Aggregation of Aβ amyloid fibrils into plaques in the brain is a universal hallmark of Alzheimer’s Disease (AD), but whether plaques in different individuals are equivalent is unknown. One possibility is that amyloid fibrils exhibit different structures and different structures may contribute differentially to disease, either within an individual brain or between individuals. However, the occurrence and distribution of structural polymorphisms of amyloid in human brain is poorly documented. Here we use X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid. Our observations indicate that (i) tissue derived from subjects with different clinical histories may contain different ensembles of fibrillar structures; (ii) plaques harboring distinct amyloid structures can coexist within a single tissue section and (iii) within individual plaques there is a gradient of fibrillar structure from core to margins. These observations have immediate implications for existing theories on the inception and progression of AD. But only a limited number of methods can carry out analyses of fibrils in situ. As we will report here, X-ray microdiffraction is amenable to in situ studies because it utilizes a micron-sized x-ray beam to collect data from very small scattering volumes making possible the targeting of regions with high amyloid concentration and low abundances of other constituents. Here, we use it to generate detailed structural information about amyloid fibrils in situ, map the spatial distribution of structural heterogeneities and identify differences in amyloid structure from subjects with different clinical histories.Detailed study of synthetic Aβ(1–40) fibrils in vitroX-ray scattering from amyloid fibrils is largely confined to equatorial scattering in the small angle and ~10 Å regimes; and meridional scattering in the wide angle regime - in particular the strong ~4.7 Å layer line which, because of poor orientation is often reported as a single reflection. The combination of equatorial 10 Å peak and meridional 4.7 Å peak was used to make the original assignment of a cross-β structure to amyloid27et al.ex vivo and two synthetic amyloid fibril preparations Sunde et al.3339The shape and breadth of the 4.7 Å peak is highly variableet al.et al.A more likely interpretation of the ‘doublet’ nature of the 4.7 Å peak is that it represents a visualization of a pair of reflections on the 4.7 Å layer line. Because of poor orientation of fibrils, the intensity along the layer line is arced about the center of the pattern transforming distances along a layer line into apparent radial separations. The lower angle peak (~4.75 Å) corresponds to a meridional or near-meridional reflection; the wider angle peak (~4.65 Å) to an off-meridional reflectionin situ, we used X-ray microdiffraction of histological sections of human tissue to map the abundance, orientation and structural heterogeneities of amyloid within individual plaques; among proximal plaques and in subjects with distinct clinical histories.In order to assess the degree of polymorphism present in amyloid fibrils Brain tissue from the amygdala of three AD subjects; one mismatch case, two age-matched controls and a subject diagnosed as having Pick’s disease were processed by standard histological methods (see Methods section) and spread onto 12 μ thick mica films to act as substrates to hold the samples in place for x-ray scattering. Serial sections, immediately adjoining the x-ray samples were captured on glass microscope slides and immunostained for presence of Aβ peptides. In all cases care was taken to collect x-ray data from regions expected to have highest plaque burden on the basis of microscopic examination of the immunostained sections since scanning microdiffraction cannot be readily used to survey large regions of tissue. For the age-matched controls and Pick’s subject no evidence of Aβ peptides was observed with immunostaining.Presence of amyloid was judged on the basis of the observation of a sharp 4.7 Å reflection known to be characteristic of cross-β structure. The proportion of diffraction patterns exhibiting this signature reflection was highly variable among fields of view even in the same subject. The number of diffraction patterns exhibiting this reflection is reported for each subject in The distribution of amyloid across a 250 × 250 μ field of view was mapped by collecting 2500 diffraction patterns using a 5 μ beam scanned over a 5 μ grid and automatically capturing the intensity of the sharp 4.7 Å reflection in each pattern. Scanning microdiffraction was carried out on Congo Red stained tissue from an AD subject. Congo red dye binds specifically to amyloid and has been used for years as a stain for locating regions with high amyloid concentration. Scans of tissue from two additional AD cases are contained in the A scan of tissue from a Pick’s disease subject that exhibited no sign of Aβ peptides but abundant presence of Tau by immunohistology was carried out in an attempt to identify the diffraction signature of neurofibrillary tangles. However, the scan showed scant evidence of a sharp 4.7 Å peak in only 4 of 2500 diffraction patterns, providing no substantive information about the nature of scattering from tangles in these samples.In many of the diffraction patterns harboring a sharp 4.7 Å peak, there is variation in the 4.7 Å peak intensity as a function of angle about the center of the diffraction pattern. In these cases, the angle at which the highest intensity of the peak occurs indicates the most common orientation of fibrils in the scattering volume. In 43The 4.7 Å peak usually appears as a doublet as seen in In both AD and mismatch cases, there are variations in fibril structure between plaques in the same tissue section. in situ within sections of human tissue prepared using standard histological methods. These preparative methods have the potential to alter the structure of amyloid and, indeed, destroy much of the other macromolecular structures present in the tissue. Nevertheless, x-ray scattering from these samples exhibited the properties expected for scattering from amyloid: (i) We observed a 4.7 Å peak in a small percentage of the diffraction patterns taken from tissue shown by neuropathology to have a heavy plaque burden. (ii) This 4.7 Å reflection falls at the scattering angle expected for scattering from amyloid. (iii) The diffraction patterns exhibiting this 4.7 Å peak are clustered into regions of size typical of amyloid plaques. (iv) Clusters of diffraction patterns exhibiting this peak are also observed at the margins of vasculature in some samples. (v) The 4.7 Å peak has a doublet character that has been reported extensively in studies of amyloid33Here we have presented evidence of the existence of structural polymorphisms in amyloid fibrils within and among plaques of a single individual and the existence of distinct differences in the organization of amyloid in subjects with different clinical presentations. X-ray microdiffraction was demonstrated to be an effective approach to investigating the structure of amyloid Orientation of the fibrils appears to be correlated over 10’s of microns, is preferentially tangential to the edge of blood vessels and relatively disorganized in smaller plaques. Congo red staining enhanced the detectability of orientation but did not appear to alter the general patterns observed. In some cases, the orientation at the center of a plaque appeared less well defined than along the periphery. This may be due to intrinsic disorder within the core; to multiple preferred directions occurring through the thickness of the section; or to a preferred orientation perpendicular to the section . There appears to be longer-range correlation in fibril orientation in larger plaques than in smaller. This might suggest that fibrils progressively pack tighter and with longer-range spatial correlations as plaques mature.Within a single section of tissue from one AD subject, we observed both perivascular amyloid deposits and plaques not associated with the vasculature. No distinct differences in fibril structure or structural variations were observed between the fibrils in these two environments. It is well known that plaques can exhibit a variety of morphologies that have been classified by light microscopyet al.o would be consistent with off meridional intensity at about the position of the ~4.65 Å peak. When spread around the center of the diffraction pattern by disorientation of fibrils, this off-meridional intensity will appear at wider angles than the meridional intensities, thus leading to the doublet nature of the 4.7 Å reflection. Consequently, the ratio may provide some measure of the tiling of β-strands in these fibrils.The systematic variation in the shape of the 4.7 Å peak reported here may reflect a systematic variation in fibril structure, perhaps related to packing, assembly or processing of fibrils within plaques, or to distinct differences in quaternary organization of peptides within fibrils. Detailed interpretation will require comprehensive modeling. However, basic principles of helical diffraction theory provide some guidance as to the meaning of the observed shifts in intensity. The doublet form of the peak arises from a combination of the intensity distribution along the 4.7 Å layer line and the significant disorientation common to virtually all diffraction patterns from these tissues. The lower angle peak (~4.75 Å) corresponds to a meridional or near meridional intensity peak on the 4.7 Å layer line. The wider angle peak (4.65 Å) corresponds to an off-meridional peak. Every peak in an x-ray pattern can be thought of as arising from a periodic variation of electron density that acts like a diffraction grating within the sample. A peak on the meridian corresponds to scattering from a diffraction grating with structural elements perpendicular to the fibril axis and periodic along the axis. In a cross-β structure like amyloid, these elements are β-strands and, they pack approximately 4.7 Å apart, constituting the structural elements that give rise to the 4.7 Å layer line. When most of that intensity is meridional, it suggests that the β-strands are perpendicular or nearly perpendicular to the fibril axis. If the β-strands tilt relative to the fibril axis (become less perpendicular), this has the effect of shifting intensity off-meridian. The effect that tiling of β-strands has on scattering from amyloid fibrils has been discussedCongo Red dye appears to enhance the meridional intensity at the expense of the off-meridional. This suggests an enhancement of structural elements perpendicular to the fibril axis, perhaps due to the orientation of the bound CR molecules parallel to the β-strands as has been observed in crystals of amyloid protein pig insulin46within individual plaques suggests that fibrils packed in the dense cores have β-strands more nearly perpendicular to the fibril axis than those on the periphery. This variation may reflect an ensemble of structures related to one another by a low energy structural transition such as a variation in fibril twist that might be influenced by side-to-side packing of fibrils. Variation of the intensity ratio among plaques follows the same pattern - the denser plaques exhibiting peak shapes consistent with β-strands more nearly perpendicular to the fibril axis. Diffuse plaques exhibit scattering more similar to the margins of denser plaques.The variability in the intensity ratio of the 4.75 and 4.65 Å peaks Plaques in the mismatch case appear to fall into two classes - those with amyloid scattering distinctly different from those in typical AD cases and others that appear similar to the plaques we observed in typical AD. Fibrils in regions where the 4.7 Å peaks are observed to take on an atypical shape (4.65 Å sub-peak stronger than 4.75 Å sub-peak) may have an atypical molecular structure. The distribution of amyloid in plaques giving rise to these atypical reflections appears to be relatively diffuse, for the most part not coalescing into tight cores. Although the pattern of structural variation in the mismatch case is similar to that seen in typical AD, the observed ensemble of peak shapes is shifted beyond that observed in even the most diffuse plaques in typical AD . It seemDeeper characterization of these atypical fibrils may provide insight into their role in disease. If the fibrils themselves are the toxic substance in AD, perhaps a different organization of Aβ peptides decreases their toxicity. Alternately, if Aβ oligomers are the actively toxic component, do the atypical fibrils in mismatch cases bind to oligomers, sequester them into large, inactive aggregates, and thereby slow or prevent their associated toxicity? Given the marked difference in neuropathology - which includes the presence of amyloid plaques and tangles but relative preservation of neurons and synaptic markersThese results document polymorphisms in amyloid fibril structure within individual plaques, among plaques in the same tissue and in tissue derived from subjects with different clinical histories:in vitro observations of fibril polymorphisms14151617181920212223The observation of polymorphism in amyloid fibril structure within individual plaques complements extensive We show in two cases that plaques harboring distinct ensembles of amyloid structures coexist within a single tissue section. This could be evidence for multiple strains of amyloid within a single brain or could reflect different stages of plaque maturation. The existence of multiple strains would contradict the suggestion that individual brains may accommodate a single predominant structure formed by self-propagation following a single seeding event1249et al.We also demonstrate that tissue derived from subjects with different clinical histories may harbor different ensembles of fibrillar structures. This observation appears to support the conclusion of Lu In summary, these results suggest that each brain accommodates an ensemble of amyloid structures, that the ensemble may exhibit significant variation among subjects and that the nature of the ensemble and its constituent fibrils may determine clinical outcome.o C for a minimum of 12 hours and then washed for 5 minutes each with 100% xylene, 100% ethanol, 10% ethanol and then water. Immediately before or after a section was cut for x-ray analysis, a serial section was cut to 5 μ thickness, captured on a glass microscope slide, processed for histological examination and immunostained with 10D5 antibody  to locate amyloid plaques. These serial sections made possible location of regions with high probability of containing plaques. Small blood vessels crossing serial sections served as useful fiducial marks for locating these regions in the sections to be studied with x-rays.Human brain tissue was obtained through the Massachusetts Alzheimer’s Disease Research Center that maintains a brain tissue bank at the Massachusetts General Hospital (MGH). The brains collected by MADRC are handled, dissected, and stored in a uniform fashion. Brains are divided in half with one half fixed, processed in paraffin for neuropathological analysis and immunohistochemical studies and the other half generally snap-frozen in coronal slices. Age matched controls were between 85 and 95 years of age and showed no overt signs of dementia at time of death. AD subjects were chosen for high plaque burden as were regions of the brain for analyses. The samples described in detail here were all from the amygdala. Thin 18 μ sections of fixed, paraffin-embedded tissue were cut and captured on 12 μ thick mica sheets. They were then de-paraffinized by heating to 75X-ray diffraction data were collected at beamline 23ID-B at the APS using the scanning microdiffraction capability developed for use in macromolecular crystallography−1. Regions in which the estimated peak heights were < 3σ were deemed to be devoid of amyloid .


Studies using positron emission tomography to image striatal dopamine function, have demonstrated that individuals with schizophrenia display increases in presynaptic function. Mesolimbic dysfunction specifically, has previously been suggested to underlie psychotic symptoms. This has not been directly tested in vivo, and the precise anatomical locus of dopamine dysfunction within the striatum remains unclear. The current article investigates the magnitude of dopaminergic abnormalities in individuals with schizophrenia, and determines how the magnitude of abnormality varies across functional subdivisions of the striatum.EMBASE, PsychINFO, and MEDLINE were searched from January 1, 1960, to December 1, 2016. Inclusion criteria were molecular imaging studies that had measured presynaptic striatal dopamine functioning. Effects sizes for whole striatum and functional subdivisions were calculated separately. The magnitude of difference between functional subdivisions in patients and controls was meta-analyzed.g = 0.68, P < .001) demonstrated elevated presynaptic dopamine functioning compared to controls. Seven studies examined functional subdivisions. These demonstrated significant increases in patients compared to controls in associative  and sensorimotor  regions, but not limbic . The magnitude of the difference between associative and limbic subdivisions was significantly greater in patients compared to controls .Twenty-one eligible studies were identified, including 269 patients and 313 controls. Individuals with schizophrenia (Hedges’ In individuals with schizophrenia dopaminergic dysfunction is greater in dorsal compared to limbic subdivisions of the striatum. This is inconsistent with the mesolimbic hypothesis and identifies the dorsal striatum as a target for novel treatment development. Previous meta-analyses of these imaging studies have found that the major dopaminergic abnormality in schizophrenia is increased presynaptic activity in the striatum.3 While an elevation of postsynaptic D2 receptors has also been proposed, meta-analytic findings have been less convincing,1 although the presynaptic results raise the possibility that receptor differences may be masked by increased endogenous dopamine levels.7–9Dysfunction of the dopamine system is one of the most well established findings in schizophrenia.11 the main anatomical focus for investigations of dopamine dysfunction has been the striatum. Animal research has demonstrated that the striatum can be divided into 3 distinct subregions based on function and the predominant topography of brain projections from limbic, associative, and sensorimotor cortical areas to the striatum , to December 31, 2016. Titles and abstracts were searched for the words: (“schizophrenia” or “psychosis” or “schizophreniform”) AND (“Positron Emission Tomography” or “PET” or “Single photon emission tomography” or SPET or “Single Photon Emission Computed Tomography” or SPECT) AND (Dopamine).25 and a control group; (2) reporting molecular imaging measures of presynaptic dopaminergic function  studies of patients with schizophrenia diagnosed in accordance with criteria specified in the Diagnostic and Statistical Manual for Mental Disorders (DSM), or the International Classification of Diseases (ICD)26–29Studies reporting data on dopaminergic functioning in individuals with treatment resistant schizophrenia, or co-morbid substance dependence, were excluded. This is because the primary neurobiological abnormality in these patients may not involve striatal hyperdopaminergia.The primary outcome of interest was the dopamine imaging parameter reported for the patient and control groups. For studies using labeled L-DOPA this was the influx constant in the region of interest relative to uptake in the reference region, while for studies using a release or depletion paradigm this was percent change in binding potential. In addition, author, year of study, number of participants, participant age and gender, illness duration, antipsychotic treatment, symptom scores, scan length, and whether an arterial input function was used were extracted.30 reporting data in individuals with schizophrenia were not included due to sample overlap with Howes et al 2013.31 Where values for the whole striatum were not given but data for the caudate and putamen were reported, whole striatum values were calculated as described previously1 by weighting these values by their volumes as reported in the Oxford-GSK-Imanova Structural–Anatomical Striatal Atlas (43% and 57% respectively). If the ventral striatum was also reported the following weightings were used to derive a summary outcome for the whole striatum: caudate – 36%, putamen-putamen – 48%, ventral striatum – 16%.32Two studiesg) for individual studies were estimated. The individual study effect sizes were then entered into a random effects meta-analytic model using restricted maximum likelihood estimation. I2 values were calculated to estimate between study heterogeneity. Where there were at least 10 studies included in a meta-analysis, funnel plots were constructed and visually inspected, and Egger’s regression test performed to check for the possibility of publication bias.33 Secondary subgroup and meta-regression analyses were undertaken to investigate the relationship between dopaminergic function and antipsychotic treatment (studies where ≥75% of patients were antipsychotic naïve were grouped as studies of predominantly antipsychotic naïve patients), scan length, paradigm type, modeling techniques, patient age and severity of symptoms.34 The statistical significance of differences between subgroups was tested for by fitting separate random effects models for each subgroup, and then comparing the subgroup estimates in a fixed effects model with a Wald-type test. A significance level of P < .05 (2-tailed) was used for all analyses.All statistical analyses were carried out using the “metafor” package (version 1.9–9) in the statistical programming language R (version 3.3.1). A minimum of 3 studies was required for meta-analysis. Standard effect sizes  comparison of the magnitude of intra-group subdivision differences . This approach employs methods used to quantify the propagation of errors.36 We estimated this correlation coefficient from individual data for 37 subjects .37 This showed Pearson’s coefficients of 0.72, 0.84, and 0.87 for correlations between sensorimotor-limbic, associative-limbic, and associative-sensorimotor divisions respectively. To be conservative the lowest of these values (0.72) was used for all comparisons. For example, to calculate the standard deviation of the limbic-associative difference in a patient group35:In order to calculate the standard deviation of this mean difference, a correlation coefficient for presynaptic functioning between subdivisions is required . Egger’s regression test was not significant , indicating publication bias was unlikely. Visual inspection of the funnel plot potentially suggested asymmetry . Studies using a challenge or depletion paradigm  showed a greater effect size when compared to those using labeled L-DOPA , and this difference was statistically significant  nor the use of an arterial input function (P = .55) was significantly associated with magnitude of effect size in the labeled L-DOPA studies. Meta-regressions of effect sizes against age (P = .29), total symptoms (P = .16), and positive symptoms (P = .39) were not significant.21 studies of individuals with schizophrenia met inclusion criteria , and 174 controls. All 7 studies used the subdivision definitions proposed by Mawlawi et al,g = 0.73, P = .002) and sensorimotor  subdivisions, but not for the limbic subdivision  (see I2 = 58.3%), with sensorimotor (I2 = 37.7%), and limbic subdivisions (I2 = 29.5%) showing relatively low levels of heterogeneity.Significant differences were found between patients and controls for associative (schizophrenia – .09) see . The resg = 0.38, P = .004). Presynaptic dopaminergic function in schizophrenia was also significantly greater in the sensorimotor compared to the limbic subdivision compared to the difference in controls . There were no significant patient-control differences as regards the comparisons between dopamine function in the associative and sensorimotor subdivision . These comparisons showed low levels of heterogeneity .In individuals with schizophrenia, the difference between associative and limbic subdivisions was significantly greater in patients compared to controls  relative to limbic striatum compared controls . Moreove58 Some studies included antipsychotic treated patients. However, our sub-analysis in antipsychotic free/naïve patients showed no statistically significant difference between these groups, and the elevation in presynaptic dopamine function was numerically larger in naïve patients than in antipsychotic treated patients, indicating antipsychotic treatment is unlikely to account for the elevation we see. Moreover chronic antipsychotic treatment may reduce dopamine synthesis capacity in some patients.59Moderate heterogeneity was seen in the studies of individuals with schizophrenia. Methodological factors such as differences in the resolution of scanners, measurement time, experimental paradigm, and modeling technique may contribute to this heterogeneity. In addition, differences in the clinical characteristics of patients could contribute to between study heterogeneity, given findings that increased dopaminergic activity is linked to acute psychosis.61 it should be recognized that these measures are indexing different, albeit related, aspects of dopaminergic function, and could be influenced by different factors. Interestingly our sensitivity analysis found that effects were greater for the challenge/depletion studies  for the sensorimotor-limbic measure  studies where the variance of the limbic measure, is noticeably greater than the variance of the associative measure.The limbic striatum has a smaller volume than either the associative or sensorimotor subdivisions. As a result it is more susceptible to partial volume effects whereby its true activity may be diluted by spill over and spill in from adjacent regions.ect size  could beNeither partial volume effects, nor reduced signal-to-noise, however, would account for the patient-control differences found when examining subdivision differences directly . In this1 Moreover, our meta-analysis extends understanding of the nature of dopamine dysfunction in psychosis by showing that the degree of dopaminergic dysfunction varies across the striatum, and identifies the dorsal striatum as the predominant locus of dopamine dysfunction in psychosis. Although patients showed no significant alteration in the limbic striatum relative to controls, we cannot rule out the possibility of a small difference in this subdivision. Nevertheless, in patients the dorsal to ventral balance was significantly shifted dorsally in patients when compared to controls. While a small mesolimbic abnormality may exist, overall these findings are not consistent with a hypothesis which proposes that the predominant locus of dopamine dysfunction is the limbic striatum.Our meta-analysis confirms, using a larger sample, the previous meta-analytic findings of increased presynaptic dopamine functioning in schizophrenia in the striatum.29 The associative subdivision receives dopaminergic innervation from the substantia nigra,12 suggesting that nigrostriatal pathways may be disrupted in schizophrenia. This hypothesis is in keeping with findings of increases in some,48 although not all,10 aspects of dopamine functioning within the substantia nigra in schizophrenia. The elevation was greatest in the associative striatum, although this was not significantly greater than the elevation in the sensorimotor striatum.Our findings thus suggest that models highlighting a primary role for excessive mesolimbic dopamine transmission in psychosis may need to be revised.5 The associative part of the dorsal striatum receives projections predominantly from dorso-lateral prefrontal cortex.13 Thus the dorsal locus of dopamine abnormality is consistent with the hypothesis that frontal cortical dysfunction underlies striatal dopamine abnormalities,69 although causality remains to be established in clinical studies.It should be noted, that while our findings support the hypothesis that dopaminergic functioning within the associative striatum may be abnormal in schizophrenia, this does not preclude the possibility that the primary site of dysfunction exists in another brain region.70 and suggest instead that selectivity for the dorsal, particularly associative, striatum may show advantages in both efficacy and tolerability. Treatment strategies may be able to make use of the neurochemical distinctions found across striatal subdivisions. For example, dopamine transporter densities are greater in the ventral, compared to dorsal, striatum.71 Due to this variable distribution, combination therapy with a dopamine reuptake inhibitor and D2 antagonist could potentially reduce dopaminergic neurotransmission to a greater degree in the dorsal, as opposed to ventral striatum. There are potential risks to this approach, but evidence suggests that in some patients it may have benefits for the amelioration of negative symptoms.72Our findings also question the proposal that mesolimbic selectivity is a desirable property for pharmacological treatments of schizophrenia,In conclusion, current molecular neuroimaging studies suggest that in individuals with schizophrenia the major locus of dopamine dysfunction is the dorsal striatum, and significant elevations were not seen in the limbic striatum. These findings are inconsistent with the mesolimbic hypothesis of schizophrenia, and suggest treatments showing nigro-striatal rather mesolimbic selectivity may have better efficacy and tolerability.Schizophrenia Bulletin online.Supplementary material is available at Supplementary DataClick here for additional data file.This work was supported by Medical Research Council-UK (no. MC-A656-5QD30), Maudsley Charity (no. 666), Brain and Behavior Research Foundation, and Wellcome Trust (no. 094849/Z/10/Z) grants to O.D.H., a Wellcome trust (no. 200102/Z/15/Z) grant to R.M., and the National Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.


Primary angiitis of the central nervous system (PACNS) is a rare autoimmune vasculitis limited to the CNS often causing substantial disability. Understanding of this disease is impaired by the lack of available biomaterial. Here, we collected cerebrospinal fluid (CSF) from patients with PACNS and matched controls and performed unbiased proteomics profiling using ion mobility mass spectrometry to identify novel disease mechanisms and candidate biomarkers. We identified 14 candidate proteins, including amyloid-beta A4 protein (APP), with reduced abundance in the CSF of PACNS patients and validated APP by Enzyme-linked Immunosorbent Assay (ELISA) in an extended cohort of patients with PACNS. Subsequent functional annotation surprisingly suggested neuronal pathology rather than immune activation in PACNS. Our study is the first to employ mass spectrometry to local immune reactions in PACNS and it identifies candidates such as APP with pathogenic relevance in PACNS to improve patient care in the future. Primary angiitis of the central nervous system (PACNS) is a rare form of vasculitis with approximately 700 reported cases published in the literature worldwide . PACNS iThe median age at onset of PACNS is around 50 years and men are more often affected then women. Clinical presentation displays a considerable heterogeneity. Initially, PACNS patients may present with unspecific symptoms such as headache and encephalopathy. Fever, seizures or focal symptoms are less common, but can also occur. Moreover, strokes, transient ischaemic attacks, and hemorrhages occur in 30–50% of patients . Very li+ T cell-mediated disease. In fact, the CSF of a patient with ABRA contained a substantial fraction of activated CD69+ CD4+ T cells, even remote from the primary site of inflammation. CD4+ T cells were present in close proximity to MHC class II–expressing epithelioid macrophages and multinucleated giant cells  the presence of an acquired otherwise unexplained neurological or psychiatric deficit, (2) the presence of either classic angiographic or histopathological features of angiitis within the CNS, and (3) no evidence of systemic vasculitis or any disorder that could cause or mimic the angiographic or pathological features of the disease .T1 weighted images, may also be useful in the assessment of PACNS  imaging can reveal ischemic and hemorrhagic lesions in cortical, subcortical, and deep gray matter structures . CNS angof PACNS –12. FinaIn summary, due to the small number of cases and limited amount of available biomaterial  it has been difficult to identify molecular mechanisms to better understand the immunological signatures of PACNS and to articulate hypotheses regarding pathomechanisms. High throughput and unbiased proteomics may provide new molecular insights in rare diseases –15. MassHere, we analyzed CSF cells by flow cytometry and non-cellular CSF using ultrahigh-definition mass spectrometry followed by multivariate statistics in patients diagnosed with PACNS in comparison to a control group . FunctioT1 weighted sequences, which showed contrast enhancement in the vessel walls . Four patients diagnosed with PACNS were included. Diagnosis was based on clinical symptoms, MRI and angiography findings and after having ruled out systemic vasculitis. The imaging diagnosis of PACNS was based on digital subtraction angiography and MR imaging using black-blood contrast-enhanced 6-053-f-SThe patients were followed-up in our department for at least 9 months after diagnosis was made. During this period consecutive MR imaging was performed and due to the diseases activity immunosuppressant therapy was started in all patients.In addition, all CSF cell samples and matching blood samples were analyzed by flow cytometry using a predefined staining panel (see below) and the non-cellular CSF fraction was processed for consecutive proteome analysis. For this, CSF samples were centrifuged at 350 g for 10 min, the CSF supernatant was removed and stored at −80°C until further proteomics processing. Maximum storage time for any sample was 21 months with an average of 11.38 ± 5.50 months. For ELISA analysis an additional four patients diagnosed as described above as well as four controls with IIH were selected from our biobank (see Supplementary Table +CD4+ (T helper cells), CD3+CD8+ (cytotoxic T cells), CD3+HLA-DR+ (activated T cells), CD3−CD56+ (NK cells), CD3−CD19+ (B cells), CD3−CD19+CD138+ (plasma cells), whereas monocyte subsets were selected as CD14+CD16− , CD14+CD16+  cells.CSF and EDTA blood samples were obtained from all patients and processed as described previously . BrieflyProteoMiner Small Capacity Protein Enrichment Kit from Bio-Rad  according to the instructions of the manufacturer. For the second depletion, the eluate of the first depletion was re-buffered using Zeba-Spin desalting columns 7 kD (ThermoFisher) and the ProteoMiner beads were washed using 20% ethanol. The second depletion then proceeded in the same way as the first.For proteome analysis, CSF samples (1 ml each) were depleted using a On filter-digestion and liquid-chromatography mass spectrometry (LC-MS) was performed as described previously . BrieflyAPP concentrations in the undepleted CSF samples were measured using a Human APP DuoSet ELISA kit from R&D Systems, Minneapolis USA, according to the manufacturer's instructions with a minimum detection limit of 0.625 ng/ml and using a protein standard curve for quantification  using the UniProt human database (downloaded October 2015) generating normalized protein abundances for each sample.http://www.R-project.org/)  . The Repct.org/) , 22. To ct.org/) .http://www.networkanalyst.ca/faces/home.xhtml. Using this online resource, a IMEx interactome based on the InnateDB platform was created , gender (p = 0.47), and body mass index (p = 0.34). Only one patient had been previously treated with two immunosuppressants and diagnosis had been established 54 months before sampling . To reduce the false positive rate, we applied very stringent selection criteria. After FDR correction, 14 proteins remained significantly different in PACNS (adjusted p < 0.05)  detected proteins were related to neuronal or nervous system function rather than immune system function illustrated by proteins such as brain abundant membrane attached signal protein 1 (gene BASP1), amyloid beta A4 protein (gene APP), and 14-3-3 proteins (genes YWHAB and YWHAH). This indicates that our screening approach preferentially detected abundance changes in proteins indicative of nervous system damage rather than reflecting immune cell activation or infiltration into the CSF compartment. Compared to candidates identified by mass spectrometry analysis of CSF in multiple sclerosis (MS) and Alzheimer's disease (AD) as well as human brain extracellular fluids in acute stroke overlap was modest. Only APP was also discovered to be changed in MS and AD (First we depleted CSF samples of most abundant proteins to improve the sensitivity for detecting low abundance proteins. Then we performed non-hypothesis driven label-free liquid-chromatography ultra-high definition mass spectrometry (LC-UDMSe) analysis comparing the CSF of four patients with PACNS to four controls with IIH. We detected 591 quantifiable proteins in all CSF samples (Supplementary Table ) Figure . NotablyS and AD –29.t(14) = 5.61, p = 0.0000641  = −2.59] as an important hub having several core connections in the newly defined network  , CD62L/L-selectin (gene SELL) , brain acid soluble protein 1 (gene BASP1) , 14-3-3 protein beta/alpha (gene YWHAB)  and 14-3-3 protein eta (gene YWHAH) . Therefore, changes of the CSF proteome in PACNS might reflect secondary nervous system pathology rather than immune activation. Nevertheless, these changes differ distinctly from findings in neurodegenerative disease . By depicting a PPI network we rather aimed to put the identified proteins into cellular and functional context to search for important nodes (hubs), which might be useful as biomarkers . We idenk Figure . The net disease , 31.The objective of this study was to generate novel hypotheses regarding the molecular mechanisms driving PACNS—a rare but particularly disabling neurological disorder. We describe the first CSF-focussed unbiased proteomics screening approach to study local immunity in the vicinity of the CNS. In fact, we identified a number of proteins with differential abundance in PACNS Figure . InteresWe performed down-stream analysis building a PPI network based on published evidence. This emphasized APP as central interaction hub and therefore potential key player in PACNS pathology. APP is one of the central proteins involved in CNS pathology in Alzheimer's disease (AD) . APP accStudies concerning APP levels in AD have been inconsistent . A role Downregulation of TFF1, which has been proposed as an anti-inflammatory agent in the brain, and downregulation of soluble L-Selectin having anti-inflammatory properties may reflect altered immune response in PACNS , 38. BAS+ T cells in the CSF of a patient with ABRA were increased .TR and GMzH designed the study, recruited patients, performed statistical analyses, and wrote the manuscript. JW performed the ELISA and data analysis. SK performed the LC-UDMSe work including bioinformatic data analysis using Progenesis QIP. AS-M and CG performed the FACS work. TR and MG did the network as well as additional statistical analyses of the proteomic data. GMzH, SM, and HW supervised the study and were involved in its design. All authors contributed to and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Metagenomics sequencing provides deep insights into microbial communities. To investigate their taxonomic structure, binning assembled contigs into discrete clusters is critical. Many binning algorithms have been developed, but their performance is not always satisfactory, especially for complex microbial communities, calling for further development.k-tuples directly, but this represents an absolute, not relative, sequence composition. Therefore, we attempted to model contigs using relative k-tuple composition, followed by measuring dissimilarity between contigs using k-tuples. To evaluate the performance of k = 6 under the independent identically distributed (i.i.d.) background model. Using the metrics of recall, precision and ARI (Adjusted Rand Index), https://github.com/kunWangkun/d2SBin.According to previous studies, relative sequence compositions are similar across different regions of the same genome, but they differ between distinct genomes. Generally, current tools have used the normalized frequency of Experiments showed that The online version of this article (10.1186/s12859-017-1835-1) contains supplementary material, which is available to authorized users. Metagenomics sequencing provides deep insights into microbial communities . A key sThe binning of contigs remains challenging owing to repetitive sequence regions within or across genomes, sequencing errors, and strain-level variation within the same species . Many stk-tuples (k-mers) with k= 2–6 as genomic signatures of contigs. MetaWatt [k-tuple frequency and Spearman correlation between the k-tuple frequency vectors. LikelyBin [4-tuple frequencies and scaffold coverage levels to populate the genomic bins using single-copy marker genes and an Expectation Maximization (EM) algorithm. MyCC [Contig binning tools based on a single sample generally follow one of three strategies. 1) Sequence composition. It is usually denoted as frequencies of MetaWatt  and SCIMMetaWatt  built muMetaWatt  clustereikelyBin  utilizedikelyBin  estimateikelyBin  package ikelyBin  combinedhm. MyCC  combinedk-tuple composition is based on the observation that relative sequence compositions are similar across different regions of the same genome, but differ between distinct genomes [k-tuples is one of the representation of sequence composition. In general, current tools use the frequency of k-tuples directly, but this represents absolute, not relative, sequence composition. Here, “absolute” frequency refers to the number of occurrences of a k-tuple over the total number of occurrences of all k-tuples. On the other hand, “relative” frequency refers to the difference between the observed frequency of a k-tuple and the corresponding expected frequency under a given background model. Contigs in the same bin are from the same taxonomic group, such as one class, species or strain. Therefore, contigs from the same bin are expected to obey a consistent background model. Several sequence dissimilarity measures based on relative frequencies of k-tuples have been developed such as CVTree, k-tuple frequencies. Therefore, in the present study, we attempted to model the relative sequence composition and measure dissimilarity between contigs with k-tuples. On the other hand, many binning tools based on absolute k-tuple frequencies and the results from such methods are reasonable. Still, these tools and methods can be improved by using k-tuple frequency vector. The bin’s center is represented by the averaged k-tuple frequency vectors of all contigs in this bin and is also modeled with a Markov chain. Then, https://github.com/kunWangkun/d2SBin.Contig binning using  genomes , 22. The studies –27 have  genomes  using Ma genomes , 25, met genomes  and meta genomes , 27. Howk = 6 and the independent identically distributed (i.i.d.) background model  are frequently the optimal parameters for recall, precision and ARI (Adjusted Rand Index).We selected six synthetic and real datasets that had originally been used to evaluate existing tools as testing datasets. uster3.0 , MetaWatmentclasspt{minimauster3.0 ) and theuster3.0 , MyCC [2uster3.0 ) based ok-tuple frequency vector. For each bin, the bin’s center is also modeled with a Markov chain based on the averaged frequency vector of all contigs in this bin. The The framework of mentclass2pt{minimk-tuple frequency vectors from two sequences X and Y, respectively, where cX , i is the occurring times of the ithk-tuple in sequence X and i = 1 ⋯ 4k. At each base in the tuple, there are four possible nucleotides, that is A, C, G, and T, for nucleotide sequences. So there are 4k combinations when tuple length is k.The equences . Let \dopi• ,  is the probability of the ithk-tuple under the Markov model with order r = 0 − 3 for one long sequence or set of reads and X or Y is the sum of occurrences of all k-tuples. The value of pX , i is the probability of the ithk-tuple under the background sequence for X. The pX , i can be the probability under the i.i.d. model, or under the Markov chain of different orders. The ithk-tuple is denoted as w = w1w2⋯wk. Under the rth order Markov chain Mr, the probability of the k-tuple w, namely the expected frequency, can be computed asp(wj) is the probability of wj estimated by the ratio of the number of occurrences of wj over the number of all nucleotides. The value of p(w1w2⋯wr) is estimated by the ratio of the number of occurrences of w1w2⋯wr over all the number of r-tuple occurrences. The value of p(wj + r| wjwj + 1⋯wj + r − 1) is estimated by the fraction of occurrences of wj + r conditional on the previous occurrences of wjwj + 1⋯wj + r − 1.The S = {S1, S2, ⋯Sl} be the partition of all contigs into l bins. Contig X is represented as k-tuples within the contig. The center of bin Sj is represented as the average frequency vector,Xi is the contig currently in Sj and nj is the number of contigs in Sj. The value of X and bin Sj.Let In our study, when the number of bins is fixed, the metrics of binning call for minimizing the within-bin sum of squares based on We then used the K-means clustering algorithm to optimize Eq. .The purpose of our study is to improve binning results using mentclass2pt{minim4-tuple frequency vectors and bins contigs with the K-median algorithm. The MetaCluster4.0 [4-tuple frequency vectors of contigs and assigns them with an EM algorithm, taking scaffold coverage levels into consideration. MyCC [The axBin1.0 , MetaCluaxBin1.0 , MetaWatmentclasspt{minimaaxBin1.0  and SCIMaxBin1.0  to evaluaxBin1.0  measuresuster4.0  and 5.0 uster4.0  were desmentclasspt{minimauster4.0  build inuster4.0  measureson. MyCC  combines20× and 80× average coverage, respectively. For the metagenomes containing 100 genomes, 100 million paired-end reads were sampled with three settings to create simLC+, simMC+ and simHC+. The three datasets represent microbial communities with different levels of complexity, which mimicked the setting of the previous study [simLC simulates low-complexity communities dominated by a single near-clonal population flanked by low-abundance ones. Such datasets result in a near-complete draft assembly of the dominant population in, for example, bioreactor communities [simMC resembles moderately complex communities with more than one dominant population, also flanked by low-abundance ones, as has been observed in an acid mine drainage biofilm [Olavius algarvensis symbionts [simHC simulates high-complexity communities lacking dominant populations, such as agricultural soil [MaxBin1.0  used the MetaSim  and asse MetaSim . The con MetaSim . For theus study : simLC smunities . simMC r biofilm  and Olavymbionts . These tral soil , where nral soil  and usedral soil .Sharon’. All metagenomic sequencing reads from the 11 samples were merged together, and 5579 contigs were assembled. The contigs were annotated with TAXAassign [This dataset was applied to test the binning tools COCACOLA  and CONCXAassign , and 261The above datasets cover various species diversity, species dissimilarity, sequencing depth, and community complexity. They include synthetic and real data. Therefore, testing on these datasets would yield a comprehensive evaluation of recall, precision and ARI (Adjusted Rand Index), were applied in our study. As described in COCACOLA [K × S matrix A = (aks) with K bins on S species where aks indicates the shared number of contigs between the kth bin and the sth species. Each contig binning tool filters out low-quality contigs; therefore, N is the total number of contigs passing through the filter and binned by the tools.To evaluate the performance of  studies , 5, 17, Recall: For each species, we first find the bin that contains the maximum number of contigs from the species. We then sum over the maximum number of all species and divide by the number of contigs.Precision: For each contig bin, we first find the species with the maximum number of contigs assigned to the bin. We then sum the maximum numbers across all bins and divide by the number of contigs.ARI (Adjusted Rand Index): ARI is a unified measure of clustering results to determine how far from that perfect grouping a bin result falls. ARI focuses on whether pairs of contigs belonging to the same species can be binned together or not. The detailed descriptions can be found in [ak∙ = ∑saks, as∙ = ∑kaks .found in , 5.9\dock-tuple and Markov order for the background sequences are required. Based on previous studies [k was generally set to 4–7 tuples, and the order of Markov chain was generally set as 0–2, as in previous applications, to analyze metagenomic and metatranscriptomic samples [k = 6 represent superior performance. The best performance was achieved under the i.i.d. background model of 6-tuples. All three criteria dropped suddenly at k = 8. The experiment offered initial guidance for the selection of tuple length and Markov order.In the calculation of  studies , 5, for  samples , 26. The10genome 80× and 100genome-simHC+ were selected to test the effect of tuple length on the performance of precision, recall and ARI for all five tools. Figures k = 4–6 because the severe dropping in performance with k = 7, 8 led to an excessively wide Y-axis coordinate range, and the curves of k = 4–6 appeared to aggregate, making it hard to display the superiority of k = 6. Therefore, we set k = 6 with According to Table entclass1pt{minimak = 6 and applied 0-2nd order Markov chain to calculate 10genome 80× and 100genome-simHC+ on the output of five contig-binning tools. As shown in Figs. Precision, Recall and ARI on all five tools. According to our previous studies about applying k = 6 and the i.i.d. model in To obtain the most suitable Markov order for the background genome, we fixed the tuple length agenomic  and metaagenomic , \documeRecall, Precision and ARI of the output of the five existing tools and after the adjustment of The contig-binning tools Maxbin , Metaclumentclass2pt{minimrecall, precision and ARI from 97.21%, 97.21%, and 95.58% to 97.75%, 97.75% and 96.16%, respectively. MaxBin, MetaCluster and MyCC assigned the contigs into 10 bins. MetaWatt and SCIMM obtained 27 and 8 bins, respectively, but From Fig. 10 genome 80× data, 10 genome 20× data have only 5 million reads for the 10 genomes. Fig. precision, but not recall or ARI, on MetaCluster. In order to have a deep insight on the deterioration of binning performance, we list the number of contigs from the 10 genomes in each bin, as shown in Additional file Flavobacterium branchiophilum, Halothiobacillus neapolitanus, Lactobacillus casei and Acetobacter pasteurianus with at least 100 contigs. But MetaCluster only output three bins: the contigs from Flavobacterium branchiophilum, Halothiobacillus neapolitanus and Lactobacillus casei are dominant in the three bins, and the contigs from Acetobacter pasteurianus are scattered into the three bins. After adjustment by Acetobacter pasteurianus were merged into the same bin as Halothiobacillus neapolitanus. Acetobacter pasteurianus and Halothiobacillus neapolitanus are both from the phylum Proteobacteria. Therefore, Acetobacter pasteurianus is phylogenetically closer to Halothiobacillus neapolitanus than to the other two genomes. From this point of view, Halothiobacillus neapolitanus were assigned to bin 1 and bin 4 and bin 1 included Halothiobacillus neapolitanus and Lactobacillus casei, which lead to the low ARI index as 24.76%. After using Halothiobacillus neapolitanus were assigned to bin 4, and bin 1 mainly included contigs from Lactobacillus casei. The ARI was increased to 70.48%. The result demonstrates that Compared with 20 million reads in simHC+ has evenly distributed species abundance levels with no dominant species. According to Fig. recall, precision and ARI, respectively. After recall, precision and ARI were improved for all of them by precision and ARI, but simMC+ [precision and ARI. However, recall, precision and ARI. The exception was MetaWatt. Among the 11,987 clustered contigs, MetaWatt isolated 41 bins. In this case, extracting contigs from the dominant genome from each bin would leave only 7978, meaning that one-third of the contigs would remain to interfere with the modeling of the 41 dominant genomes, in turn leading to decreased performance for precision and ARI.According to Fig. f simMC+ , only ~1recall, precision and ARI from 70.99%, 46.29% and 32.64% to 76.42%, 65.46% and 55.24%, respectively, which represents the best performance among the five tools.Sharon originally. As shown in Fig. Recall, Precision and ARI for all tools. (2) We applied CheckM [recall (completeness) threshold with precision (lack of contamination) > 80%. Although the tools identified 10–23 bins among the 21 species in the Sharon dataset, only 4–6 genome bins were recovered with precision > 80%. recall and precision. For MetaWatt and MyCC, precision > 80%. For MetaCluster and SCIMM, precision > 80% but also increased the number of bins with recall > 90%. The recall of each bin for MaxBin and MyCC. Figure precision thresholds with recall > 80%. For all tools, recall > 80%. For MaxBin and MyCC, the number of bins with precision > 90% is also increased by For this real dataset, the ground truth of binning was not available. The following two evaluations were implemented: (1) We only binned the 2614 contigs with unambiguous labels belonging to 21 species, and the annotations were considered as the ground truth. MaxBin, MetaCluster, MetaWatt, SCIMM and MyCC isolated 11, 10, 23, 19 and 16 bins for d CheckM  to estimTesting on these synthetic and real datasets showed that In order to evaluate the convergence of K-means iteration on https://github.com/kunWangkun/d2SBin.The code of Our experiments demonstrate Currently,  ISODATA , requirek-tuple sequence compositions.The ability of  genomes , 22. \dok = 6 under the i.i.d. model with no need to search for the optimal parameters.Our experiments demonstrate that


Lyme neuroborreliosis (LNB) is a frequent manifestation of Lyme disease in children and its current diagnosis has limitations. The elevation of the chemokine CXCL13 in the cerebrospinal fluid (CSF) of adult patients with LNB has been demonstrated and suggested as a new diagnostic marker. Our aim was to evaluate this marker in the CSF of children with suspected LNB and to determine a CXCL13 cut-off concentration that would discriminate between LNB and other central nervous system (CNS) infections.For this single-center retrospective case-control study we used a diagnostic-approved ELISA to measure CXCL13 concentrations in the CSF of 185 children with LNB suspicion at presentation. Patients were classified into definite LNB (cases), non-LNB (controls with other CNS affections), and possible LNB. A receiver-operating characteristic curve was generated by comparison of cases and controls.p < 0.001). A cut-off of 55 pg/ml resulted in a sensitivity of 96.7% and a specificity of 98.1% for the diagnosis of definite LNB and the test exhibited a diagnostic odds ratio of 1525.3. Elevated CSF CXCL13 levels were also detected in three controls with viral meningitis  while other CNS affections such as idiopathic facial palsy did not lead to CXCL13 elevation. Of the 41 patients with possible LNB, 27% had CXCL13 values above the cut-off of 55 pg/ml (median 16.7 pg/ml).CXCL13 was significantly elevated in the CSF of 53 children with definite LNB (median 774.7 pg/ml) compared to 91 control patients (median 4.5 pg/ml, CSF CXCL13 is highly elevated in children during early LNB as previously shown in adults. CXCL13 is a highly sensitive and specific marker that helps to differentiate LNB from other CNS affections in children. Borrelia burgdorferi sensu lato spirochetes [Lyme disease is the most common vector-borne disease in the Northern Hemisphere , 2. It irochetes , which arochetes . It presrochetes . The mosrochetes –8. Signsrochetes .Borrelia burgdorferi (B.b.) must be proven to confirm the diagnosis of definite LNB. Therefore determination of Borrelia-specific antibody index (AI) in the CSF has become the traditional diagnostic gold standard, despite limitations such as low sensitivity in the very early phase of the disease [Borrelia burgdorferi infection have negative AI [Laboratory confirmation of LNB is hampered by the low yield of bacterial culture and the poor sensitivity of polymerase chain reaction (PCR) in the cerebrospinal fluid (CSF) , 11. Acc disease , 13, 14  disease , 16. In ative AI . In suchative AI , causingative AI . AdditioRecently, measurement of the chemokine CXCL13 in CSF samples of suspected LNB patients has been identified as a new potential diagnostic tool –28. CXCLThe objective of our study was to evaluate the clinical relevance of measuring CXCL13 in the CSF of children with suspected LNB in the endemic region of Bern, Switzerland, and to establish a cut-off value for the diagnosis of early LNB in children. Using specific antibody index as a gold standard, we compared concentrations of CXCL13 among CSF samples of patients with definite LNB, possible LNB, and non-LNB controls.This is a single-center retrospective case-control study in the endemic region of Bern, Switzerland, over the period of January 2012 to December 2016. Children (0 to 18 years of age) with suspicion of neuroborreliosis and for whom frozen CSF samples were available, were retrospectively identified using the electronic database of the Institute for Infectious Diseases of the University of Bern, Switzerland. Clinical data were obtained from the archived charts or the electronic database of the Children’s University Hospital Bern, Switzerland. Patients were divided into the following subgroups according to the European Federation of Neurological Societies (EFNS) guidelines [Definite LNB was diagnosed using the following criteria: (i) patients had symptoms consistent with LNB and other relevant differential diagnoses were excluded, (ii) presence of mononuclear pleocytosis , and (iii) intrathecal production of Borrelia-specific antibodies. Possible LNB was diagnosed if only two out of three criteria were fulfilled. When less than two criteria or when a relevant differential diagnosis was present, LNB was excluded and the case was moved to the control group (non-LNB). Relevant differential diagnoses were defined as: spondylodiscitis, Guillain-Barré syndrome, mastoiditis, hereditary neuropathy, muscular atrophy, stroke, esophoria, erythromelalgia, infections with enterovirus, tick-borne encephalitis virus (TBEV), or varicella-zoster virus (VZV). Patients with malignancies or with CNS symptoms longer than 6 months were excluded from the study. LNB symptoms were defined by one or several of the following symptoms: facial palsy, headache, cranial nerve palsy, meningism, radiculitis, myelitis, ataxia, hemiparesis, fever, blurred vision, and cauda equina syndrome. Known antibiotic pre-treatment was not considered an exclusion criteria since patients who received antibiotics prior to lumbar puncture were not exhaustively identified in our data bank.Routine CSF and serum laboratory analyses were performed at the University of Bern, Switzerland, respectively, in the Center for Laboratory Medicine  and in the Institute of Infectious Diseases (serology and antibody index). Serum and CSF samples were subsequently stored at −20 °C at the Institute of Infectious Diseases. A screening test for Borrelia was first performed on fresh serum samples using enzyme-linked immunosorbent assays (ELISA) for IgG and IgM  according to the manufacturer’s instructions. In case of positive screening test or persistent suspicion of early neuroborreliosis, CSF/serum antibody index was determined. In this case, the presence of Borrelia-specific antibodies in simultaneously sampled serum and CSF probes was determined using IgG and IgM ELISA according to the manufacturer’s instructions. Two different ELISA kits were used over the period of the present study . The antibody index (AI) was considered positive when ≥0.3  or when ≥1.5 (Virion/Serion).CSF CXCL13 concentrations were measured by the first diagnostic-approved CXCL13 ELISA  according to the manufacturer’s instructions. CSF samples which had been stored for maximum 3 years at −20 °C were allowed to thaw at room temperature and measurements were performed neat in duplicates (intra-assay variation coefficients <15%). In a few cases, a dilution 1:5 was needed to analyze the samples due to low available volume; this had no influence on the test result. Incubations were done at room temperature. CXCL13 values below the detection limit of 4.6 pg/ml were assigned a value of 4.5 pg/ml. When optical density (OD) values exceeded the OD value of the highest standard, samples were further diluted 1:10 and 1:100 to obtain accurate concentrations of CXCL13. Results were obtained within 4 h.P values <0.05 were considered significant.Statistical analyses and ROC curves were performed using GraphPad Prism 6.0 . Diagnostic test evaluation was performed using MedCalc online calculator . Data are presented as a median (range) value or a number (%) value. Comparison of means was performed using non-parametric Mann-Whitney test. n = 4), enterovirus (n = 20), or tick-borne encephalitis virus , while others had inflammatory diseases (n = 8), idiopathic facial palsy (n = 19), primary headache (n = 11), or other diseases (n = 29). Inflammatory diseases included three Guillain-Barré Syndrome, three retrobulbar optic neuritis, one acute disseminated encephalomyelitis (ADEM), and one parainfectious brainstem encephalitis.Initially, 207 children with suspicion of Lyme neuroborreliosis at presentation were retrospectively identified for the present study, of which 22 had to be excluded due to malignancy or prolonged symptom duration. The remaining 185 patients were retained, and their CSF was analyzed for CXCL13. Details on sex, age, clinical presentation, and duration of symptoms until diagnostic lumbar puncture are presented in Table Forty-one additional patients (22%) were classified as “possible LNB” as they fulfilled only two out of three criteria for LNB . This inp < 0.001, Fig. The median CXCL13 concentration in the CSF of patients with definite LNB was 774.7 pg/ml and values ranged from 58.9 to 13487.0 pg/ml with 1 outlier at 4.5 pg/ml  curve was drawn for cases (definite LNB) versus controls  had a CSF CXCL13 concentration above the 55 pg/ml cut-off Table , Fig. 2.n = 27), of which nine (33%) had elevated CSF CXCL13 concentration. Another group of 13 possible LNB patients had pleocytosis but no determined antibody index due to a negative serology at initial work-up . A cut-off at 250 pg/ml had been twice suggested , 26. Thefections  have strWithin our definite LNB cases, only one patient had a CSF CXCL13 concentration lower than the defined cut-off of 55 pg/ml. She was a 3-year-old girl who was treated for an erythema migrans with amoxicillin 2 weeks before the onset of an acute facial palsy. She had a positive AI but a low CSF cell count of eight cells per microliter and her CXCL13 value was 4.5 pg/ml. It is known that CSF CXCL13 levels quickly decline after the initiation of an antibiotic treatment with ceftriaxone or doxycycline , 39, 40;Patients with possible LNB and a negative AI had a very short history of symptoms (median 3 days), compared to 9 days for patients with definite LNB. Such short symptoms durations could explain the lack of intrathecal antibody production , 20 and Our study also shows for the first time the expected percentage of mononuclear cells in the CSF of children with definite LNB  had already been observed in previous studies , 25, 26 The strength of the CXCL13 diagnostic test  combined with pre-test probabilities should enable rapid exclusion of LNB in case of negative CSF CXCL13 results and absence of previous antibiotic treatment, while the high positive likelihood ratio (29.8) will also give a strong diagnostic lead , 37. The


Recent in vitro experimental work in brain slices and neuronal cultures has shown that human CSF indeed contains neuromodulators that strongly influence neuronal activity. Here we briefly summarize these new findings and discuss their potential relevance to neural circuits in health and disease.The cerebrospinal fluid (CSF) occupies the brain’s ventricles and subarachnoid space and, together with the interstitial fluid (ISF), forms a continuous fluidic network that bathes all cells of the central nervous system (CNS). As such, the CSF is well positioned to actively distribute neuromodulators to neural circuits  Figure 1A), and forms a continuous fluidic network together with the interstitial fluid (ISF) of the parenchyma . Its composition is characterized by low protein (∼1% of blood) and high salt (>150 mmol/L) content, and is influenced by multiple sources including blood, the choroid plexus, ventricular ependymal cells, neurons and glia  system is an evolutionarily preserved feature of animal brains  and provand glia .in vitro showing that endogenous neuromodulators in human CSF potently influence the function of pyramidal cells and interneurons in the rat hippocampal brain slice, and in rat cortical neuronal cultures. The potential significance of this neuromodulation is discussed in health and disease, and an outlook on the future advancement of this research field is provided.Communicating freely with the ISF , the CSFin vitro platform to study synaptic, cellular and network aspects of neuronal activity (Table 1) whose collective influence on cellular and synaptic properties has been poorly investigated.Since its introduction in the early 1970s, the hippocampal brain slice preparation has remained a major activity . HoweverFigures 2A,B). Moreover, hCSF depolarized the resting membrane potential of CA1 pyramidal cells  and lowered their firing threshold  through apparent G-protein signaling-dependent mechanisms , leading to a left shifted frequency-current (input–output) relationship . In extracellular field recordings from CA3–CA1 synapses in stratum radiatum, hCSF caused a large increase in evoked excitatory synaptic transmission that was accompanied by an apparent increase in presynaptic release probability . In attempt to isolate the active neuromodulatory fraction of hCSF based on molecular size, the authors dialyzed hCSF samples to remove all constituents larger than 8 kDa. Compared to untreated hCSF, evoked excitatory synaptic responses were strongly reduced in the dialyzed hCSF, indicating a large contribution from low molecular weight (≤8 kDa) substances to these effects . Overall the study revealed strong neuromodulatory influence of hCSF on hippocampal CA1 pyramidal cells, suggesting that such neuromodulation could be of relevance to pyramidal cell function in vivo.In a recent study, Figures 3A,B,D,I,J,L). However, in contrast to CA1 pyramidal cells, hCSF had no effect on the resting membrane potential of interneurons , indicating cell specificity of the neuromodulatory effects. Similar to pyramidal cells, hCSF caused a left-shift in the frequency-current relationship of both groups of interneurons, increasing their responsiveness to excitatory input . Moreover, both interneuron groups displayed increased firing in response to sinusoidal current injections at theta and gamma frequencies in hCSF. Together with strong excitation of pyramidal cells, these effects are likely to promote hippocampal oscillatory network activity .Another recent study examined the effects of hCSF on GABAergic interneurons in CA1 stratum pyramidale of rat . Corticain vitro, (ii) some of these neuromodulatory effects are cell-type specific and act through G-protein coupled receptors (GPCRs), (iii) pyramidal cells appear to be more strongly modulated than the two examined groups of GABAergic interneurons, (iv) active neuromodulators are largely ≤ 8 kDa in size and (v) the presence of neuromodulators in physiological brain extracellular fluid such as hCSF can help explain differences in the amount of spontaneous neuronal activity observed in vivo vs. in typical in vitro brain slice recordings.Together these studies show that (i) hCSF contains physiologically active neuromodulators that potently increase the excitability of both pyramidal cells and interneurons Figure 4). MEAs are composed of tens of electrodes embedded into a glass or plastic substrate. On this electrode array, neural cells can be cultured and brain slice preparations can be placed to study neuronal activity at the single cell and network level for minutes up to several months (Multielectrode arrays (MEAs) are extracellular recording devices allowing the detection of neuronal network activity generated by cultured neurons or within brain slice preparations . Nevertheless, hippocampal or cortical neuronal cultures exposed to healthy hCSF show a strong increase in the number of population bursts . Thus, all together these studies show that hCSF enhances neuronal activity and induces synchronicity in the network  we were unable to obtain a CSF pool large enough to allow proper electrophysiological experimentation. Since neuromodulatory systems architecture is known to be well conserved across species , we woulAnother issue relates to the artificial manner in which neurons were exposed to neuromodulators in these studies. hCSF was introduced directly to brain slices and cultured neurons via an artificial perfusion system, allowing neuromodulators to bypass any natural diffusion barrier. It therefore remains unclear whether certain neuromodulators in hCSF would ever reach the corresponding areas in the intact brain. Moreover, it is possible that the neuromodulatory composition of hCSF could differ between brain and spinal compartments. All hCSF used in above described studies was sampled from the lumbar subarachnoid space, raising the question of whether hCSF sampled, e.g., from one of the ventricles could have produced somewhat different effects.Finally, a number of studies suggest that the CSF system during development is more compartmentalized than in the adult and young adult brain due to the presence of strap junctions between ependymal cells that largely limit the exchange of molecules between the CSF and ISF . Thus, iThe essential finding that hCSF contains physiologically active neuromodulators at ambient levels can be interpreted in light of two separate, although not mutually exclusive, scenarios. One possibility is that the neuromodulators, whose specific identities and relative functional importance are yet to be fully established, were selectively secreted into the CSF system in order to exert effects at downstream target areas. In this case, one could consider the CSF as an ‘active’ vessel or distributor of neuromodulatory signals within the brain, as has been previously suggested . An altein vivo or not, their remarkably potent effects on cortical neurons in recent studies have interesting implications for neuromodulation and neuronal communication in the brain. These findings show that (i) there exists neuromodulatory substances in brain extracellular fluid that retain physiological function over prolonged time periods  and (ii) there are neuromodulatory GPCRs (not excluding contributions from other receptor types) in cortex with sufficient affinity for these neuromodulators to mediate observed effects on neuronal activity. Thus, these results provide strong support for the concept of long distance (non-synaptic) volume transmission (in vivo would likely operate on a slow (minutes to hours) timescale with broad spatial distribution, targeting neuromodulatory GPCR families , their collective influence on neural circuits should be considered. In such a paradigm, the functional impact of a given neuromodulator would depend on its local concentration, target receptor affinity, expression and distribution, as well as sensitivity to enzymatic degradation and/or uptake by transporter proteins. Moreover, parameters affecting diffusion through extracellular space, such as local tissue tortuosity and chemical properties of the neuromodulator , are likely to be important . Depending on the experimental readout of neuronal activity , and the type of neuronal population or brain area examined, the relative importance of specific neuromodulators in CSF and their various receptor signaling pathways may vary.Our work so far has focused largely on characterizing the functional impact of CSF neuromodulators on cortical neurons. While we believe this to be an important effort, the specific identities of active neuromodulators in hCSF should also be thoroughly addressed. There is at present evidence suggesting critical contribution from a distinct receptor subfamily in promoting hippocampal network oscillations in vivo degradation and reuptake, and experimental handling such as thawing and freezing. Neuropeptides may display long half-lives in brain extracellular fluid and are active at nanomolar concentrations due to their high affinity GPCRs  is the defining feature of the disorder, which has translated into a clinically established therapy based on levodopa/carbidopa treatment . Given tO-methyltransferase, CSF HVA depends on uptake and intracellular O-methylation in non-dopaminergic cells. Thus, in Parkinson’s disease, the striatal content of HVA is not as severely decreased as that of dopamine , Alzheimer’s disease (acetylcholine), neuroinflammatory conditions, including multiple sclerosis (cytokines and interleukins), schizophrenia (dopamine and its metabolites) and affective disorders (serotonin and its metabolites). CSF orexin concentration is used as an adjunct in the criteria of the disease [CSF orexin concentration is low in the disease due to the loss of orexin-producing neurons ]. For Alin vivo and, if so, how this neuromodulation impacts neural circuit function, e.g., during different brain states such as sleep and wake. To experimentally confirm such active role of the CSF system would require in vivo demonstration of (i) active release of neuromodulator into a CSF compartment, (ii) neuromodulator utilization of the CSF/ISF system to distribute within the brain and (iii) a functional impact of the neuromodulator(s) on a distant neuronal population. Moreover, it would require the capacity to distinguish between functional effects caused by CSF-derived and locally released neuromodulator.Neuromodulation encompasses a vast variety of biochemical processes, occurring at various spatiotemporal scales, which tune the excitability and function of central neurons. In this review, we have considered how long distance neuromodulation via the CSF might influence the function of neurons and neural circuits in health and disease, in light of new experimental findings. Further efforts are needed in order to establish whether the CSF acts as an ‘active’ neuromodulatory channel in vivo combined with use of, e.g., fluorescent GPCR activation reporters and genetically encoded calcium/voltage indicators should provide new and better means to address the functional relevance of neuromodulation via the CSF system in health and disease. At present, further in vitro studies examining the neuromodulatory influence of healthy and pathological CSF on central neurons and circuits are warranted.A conceivable path toward elucidating the role of the CSF system in neuromodulation involves further research into the brain’s CSF-contacting neurons. Identification of cell type-specific markers enabling selective manipulation of CSF-contacting neurons that harbor neuromodulatory release machinery would greatly aid in this effort. The development of better techniques for detecting neuromodulator release and diffusion All authors took part in designing, writing and revising the manuscript. AB and SI provided the data and prepared the figures and tables.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer GK and handling Editor declared their shared affiliation.


The aim of this study was to emphasize the significance of internal audits of the blood donor selection process and documentation in a resource-limited country by assessing compliance with the established protocols, and to identify weak areas in the process.This audit reviewed the donor selection process at the blood bank of Liaquat National Hospital & Medical College, Karachi, over a 6-month period. Seven variables selected as performance indicators were graded as very good (%90-100%), good (80%-89%), satisfactory (70%-79%), or unacceptable (<70%). Blood bank staff was asked for feedback and suggestions.Documentation of donor demographics was not within the acceptable range (documentation rates of 65.14%), donor status records were satisfactory (77.64%), and donor physical exam records were graded as good (86.34%). Five performance indicators were graded as very good (90%-100%).The audit proved productive in identifying major causes of irregularities in documentation and in making valuable suggestions for their rectification. Documentation and record-keeping play integral roles in transfusion medicine from every step of the vein-to-vein chain of blood donation to the dispatch of blood components. Regular medical audits are a part of quality assurance programs in transfusion medicine and a means of continuous assessment and improvement of existing systems. For conducting audits of clinical laboratories, a written set of questions in the form of a checklist is used, evaluation of which indicates whether the laboratory is performing its procedures according to its documented policies and standard operating procedures and on time. Historically, audits done in blood banks were focused on clinical uses of blood components to ensure appropriate use, minimize wastage, and reduce the risk of transfusion-transmissible diseases. Developing countries like Pakistan depend heavily on non-remunerated blood donors as only 10% of blood donations are collected from voluntary donors . The WorThe objective of our study is to assess compliance with the established protocols for blood donor selection processes and documentation, to identify weak areas in these processes, and to recommend improvements in the system based on feedback obtained from blood bank staff.As a part of quality system improvement we planned a prospective 6-month internal audit of the donor recruitment process and documentation at the blood bank of a tertiary care hospital in Karachi, Pakistan, from January to June 2014. An audit plan was devised and checklists were prepared with the help of a toolkit developed by the Directorate General of Health Services, Dhaka WHO, July 2008 [Donor records were grouped into group A (donors deferred before donation), group B (donors rejected after donation: seropositive cases), and group C (donors selected for donation: seronegative cases). We selected documentation of 7 parameters as performance indicators: donor demographics, donor status, general physical exam, hemoglobin estimation, informed consent, reason of deferral, and notification of seropositive cases. For each group, performance was graded as very good, good, satisfactory, or unacceptable by maintaining a high level of scoring documentation rates of 90%-100%, 80%-89%, 70%-79%, and <70%, respectively. Based on the results, feedback was obtained from the blood bank staff responsible for conducting interviews of donors to determine common causes of nonconformance. A list of recommendations for appropriate changes in the current system was designed and submitted to the head of the blood transfusion services of the institute at the end of this exercise.The study was conducted after obtaining approval from the institute’s ethics review committee.Data analysis was performed using descriptive statistics. Numerical data are shown as percentages.In this study, inspection of the donor area was found to be satisfactory as almost all the prerequisites mentioned in the checklist were met. Equipment and materials were being properly maintained as the department is ISO-9001:2000 certified. Exceptions were absence of privacy for asking questions related to sexual behavior and lack of written material for donor self-deferral. Staff members were qualified and trained. Review of donor records from January 2014 to June 2014 showed that out of 10,041 prospective blood donors, 1027 donors belonged to group A, 496 to group B, and 8518 to group C. Donor demographic records were inadequately maintained (documentation rate: 65.14%) as donor identification card numbers and area of residency were not always documented in all 3 groups. This was followed by donor status (documentation rate: 77.64%) and vital statistics (documentation rate: 86.34%) in that order. See Among deferred donors, the reason of deferral was mentioned in all cases but the donor notification rate was 89.51%. The main reason cited for not documenting the identification card number was not asking for it due to its nonavailability at the time of donation, and the area of residency was missed due to ignorance about the exact zonal divisions of the city. Irregularities in documentation of donor’s vital statistics were mainly due to bypassing the set standard operating procedures.This audit gave insight into the existing practices of the donor selection process at our institute in particular and in developing countries in general. Random checks were conducted to evaluate the premises and processes using checklists and direct observations. Although the overall performance and documentations were good, some important issues were highlighted. Lack of privacy for conducting donor interviews was a concern identified in our study and mentioned by Kumar et al. in a similar study from India . AbsenceReview of donor records showed some nonconformance in donor demographics including irregularities in donor identification numbers and area of residency in all 3 groups. The purpose of the former is donor traceability and that of the latter is use for epidemiological data. Use of mobile numbers for contacting donors has become the norm as it is much easier and a quick method for donor notification that can safely replace identification numbers. However, its documentation rate needs to be 100%, especially in deferred donors with seropositive status (group B); in our case, this rate was 94.50%. Area of residency has also lost credibility as people lack awareness of exact zonal locations due to formation of new localities and the constant expansion of the city.Our study showed a shortcoming as per documentation rate for donor status (77%). Omission of data regarding donor status can be overcome by using different-colored forms for voluntary donors or by keeping separate registers. In this way, the focus can be directed towards voluntary donors with regular reminders for donations, thus facilitating the donor recruitment program. Documentation rate of vital signs collectively was good (86%). Documentation of the remaining 4 indicators was satisfactory.Feedback from blood bank staff was obtained to determine the most likely causes for omitted data. Failure to document identification number and area of residency was due to a silent understanding among staff about their triviality; hence, this information was not being collected from donors. Donor status was not noted mostly due to inattention and the incongruous location of the question window in the proforma according to the staff. Documentation rate of vital statistics was selectively poor in group A (documentation rate: 59.59%). This was attributed to bypassing of normal standard operating procedures of conducting a physical exam first, followed by hemoglobin estimation, by some new staff members due to ignorance of the protocol. Notifying donors about the potential presence of transfusion-transmissible disease is a major responsibility of blood banks. In our study, the donor notification documentation rate was good (94.5%). Failure to inform donors were due to no response when called, wrong mobile numbers, and failure to document the mobile number, in that order. Thus, instead of identity card number, at least two contact phone numbers should be noted to ensure a 100% donor notification record.The donor selection process is a vital link in the chain of blood collection, screening, and transfusion. A detailed audit of this program showed certain gaps in the documentation process. The following recommendations are made to minimize chances of lacunae and further improve the system:• Privacy must be provided for conducting donor interviews.• Donor identity card number and area of residency may be removed from the donor pro forma and replaced by two valid contact phone numbers.• Separate registers or color-coded forms can be used for voluntary blood donors.• Mini-audits of selected areas must be done apart from the annual external audits to improve quality.• Refresher courses for blood bank staff should be conducted regularly.• Introduction of electronic record-keeping in blood banks is vital for easy data retrieval.Ethics Committee Approval: Ethical Review Committee, Liaquat National Hospital, Institute for Postgraduate Medical Studies and Health Sciences, Karachi, Pakistan (App No. 0181-2014 LNH-ERC). Informed Consent: It was taken.