAM, NCAM-1, AQP4, and total protein were investigated in the CSF of children with and without congenital hydrocephalus. Of these candidate CSF biomarkers, all but CSF TP, AQP4, and NCAM-1 showed a robust association with CHC. Normalized sAPPα demonstrated a particularly strong relationship with infantile CHC, with high sensitivity and specificity. Soluble APPβ, tau, APP, and L1CAM were also able to discriminate CHC, though not as strongly as sAPPα. While CSF TP showed an association with ventricular size in controls and CHC, it did not exhibit predictive ability for CHC.In the current study, levels of APP, sAPPα, sAPPβ, Aβ42 and tau, correlate well with clinical signs and symptoms [The pathophysiology of hydrocephalus is multifactorial with a pervasive impact on the brain. Previous studies of CSF composition in hydrocephalus , signs , and ventricular enlargement on neuro-imaging. However, the symptoms and signs used in the diagnosis of hydrocephalus are nonspecific, and ventricular size may be affected by a number of conditions common to infants and children at risk for hydrocephalus, including brain malformations, intracranial hemorrhage, and hypoxia/ischemia. Clinically, the diagnosis of hydrocephalus and the optimal timing of initiation of treatment (CSF shunting or ETV) are often unclear. Once implemented, treatment is invariably challenging to assess, since imaging is frequently unreliable after shunting, and few other tools are available to provide insight into CSF shunt or ETV function or, importantly, optimizing the child’s neurodevelopmental trajectory.While the most direct effect of these novel CSF biomarkers for CHC may be their diagnostic and therapeutic potential, the results reported herein also open new lines of scientific inquiry into the pathogenesis and pathophysiology of CHC. Elevations in CSF biomarkers, APP and tau in particular, may suggest recent or ongoing neurological injury, for example axonal stretch-related injury or synaptic disruption, as has been proposed in traumatic brain injury and other conditions (reviewed in ). In parThe relationships between increased CSF proteins and CSF physiology is likely to be complex. From a traditional perspective, obstructions that decrease CSF flow and/or absorption would be expected to impair clearance and cause CSF protein concentrations to rise. Fifteen of 20 of our patients exhibited aqueductal stenosis, potentially implicating CSF stasis in high TP and biomarker levels . In addition, an increase in total protein could have impacted CSF-parenchymal osmotic gradients and promoted ventriculomegaly in the same way that and dextrose- or sucrose-induced gradients cause mild-moderate ventriculomegaly in adult rats, dogs and cats –62. FurtHigh CSF protein concentrations also could be related to alterations in the CSF glymphatic system . ThiIn addition to PHH, many of the CSF biomarkers examined in the current report have been investigated in the setting of Alzheimer’s disease, Parkinson’s disease, and notably, iNPH, among other conditions (reviewed in). For ex2, body position, fluid loss in dural opening or ventricular cannulation) impact intracranial pressure under these conditions. The relationship between intracranial pressure and CSF biomarkers is of great interest, however, and our basic science efforts are directed at delineating this relationship in an experimental model where such factors can be rigorously controlled.A number of limitations must be acknowledged in this research project. While, to our knowledge, this study represents the largest study of CSF in non-hemorrhagic CHC, it nonetheless details an investigation of a modest sample size from a heterogeneous group of patients with likely myriad etiologies for hydrocephalus. The samples themselves span a wide range of ages with relatively fewer samples coming from individuals older than two years. Also contributing to the heterogeneity is the number and variability of associated brain abnormalities, non-CNS conditions, and genetic findings in the study population. It is likely that as we learn more about genetic alterations and their relationship to hydrocephalus that many of those individuals in this study in which no genetic etiology was identified will indeed have had a genetic cause and we were simply not yet able to detect it. While narrowing the scope of the project somewhat, subjects with hydrocephalus associated with myelomeningocele were deliberately excluded from the study, since factors such as active CSF leak or fluid shifts and uncertainty of infection presented challenging confounds to the current analysis. An inevitable limitation and challenge to studies of human CSF in children is the absence of true age-matched control CSF samples; the current study relies on CSF samples from human infants and children who underwent a lumbar puncture for clinical evaluation for sepsis or other diagnostic assessment where the CSF cultures and evaluation were negative. Ethical principles clearly prohibit routine CSF collection from asymptomatic patients. Another limitation inherent to this and similar studies is the potential for bias in cross-group comparisons introduced by rostrocaudal protein gradients, since control and OND CSF samples were acquired via lumbar puncture and CHC samples were acquired by ventricular cannulation. Reiber et al have demonstrated that different sources of CSF proteins exhibit different hydrodynamics, especially in relation to CSF flux variations associated with blood-brain and blood-CSF barriers –80. For CSF biomarkers including APP, sAPPα, sAPPβ, tau, and L1CAM hold promise as biomarkers of CHC in infants and young children. Soluble APPα in particular demonstrated a strong relationship with infantile CHC, with high sensitivity and specificity. In addition to supporting potential diagnostic and therapeutic roles for the care of children with CHC, the results of this study provide insight into the pathophysiology of CHC during this critical period in neurodevelopment.S1 TableR) and corresponding p-values for normalized CSF biomarker levels and ventricular size  across all study groups.Pearson correlation coefficients ((PDF)Click here for additional data file.S2 TableR) and corresponding p-values for normalized CSF biomarker levels and age at CSF sample across all study groups.Pearson correlation coefficients ((PDF)Click here for additional data file.


Förster resonance energy transfer (FRET) is a widely used single-molecule technique for measuring nanoscale distances from changes in the non-radiative transfer of energy between donor and acceptor fluorophores. For macromolecules and complexes this observed transfer efficiency is used to infer changes in molecular conformation under differing experimental conditions. However, sometimes shifts are observed in the FRET efficiency even when there is strong experimental evidence that the molecular conformational state is unchanged. We investigate ways in which such discrepancies can arise from kinetic effects. We show that significant shifts can arise from the interplay between excitation kinetics, orientation diffusion of fluorophores, separation diffusion of fluorophores, and non-emitting quenching. R, Förster’s theory predicts an energy transfer efficiency scaling as ∼(R/R0)−6. In practice one typically has R0 ∼ 1nm [Förster resonance energy transfer (FRET) is a widely used single-molecule technique for measuring distances within and between molecules , 2. FRETR0 ∼ 1nm . ExperimR0 ∼ 1nm , 5. SincR0 ∼ 1nm .In the biological sciences, FRET has been used to report on protein-protein interactions , 12. At + ions released during neuronal activity [Many types of molecules can be used for acceptor-donor pairing in FRET. Some molecules have photophysics that result in non-emitting quenching when interacting with surrounding chemical species or intramolecular chemical groups –30. Thisactivity . In emeractivity , 8, 20.E [E. Under differing experimental conditions, such as introduction of a denaturant, shifts in the observed efficiency histogram are interpreted as changes in the molecular conformational state [In single-pair FRET (spFRET), a single pair of acceptor and donor molecules are used to measure intramolecular distances . To charE , 32. Oveal state , 23, 33.al state , 35, it al state , 35. Theal state , 37 demoal state  suggest This presents the important issue of characterizing how shifts can occur in FRET efficiency in the apparent absence of any change in the conformational state. We investigate using theory and stochastic simulations the roles played by excitation kinetics, orientation diffusion of fluorophores, separation diffusion of fluorophores, and non-emitting quenching. Our results aim to quantify the magnitude of these effects and to help identify regimes in which these factors could impact experimental measurements.κA and κD of the photon emission of the donor and acceptor asThe FRET efficiency is the fraction of energy that is transferred non-radiatively from the donor to the acceptor molecule. Initially, it will be assumed that the energy can only be emitted as a donor photon or non-radiatively transferred to the acceptor ultimately to be emitted as an acceptor photon. In this case, the transfer efficiency is related to the rates For some systems it may be important to consider also additional photo-chemical states as in , 40 or tκT depends on the donor-acceptor separation distance R asτD = 1/κD denotes the average lifetime of the excited state of the donor in the absence of the acceptor. The characteristic Förster distance R0 depends on the photophysics of the donor and acceptor molecules throughNA is Avogodros’ number, κ2 a factor associated with the donor-acceptor dipole-dipole relative orientations [D is the quantum yield of the donor fluorescence in the abscence of the acceptor, J is the overlap integral associated with the adsorption spectrum of the donor and acceptor, n is the index of refraction. For a more detailed discussion see [Förster theory predicts that the non-radiative transfer rate e-length . The τD ntations , ΦD is t128π5n4NAThe NA iR06=9(ln)κ2ΦDJ128sion see .κA = κT. The distance dependence of the FRET efficiency can then be expressed asR can be inferred from observations of E. To obtain R0 only requires in principle knowledge of a few properties of the photo-physics of the donor and acceptor molecules. This allows for FRET to be used as an effective nanoscale ruler for molecular systems [When all transferred energy is immediately emitted as an acceptor photon, we have 1+(R/R0)6Förster  systems , 27, 43.nD, nA can be counted. This allows for a ratio-metric estimate of the transfer efficiency as [E. We remark that there are a number of important considerations in practice for such experiments, such as the development of criteria for when such a sequence of emissions is to be considered a significant FRET event or when there are short durations in the focal volume or shot noise.To obtain single molecule measurements for freely diffusing molecules, the donor is typically excited by waiting for an individual molecule to diffuse into the focus of a laser beam , 23, 24.iency as , 32E=nAThe efficiency histogram provides a characterization of the relative proportions of different conformational states or sub-populations of the molecules encountered during a measurement. For the case of homogeneous molecules in the same conformational state, the efficiency histogram is expected to exhibit a narrow peak around the characteristic FRET efficiency corresponding to the donar-acceptor separation of the conformation. It is then natural to consider changes in the conformational state of the molecule by looking for shifts in the location of the peak in the FRET histogram. This is widely used in experimental practice to characterize biomolecular systems , 14, 22.However, in recent experiments by Lipman et al. , 35, it κD = 1/τD. The τD is the mean donor excitation life-time in the absence of the acceptor. A donor molecule in the excited state either relaxes by emitting a photon at the rate κD or by transferring energy to the acceptor molecule at the rate κT in accordance with κT depends on a number of factors. This includes the separation distance R between the donor and acceptor. This also depends on the relative orientations of the donor and acceptor which is captured by the κ2 term in We consider the role of the kinetics of donor and acceptor excitation, energy-transfer, and relaxation. We model the event of donor excitation as occurring at the rate κT and observed FRET transfer efficiencies E.We investigate how such dependence of the energy transfer on the donor and acceptor configurations competes with the other excitation and relaxation kinetics. For this purpose we develop a stochastic model of the excitation-relaxation kinetics and perform simulations of the rotational and translational diffusion of the acceptor and donor molecules. We investigate the impact of these effects on the effective κ2 that contributes in κ is given by [The relative orientation of the dipole moments of the donor and acceptor molecules can significantly influence the efficiency of energy transfer , 44, 45.given by , 42κ=d^κ2〉 = 2/3, [κ2 values which can range between 0 and 4. This is sampled from a distribution with irregular and asymmetric features, see Contributions from orientation effects are often approximated by averaging assuming that the orientation rapidly diffuses isotropically on a time-scale much longer than the donor excitation time. The averaged orientation factor is often used 〈〉 = 2/3, , 42. How〉 = 2/3, , 44, 46.DR denotes the diffusion coefficent on the surface and ρ the radius of the sphere. The equations are to be interpreted in the sense of Ito Calculus [ρ, a configuration associated with the spherical coordinates  are to be interpreted in cartesean coordinates as Xt = ρ sin(Θt)cos(Φt), Yt = ρ sin(Θt)sin(Φt), and Zt = ρ cos(Θt).We investigate the role of orientation diffusion and its role on observed FRET efficiencies leading to possible shifts. Since only the relative angle between the donor and acceptor is relevant, we can model rotational diffusion by a Brownian motion on the surface of a sphere . This caCalculus , 49. TheWe perform simulations by numerically computing time-steps approximating the stochastic process in e sphere .τR = 4π2ρ2/DR. We use for the dye length ρ = 1nm and the sphere circumference 2πρ. The sphere circumference serves as the reference length-scale for the diffusion time-scale τR. We perform stochastic simulations using these parameters with a time step at most Δt = τR/500.We characterize the time-scale of the rotational diffusion by R. We take R = R0 so that for perfect averaging over all of the orientation configurations the transfer efficiency is E = 0.5. We consider the rotational dynamics relative to the donor excitation life-time characterized by τD/τR.We consider the case when the acceptor and donor are free to rotate but are held at a fixed separation distance τD/τR ≫ 1, and slow rotational diffusion where only a very limited subset of configurations are sampled over the donor lifetime τD/τR ≪ 1. For slow rotational diffusion, we find that the limited sampling over the donor lifetime can result in significant shifts of the observed FRET transfer E toward lower efficiencies, see We consider both fast rotational diffusion where most configurations are well-sampled over the donor lifetime κ2 contributes linearly to the transfer efficiency in κ2 values toward the smaller values corresponding to less efficient transfer when the rotational diffusion is slow relative to the donor lifetime, see The orientation configurations are all equally likely and the factor τR/τD = 19.5, we find the energy transfer is E = 0.486. This is close to when orientation is fully averaged to yield the energy transfer E = 0.5. For a slow rotational diffusion time-scale of τR/τD = 975 we have a transfer efficiency of E = 0.403. In this case, the rotational kinetics has resulted in a shift in the average transfer efficiency of 17%.The shift in transfer efficiencies resulting from the rotational kinetics can be significant. For a relatively fast rotational diffusion on the time-scale E can exhibit a significant shift without any change in the conformational state of the measured molecule. These changes arise purely from different rates of rotational diffusion. In practice, this could arise from changes in the viscosity of the surrounding solvent or from transient binding events with molecules present in the solvent that transiently restrict rotation of the donor and acceptor. We show over a range of diffusivities the shifts that can occur from these effects in Our results indicate on way that the FRET transfer efficiency We consider the role of the relative translational diffusion of the donor and acceptor molecules. We are particularly interested in the case when the measured molecule’s conformational state involves a sampling over an ensemble of different configurations. In this case, the donor and acceptor could undergo significant translational diffusion over the donor lifetime , 52. ForR by the stochastic processγ denotes the effective drag, Φ the potential of free energy for the separation distance R, DS the effective diffusivity in separation, and Wt Brownian motion. The equation is to be interepreted in the sense of Ito Calculus [DS and take the drag γ = kBT/DS where kB is Boltzmann’s constant and T is temperature. To model what happens as the separation distance approaches zero, we avoid negative lengths by a reflecting boundary condition at zero [τS = ℓ2/DS where ℓ is the same length that appears in We model the diffusion of the separation distance Calculus . We mode at zero . We charηn is generated each time-step as an independent standard Gaussian random variable with mean zero and variance one. The time-step duration is denoted by Δt. In practice, we use a time-step with Δt = τS/104. To give some intuition for the separation fluctuations and as a validation of our simulation methods, we show numerical results for the equilibrium distribution in At equilibrium this diffusion process has the separation distributionfunction . To simufunction Rn+1=Rn-DS relative to the donor life-time τD. This can be characterized by τD/τS where τS = ℓ2/DS.We consider the role of the separation kinetics of the donor and acceptor over the donor excitation life-time. We consider the transfer efficiency for different rates of separation diffusion E = 0.723 verses for the slowest translational diffusivity considered E = 0.508. This gives a relative shift in the FRET transfer efficiency of 30%.We find that a decrease in the separation diffusivity results in a significant shift in the FRET transfer efficiency, see The ensemble of configurations is the same for both the fastest and the slowest diffusion so the shift in transfer efficiency arises purely from kinetic effects. Over the donor life-time, the diffusion influences how likely the donor and acceptor are to encounter configurations favourable to energy transfer. In the case of slow diffusion, the rate of energy transfer is primarily governed by the initial configuration of the donor and acceptor.In the case of fast diffusion relative to the donor life-time, the donor and acceptor have more of an opportunity to encounter favorable configurations for energy transfer. This difference in how often such “sweet spots” for energy transfer are encountered over the donor life-time is supported by the observed separation distances that occur at the time of energy transfer, see For the fastest diffusivity, we see that significantly smaller separation distances occur at the time of energy transfer and thus yield on average larger FRET efficiencies. In the case of the slowest diffusivity, we see that the distribution of separation distances is broader and more closely follows the equilibrium distribution of separation distances since the rate of energy transfer is largely determined by the initial configuration of the donor and acceptor. We show the shifts in energy transfer for a wide range of separation diffusivities in We also consider the case when the donor can de-excite through a non-emitting pathway . One posα gives the fraction of donor de-excitations that result in some type of non-emitting quencher event. The We take these effects into account by developing some theory for how an additional non-emitting pathway would shift the observed FRET efficiency. A non-emitting quenching pathway can be modelled in our kinetics by killing some fraction of the donor de-excitation events that would have resulted in energy transfer to the acceptor and ultimately emission of acceptor photons. For the FRET transfer efficiency this corresponds to augmenting α = 1 corresponds to the situation when no non-emitting quenching events occur. In this case we have α = 0, all observed de-excitations result in non-emitting quencher events instead of donor de-excitation through FRET transfer events and emission of acceptor photons. In this case we have The case f corresponding to the ratio of donor to acceptor emissions when there is no non-emitting quenching. The reference fraction f is related to a reference FRET transfer efficiency E by f = E−1 − 1. The percentage shift of the observed FRET efficiency that arises from quenching is given byE. In fact, the shift that occurs becomes increasingly sensitive as E decreases, see The FRET efficiency can be conveniently expressed asWe have shown a few different ways that FRET efficiency can be shifted as a consequence of kinetic effects while the underlying molecular conformational state in fact has remained the same. We consider how such kinetic mechanisms relate to some recent experiments investigating the origins of shifts in FRET efficiency , 35, 58.FRET is often used to measure conformational changes or folding of proteins as denaturant conditions are varied , 35, 53.kDa PEG polymer in denaturant GuHcl ranging in concentration from 0 − 6M molar a shift was observed in the transfer efficiency of ∼20% referenced from E0 = 0.5. For the same polymer in the denaturant urea ranging in concentration from 0 − 8M a shift was observed in efficiency of ∼24% referenced from E0 = 0.5. Similar shifts were found for experiments performed using 5kDa PEG [An interesting finding is that FRET measurements under the same conditions exhibit a significant shift in the measured transfer efficiency. For a 35kDa PEG .Our results show that significant shifts can occur in the observed FRET efficiency even when there is no underlying change in the conformational ensemble. We showed how the transfer efficiency can shift purely from kinetic effects arising from changes in the rate of diffusion of the acceptor-donor orientation, diffusion of the separation distance between the donor and acceptor, and from non-emitting quenching. For diffusion of the donor-acceptor separation distance, we found such kinetic effects can cause shifts in efficiency as large as 48%. This occurred as the distance diffusion time-scale approached that of the donor life-time, see One way to try to account for the experimentally observed shifts is to consider how the denaturant augments the viscosity of the solvent , 59. Chaurea denaturant concentration at 8M is the factor 1.66 and for GuHcl 6M the factor 1.61 according to the experiments in [D = kBT/γ. The drag is given by γ = 6πμa where μ is the solvent viscosity and a is a reference length-scale characterizing the size of the diffusing molecule. This suggests that by increasing the solvent viscosity by a factor of 1.61 reduces the diffusivity by a factor of 0.6.The purported change in bulk solvent viscosity under changes in the ments in . To relaτD/τS = 0.1, such a change in viscosity shifts the transfer efficiency by ∼12%. This contribution solely from the diffusive kinetics of the donor-acceptor separation accounts for about half the ∼24% shift observed for 8M urea and the ∼20% observed for 6M GuHcl in [In our simulations taking as the base-line case GuHcl in . This isGuHcl in  suggestiThere are a number of potential subtleties when interpreting these effects. For one the donor and acceptor molecules are comparable in size to the viscogen denaturant molecules and the changes in diffusivity could possibly be more significant owing to more complicated interactions than suggested by the use of simple bulk theory for viscosity and diffusion –63. AnotWe have shown that kinetics can play a significant role in shifting the observed FRET transfer efficiency even when there is no underlying change in the conformational state of the molecule being measured. We found that changes in the orientation diffusion can in the most extreme cases shift the transfer efficiency by up to 20%. For the considered diffusion of the donor-acceptor separation distance, we found in the most extreme cases shifts up to 48%. Our findings concerning the donor-acceptor distance are in agreement with the investigations of Makarov and Plaxco’s work . We ment


P < 0.001). The odds ratios (ORs) of NAFLD for the highest (vs. lowest) quartile of RBP4 were 1.884  for females (P < 0.001), and 2.107  for male participants (P < 0.01) after adjusting for related factors. The serum RBP4 levels were positively associated with the prevalence of NAFLD in middle-aged and elderly Chinese people, and Homeostatic model assessment-insulin resistance (HOMA-IR), trunk fat, the waist-to-hip ratio (WHR), systolic blood pressure (SBP), fasting insulin, high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) might be implicated in the pathogenesis of RBP4 in NAFLD.Previous reports on the association between retinol binding protein 4 (RBP4) and nonalcoholic fatty liver disease (NAFLD) were controversial. This study aimed to investigate the association between the serum RBP4 levels and occurrence of NAFLD in Chinese population. In total, 2938 participants aged 40–75 years were involved in this community-based cross-sectional study. General information, lifestyle factors, serum levels of RBP4 and the presence of NAFLD were determined. Patients with NAFLD had significantly higher concentrations of RBP4 (37.9 ± 6.8 μg/ml) than did non-NAFLD controls (35.0 ± 6.7 μg/ml) ( Non-alcoholic fatty liver disease (NAFLD) is defined as a condition in which more than 5% of the hepatocytes exhibit macroscopic steatosis with no cause of secondary hepatic fat accumulation such as increased alcohol consumption, the use of steatogenic medications or hereditary disordersGlut4−/− mice911The contribution of retinol binding protein 4 (RBP4) in obesity and IR was first discovered in adipose-However, the data in the literature concerning the role of RBP4 in IR and NAFLD are inconsistent. The serum RBP4 levels were not associated with whole-body or hepatic IR in Mexican Americans14Our data showed that patients with NAFLD had higher levels of body mass index (BMI), waist-to-hip ratio (WHR), trunk fat percentage and serum ALT, fasting glucose, homeostatic model assessment-insulin resistance (HOMA-IR), triglycerides (TG) and uric acid (UA) as well as a high prevalence of high blood pressure (HBP) and diabetes mellitus (DM), but they had lower physical activities and serum high density lipoprotein cholesterol (HDL-C) than did those with non-NAFLD in both the female and male groups. NAFLD patients had higher low density lipoprotein cholesterol (LDL-C) than did non-NAFLD subjects only in the female group. There were no differences in household income and serum AST and total cholesterol (TC) between NAFLD and non-NAFLD participants divided by sex .P < 0.001 for all above parameters). In addition, a negative association between RBP4 and the HDL-C levels was observed (P < 0.001). However, there was no significant association between RBP4 and SBP and fasting glucose in the male group among the variables mentioned above. A positive association between RBP4 and LDL-C was found only in female participants.We next assessed the correlation between the serum RBP4 levels and a battery of known metabolic risk factors . Our anaP < 0.001 for all of the above parameters). However, DBP and fasting glucose were just significant variables associated with the serum RBP4 levels before adjustment.To investigate the relationship between serum RBP4 and IR as well as other correlated metabolic risk factors, multiple linear regression was performed using RBP4 as a dependent variable. As presented in P < 0.001). After the adjustment for variables (included in model 3 of P < 0.001) (The serum RBP4 levels ranged from 13.3 to 67.9 μg/ml among the participants. The results demonstrated that patients with NAFLD had significantly higher RBP4 concentrations (37.9 ± 6.8 μg/ml) than did those with non-NAFLD (35.0 ± 6.7 μg/ml) (< 0.001) .P < 0.001). As shown in P < 0.01). Furthermore, these associations remained robust after adjusting for the physical and metabolic parameters in models 2 and 3. The ORs of NAFLD for the highest (vs. lowest) quartile in model 3 were 1.884  for females (P < 0.001) and 2.107  for males (P < 0.01). The ROC analysis of serum RBP4 levels for the prediction of NAFLD can be found in To explore the ability of RBP4 to predict the presence of NAFLD, multivariate logistic regression analysis was performed. We found that serum RBP4 levels were positively associated with NAFLD in all subjects .All ultrasound examinations, using a Doppler sonography machine  with a 3.5-MHz probe, were performed to diagnose NAFLD by a single experienced radiologist who was blinded to the laboratory and clinical data. The diagnosis of NAFLD was based on standard criteria issued by the Fatty Liver Disease Study Group of the Chinese Liver Disease AssociationVenous blood samples were collected from the participants after overnight fasting. The serum was separated into several aliquots and stored at −80 °C within 2 hours. Colorimetric methods were used to measure fasting glucose, TG, TC, HDL-C, LDL-C, AST, ALT and UA in a Hitachi 7600-010 automated analyzer . IR was evaluated using the homeostasis model assessment (HOMA) in which HOMA = fasting glucose (mmol/L) × fasting insulin (mU/L)/22.5. The diagnosis of virus infection is established through serological testing using chemiluminescence immunoassay. All patients with positive HbsAg or positive anti-HCV antibody were also under the detection of HBV DNA and HCV RNA in serum using polymerase chain reaction (PCR).Serum RBP4 was measured using enzyme-linked immunosorbent assay (ELISA) kit , and the absorbance was determined using a microplate spectrophotometer . The lowest level of RBP4 that can be detected by this assay is 380 pg/ml, and the intra-assay coefficients of variation for RBP4 were 3.59%.t-test was used for parametric data comparison, and the Mann–Whitney U-test was used for non-parametric data.Descriptive statistics were computed for all variables. The normality test of Kolmogorov-Smirnov was performed to assess whether the data were normally distributed. The means ± SDs or medians  were used to describe continuous factors. For categorical variables, the frequencies and percentages were estimated. Categorical data were compared using Chi-squared test. Student’s P-value < 0.05 was considered statistically significant. All statistical procedures were performed using SPSS Statistics .The association between the serum RBP4 levels and metabolic risk factors was estimated using Pearson’s or Spearman’s correlation coefficient. Logistic regression analyses were used to estimate the ORs and 95% confidence intervals (CIs) in three models for the risk of NAFLD with increasing quartiles of serum RBP4 levels, using the lowest quartile as the reference group. Analysis of covariance (ANCOVA) assessed the serum RBP4 levels of NAFLD patients compared with non-NAFLD subjects stratified by sex. A two-tailed How to cite this article: Chen, X. et al. Retinol Binding Protein-4 Levels and Non-alcoholic Fatty Liver Disease: A community-based cross-sectional study. Sci. Rep.7, 45100; doi: 10.1038/srep45100 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Epithelioid hemangioendothelioma (EHE) of the thyroid is an extremely rare disease; only three cases have been reported in the English literature to date. Here, we describe a case involving a patient with thyroid EHE successfully treated with curative surgery.A 74-year-old woman presented with a right thyroid mass. The nodule was approximately 2 cm in size and was diagnosed as an indeterminate lesion by fine needle aspiration cytology. She was treated with thyroid lobectomy. The histopathological and immunohistochemical findings indicated an EHE of the thyroid. At the latest follow-up, 3 years postoperatively, the patient showed no signs of recurrence.There is currently no standard therapy for EHE; however, our case suggests that curative resection represents an effective treatment. Epithelioid hemangioendothelioma (EHE) is a rare vascular tumor, most commonly reported to occur in the lungs, liver, and bone, along with many other sites throughout the body . The etiEHE of the thyroid is very rare, and only three cases have been reported to date in the English literature. Here, we describe a patient with EHE of the thyroid who underwent successful resection.A 74-year-old woman was referred to our hospital because of a nodule in the right lobe of the thyroid. The nodule had been found during a medical examination performed 3 years ago, but she did not undergo a detailed examination at the local hospital at that time. On physical examination, a palpable and hard nodule of approximately 2 cm in size was noted in the right side of her neck. Results of the blood examination were normal, except for mildly increased thyroglobulin 142 ng/mL) and antithyroglobulin antibody levels (59.7 IU/mL). Ultrasonography and computed tomography showed a right thyroid nodule with calcification. The nodule had no signs of extrathyroidal invasion and measured 21 mm in diameter. The lymph nodes of the neck were not swollen 34, and factor VIII; diffusely positive for vimentin; and negative for thyroglobulin, thyroid transcription factor-1, smooth muscle actin, desmin, S100, CD31, CD68, and CD163 , as did the antithyroglobulin antibody level, although it remained positive. The mild elevation of the preoperative and postoperative thyroglobulin levels might have been caused by chronic thyroiditis. The patient has been followed-up for 3 years and has had no signs of recurrence postoperatively.EHE is a rare disease, first described by Weiss et al. in 1982 . The 200Especially, EHE of the thyroid is extremely rare –9, with Of note, only one of the five patients was male (20%), which is in accordance with previous studies on EHE of other organs, in which most patients were women  .There have been several discussions on the prognostic factors of EHE, such as the presence of pulmonary lesions, multiorgan involvement, age, and sex . In addiThere is no standard treatment for EHE, although curative resection has been reported as a successful treatment with good outcomes . All fivFinally, the presence of CAMTA1-WWTR1 gene fusion in EHE was recently demonstrated . This abIn conclusion, EHE of the thyroid is an extremely rare disease. Its clinical course varies, and the prognostic factors are unclear. Curative resection may be an effective treatment, but surgical issues such as the appropriate extent of the surgical margin and use of lymph node dissection remain to be clarified. Further studies are needed to analyze the etiology of EHE and to determine the optimal treatment.