Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.An NZB-derived genetic locus  model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.Leukocytes from B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.B6.in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.The different  The strong genetic basis of SLE is sustained by a large number of polymorphisms that have been identified in recent years through association studies in large cohorts of patients and controls [Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex etiology in which the production of pathogenic autoantibodies (autoAbs) results in cellular and tissue damage. Aside from B cells, which produce these autoAbs, and CD4controls . Mouse mcontrols ,3. The gcontrols .bm12 lymphocytes are transferred into H-2b B6 hosts. Within 3 weeks of transfer, mice develop lupus-like phenotypes including lymphocyte activation and anti-nuclear autoAbs, which are dependent on interactions between donor CD4+ T cells and host autoreactive B cells [Sle2c2 mice, which are B6 mice carrying an NZM2410 (NZB)-derived genomic region on the telomeric potion of the Sle2 locus, are profoundly resistant to bm12-cGVHD induction as compared to their B6 congenic controls [Sle2c2 suppression is mediated by BM-derived cells, but not by T cells, B cells, or dendritic cells (DCs). We mapped Sle2c2 resistance to a short genomic interval that contains Csf3r, the gene encoding for the granulocyte-colony stimulation factor (G-CSF) receptor (G-CSFR) [Csf3r  carries a mutation in exon 10 (rs13477964) that results in a S378N substitution in its extracellular domain.The bm12- chronic graft vs host disease (cGVHD) model is a well-defined model of induced lupus in which B6.C-H2controls . Using m(G-CSFR) . The NZMlo MDSC population in B6 mice with established cGVHD [G-CSF is an essential factor in the recruitment and function of neutrophils . G-CSFR ed cGVHD .378S mutation in the Csf3r gene impairs G-CSFR function. This mutation is located in the solvent-exposed part of the receptor in a putative fibronectin III domain outside the solved structure of the protein [378S mutation is responsible for the differential bm12-cGVHD response between B6 and B6.Sle2c2 mice, which we tested by comparing their response to exogenous G-CSF. We reasoned that if bm12-cGVHD resistance in B6.Sle2c2 mice was mediated by a gain of function in suppressive cells such as MDSCs, G-CSF treatment would induce bm12-cGVHD resistance in B6 mice. In contrast, if bm12-cGVHD resistance in B6.Sle2c2 mice was due to a loss of function or recruitment in inflammatory neutrophils, then G-CSF treatment would alleviate bm12-cGVHD resistance in B6.Sle2c2 mice, which would develop autoAbs and lymphocyte activation comparable to that of B6. Here we demonstrate a weaker binding of mouse G-CSF to B6.Sle2c2 leukocytes as compared to B6, as well as a defective expansion of B6.Sle2c2 myeloid cells and neutrophils in response to in-vivo G-CSF treatment. Furthermore, exogenous G-CSF restored bm12-cGVHD responses in B6.Sle2c2 mice. Finally, the B6.Sle1.Sle2.Sle3 congenic mice that display a full-blown spontaneous lupus as its NZM2410 parental strain carry the Sle2c2 locus [Sle2c2 prevents the induction of systemic autoimmunity by impairing the production of inflammatory neutrophils due to a decreased ability of the G-CSFR to bind its ligand.These studies lead us to hypothesize that the N protein . It is tc2 locus . It was Sle2c2 mice carrying an NZM2410 (NZB)-derived interval located between D4Mit11 and D4Mit72 have previously been described [bm12H2/KhEg (bm12) were originally purchased from the Jackson Laboratory . Cohorts of mice within an experiment were sex- and age-matched, but the reported phenotypes were neither affected by sex or age. The NZM2410-derived triple congenic mice B6.Sle1.Sle2.Sle3 (B6.TC) have been previously described [The B6.escribed . B6 and escribed . All ani6 bm12 splenocytes were injected intraperitoneally into 2 to 4 month-old B6 or B6.Sle2c2 mice. Serum was collected for autoAb detection by ELISA as previously described [bm12-cGVHD was induced according to an established protocol . Briefly®, huG-CSF, Amgen, Thousand Oaks, CA, USA), all starting at d1 of induction. Some huG-CSF injections were repeated every 7 d resulting in 4 different treatment regimens: one injection of 120 ug (120 × 1), two or three injections of 12 ug (12 × 2 and 12 × 3), and three injections of 1.2 ug (1.2 × 3). Control mice were injected with 5% dextrose as vehicle control. Each experimental group contained at least five recipient mice per treatment per strain, and control groups were performed side by side for each treatment group. Groups of four un-manipulated mice per strain were injected with huG-CSF and blood was collected every 2 to 3 d to compare the dose-response effect of G-CSF of peripheral blood leukocytes (PBLs) between strains. Serum mouse (m) G-CSF was measured by ELISA with reagents from Peprotech  in sera diluted 1:50. B6.TC and B6 female mice were treated with six weekly injections of 1 ug of Neulasta or 5% dextrose starting at 2 months of age and were sacrificed at week 7. Serum was collected every two weeks and anti-dsDNA IgG, anti-chromatin IgG and total IgG were measured as previously described in sera diluted at 1:100 and 1:5000, respectively [bm12-cGVHD-induced mice were treated with subcutaneous injections of 1.2, 12.0, or 120.0 ug pegylated human G-CSF (Neulastab (AF6-120.1), CD11b (M1/70), CD11c (HL3), Ly6C/G (GR-1/RB6-8C5) or isotype controls, all from BD Biosciences . Biotinylated Abs were revealed by streptavidin-PercP-Cy5a. p-signal transducer and activator of transcription 3 (Stat3) (Y705) staining was performed after fixation and permeabilization (BD Cytofix/Cytoperm kit). G-CSF binding was performed by incubating splenocytes for 1 h with 25 ng of mG-CSF  biotinylated with a Biotin XX Microscale protein labeling kit , detected by flow cytometry with PE-streptavidin. As negative controls, cells were incubated only with PE-streptavidin. ROS production was detected with 1 μM dihydrorhodamine 123 . Cell staining was analyzed using a FACScalibur or LSRFortessa . At least 30,000 (PBLs) and 50,000 (splenocytes) events were acquired per sample, and dead cells were excluded based on scatter characteristics.Splenocytes or PBLs were blocked with anti-CD16/CD32 (2.4G2), then stained with pre-titrated amounts of the following FITC-, phycoerythrin (PE)-, allophycocyanin-, or biotin-conjugated Abs: CD4 (RM4-5), CD69 (H1.2F3), CD44 (IM7), CD62L (MEL-14) B220 (RA3-6B2), CD86 (GL1), CD80 (16-10A1), CD22.2 (Cy34.1), I-aSle2c2 mice (three each) were cultured in complete medium at a concentration of 106 cells/ml with 0.0, 0.1, or 1.0 ug/ml of Neulasta for 8 h. RNA was extracted and cDNA was synthesized as previously described [Itgam and Mpo relative to Gapdh was quantified by quantitative PCR measuring the incorporation of Sybergreen (Molecular Probes). The primer sequences were: Itgam 5'-CCC CAA TTA CGT AGC GAA TG-3'and 5'- TGC TGC GAA GAT CCT AGT TG -3'; Mpo 5'- TGC TCT CGA ACA AAG AGG GT-3' and 5'- CTT TGA CAG CCT GCA CGA-3'; Gapdh 5'- AGC TTG TCA TCA ACG GGA AG -3' and 5'- GTG GTT CAC ACC CAT CAC AA -3'. Results were calculated with the ΔΔ cycle threshold (Ct) method and normalized to the average values for untreated B6 samples.Splenocytes and BM cells from B6 and B6.escribed . Expresst-test or Bonferroni multiple comparison test when multiple groups were compared. Non-parametric statistics were performed when data were not normally distributed. Graphs show mean and standard error of the mean (SEM), and statistical significance is represented as *P < 0.05, **P < 0.01, and ***P < 0.001.Statistical analyses were performed with GraphPad Prism5, using the two-tailed unpaired 378N mutation in the extracellular domain of the G-CSFR-affected ligand binding, we compared in vitro binding of mG-CSF between B6 and B6.Sle2c2 splenocytes. As expected, the highest binding was found on GR1hi CD11b+ neutrophils, whereas intermediate levels were found on GR1lo CD11b+ and B cells . To test whether the decreased G-CSF binding by B6.Sle2c2 leukocytes provides functional consequences, we compared the effect of in vivo huG-CSF treatment on the expansion of myeloid cells between the two strains  expression and STAT3 phosphorylation [Ex vivo, CD11b expression on splenocytes was higher in B6.Sle2c2 than in B6 mice  on CD11b+ PBLs (data not shown). We next compared CD11b/Itgam and Mpo message expression in BM and spleen cells obtained from B6 and B6.Sle2c2 mice 4 d after treatment with 1 ug hu-G-CSF, then treated for 8 h with hu-G-CSF in vitro  [Sle2c2 Cfs3r allele, evidence of greater G-CSFR signaling in leukocytes expressing the Sle2c2 Cfs3r allele was also observed. These apparently paradoxical results should be put in perspective that very little is known about G-CSFR signaling and G-CSFR-responsive genes in primary myeloid cells, as all the studies have been performed with myeloid cell lines . Our reSle2c2 mice, we postulated that huG-CSF treatment should eliminate the difference in cGVHD response between B6 and B6.Sle2c2 mice. If resistance in B6.Sle2c2 mice is due to a G-CSFR loss of function, huG-CSF treatment should restore cGVHD response in B6.Sle2c2 mice. On the other hand, if resistance is due to a G-CSFR gain of function, cGVHD resistance would be expected in huG-CSF-treated B6 mice. Based on our time course analysis showing a maximal effect at d4 after injection that was eliminated at d7, we tested a combination of treatment protocols in which the dose  and the frequency  varied. Two treatment protocols (120 × 1 and 12 × 2) raised the production of anti-dsDNA IgG in B6.Sle2c2 mice significantly above the dextrose-treated controls . All huG-CSF treatments reduced the size of the CD4+ T cell compartment in both strains, and the 12 × 2 treatment, which was the most successful in restoring a cGVHD response in B6.Sle2c2 mice, resulted in an equivalent percentage of CD4+ T cells between strains . The 12 × 2 treatment also significantly increased the percentage of CD4+ T cell blasts in B6.Sle2c2 mice , increased CD69 expression, and reduced the naïve and expanded the effector memory CD4+ T cells in B6.Sle2c2 mice . The huG-CSF treatments (12 × 3 and 1.2 × 3) that did not restore autoAbs production in B6.Sle2c2 mice had no effect on CD4+ T cell activation (data not shown).cGVHD increases the percentage of blasts and the expression of activation markers such as class II MHC, CD22 and CD69 on B6 but not on B6. B cells . Treatme2c2 mice . B6.Sle2e Figure . The sim+ CD11c- GR1- macrophages was equivalent between B6 and Sle2c2 mice 3 weeks after cGVHD induction. huG-CSF treatments increased the percentage of macrophages in a dose-dependent manner similarly between these two strains without any correlation with autoAb production (data not shown). In contrast, the percentage of CD11b+ CD11c+ myeloid dendritic cells (DCs) was significantly lower in B6.Sle2c2 than in B6 mice, and the 12 × 2 treatment significantly expanded this cell population in the spleen of B6.Sle2c2 mice to a level similar to that of treated B6 mice . Three weeks after cGVHD induction, this difference was inverted, with B6.Sle2c2 values being significantly lower than B6 , but three weeks after cGVHD induction, this population was significantly depleted in B6.Sle2c2 mice . The same result was obtained with PBL (data not shown). Bm12-cGVHD induction expanded by over 5-fold the population of ROS+ granulocytes in both strains, and eliminated the difference between strains  oxidase complex, being associated with collagen-induced arthritis [Sle2c2 granulocytes. huG-CSF treatment increased ROS production in B6.Sle2c2 mice, which was concomitant with their production of autoAbs. These results suggest that the NZM2410 allele of Csf3r results in a higher ROS production at steady state levels of G-CSF that maintains CD4+ T cell tolerance, as it has been shown in other models [Sle2c2 mice cannot achieve the same expansion as B6 mice unless exogenous G-CSF is provided. Signaling through the G-CSFR is critical for the development and function of inflammatory granulocytes, whereas G-CSFR deficiency is protective in collagen-induced arthritis [We also found that B6.oduction . This isrthritis  and experthritis . Upon bmr models . We showr models , of whicrthritis . We therrthritis .Csf3r expression between B6 and B6.Sle2c2 mice [Csf3r locus has identified a large number of intronic polymorphisms and four coding synonymous mutations, but S378N was the only coding non-synonymous mutation identified between the NZM2410 and B6 genomes (unpublished observations). None of the genes located within Sle2c2 locus have known functions in the G-CSF pathway. This implies that the differences that we report here between B6 and B6.Sle2c2 mice in their response to G-CSF are most likely due to the S378N polymorphism. This will, however, need to be formally addressed by stringent structure/function studies.We have previously reported that there is no difference in 2c2 mice . GenomicSle2c2 suppression involves the G-CSF pathway, as it is a very robust model that leads to a rapid synchronized production of autoAbs with very little inter-individual variation. However, we have evidence that Sle2c2 also suppresses spontaneous autoimmune responses in two NZM2410-derived models [Sle2c2 [We have used the bm12-cGVHD model to test the hypothesis that d models ,31 and w [Sle2c2 .Sle2c2 and B6.TC strains offer an excellent model to identify the cellular and molecular bases on how G-CSF modulates SLE pathogenesis.An association between G-CSF treatment and disease exacerbation is supported by clinical findings, where G-CSF treatment of neutropenic SLE patients can be associated with severe flares . MoreoveNcf1-deficiency was associated with disease protection [NCF2-impaired function [Contrary to the results reported here, G-CSF treatment has been reported to promote reversal  or preveotection , contrarfunction . These rSle2c2 and B6.TC mice and both of these strains carry the same mutation in the G-CSFR. Furthermore, we have shown a defective mobilization of BM neutrophils in response to G-CSF treatment of B6.Sle2c2 mice as well as a different expression of G-CSF-responsive genes between B6 and B6.Sle2c2 mice. Overall, these data suggest that mice carrying the G-CSFR mutation are defective in mobilizing and/or activating inflammatory neutrophils, which inhibits the production of autoAbs. Further experiments will be required to demonstrate a direct role of neutrophils in this process.Exogenous G-CSF increases the production of autoAb in B6.bm12; cGVHD: chronic graft vs host disease; ct: cycle threshold; DC: dendritic cell; ELISA: enzyme-linked immunosorbent assay; FACS: representative fluorescence-activated cell sorting; FITC: fluorescein isothiocyanate; G-CSF: granulocyte-colony stimulation factor; G-CSFR: granulocyte-colony stimulation factor receptor; huG-CSF: human G-CSF; IFN: interferon; Ig: immunoglobulin; IL: interleukin; MDSC: myeloid-derived suppressor cell; MFI: mean fluorescence intensity; mG-CSF: mouse G-CSF; MPO: myeloperoxidase; NADPH: nicotinamide adenine dinucleotide phosphate-oxidase; NET: neutrophil extracellular trap; PBL: peripheral blood leukocyte; PCR: polymerase chain reaction; PE: phycoerythrin; ROS: reactive oxygen species; SEM: standard error of the mean; SLE: systemic lupus erythematosus; STAT3: signal transducer and activator of transcription 3; TLR: toll-like receptor.ANA: antinuclear antibodies; autoAb: autoantibody; B6: C57BL/6; BM: bone-marrow; bm12: B6.C-H2The authors declare that they have no conflict of interestin vitro assays. RS performed the neutrophil mobilization assays, the expression assays in G-CSF-responsive genes and the treatment study with lupus-prone mice. LZ performed the cGVHD induction and G-CSF treatments of these mice, antibody measurements and flow cytometry. CW assisted with the conception and implementation of the in vivo G-CSF treatment studies. YYZ assisted with the neutrophil mobilization assay. MA participated in the study design and coordination, as well as in data interpretation. LM conceived the study, overviewed data analysis and interpretation, and wrote the paper. All authors read and approved the final manuscript.ML performed the flow cytometry analysis and the


In hydrocephalus an imbalance between production and absorption of cerebrospinal fluid (CSF) results in fluid accumulation, compression and stretching of the brain parenchyma. In addition, changes in CSF composition have a profound influence on the development and function of the brain and together, these can result in severe life-long neurological deficits. Brain damage or degenerative conditions can result in release of proteins expressed predominantly in neurons, astroglia, or oligodendroglia into the brain interstitial fluid, CSF and blood. Determination of such products in the CSF might be of value in diagnosing cause, aetiology and/or assessing the severity of the neurological damage in patients with hydrocephalus. We therefore analysed CSF from human neonates with hydrocephalus for these proteins to provide an insight into the pathophysiology associated with different aetiologies.′ , 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were analysed through Western blotting of hydrocephalic CSF samples (n = 17) and compared with data from CSF of normal infants without neurological deficits (n = 8).CSF was collected during routine lumbar puncture or ventricular tap. Samples were categorized according to age of onset of hydrocephalus and presumed cause . Glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), vimentin and 2GFAP was significantly raised only in CSF from post-haemorrhagic hydrocephalus while MBP was significantly raised in post-haemorrhagic and in spina bifida with hydrocephalus infants. Vimentin protein was only detected in some CSF samples from infants with late-onset hydrocephalus but not from other conditions. Surprisingly, CNPase was found in all neonatal CSF samples, including normal and hydrocephalic groups, although it was reduced in infants with late onset hydrocephalus compared with normal and other hydrocephalic groups.Apart from CNPase, which is an enzyme, the markers investigated are intracellular intermediate filaments and would be present in CSF only if the cells are compromised and the proteins released. Raised GFAP observed in post-haemorrhagic hydrocephalus must reflect damage to astrocytes and ependyma. Raised MBP in post-haemorrhagic and spina bifida with hydrocephalus indicates damage to oligodendrocytes and myelin. Vimentin protein detected in some of the late-onset hydrocephalic samples indicates damage to glial and other progenitors and suggests this condition affects periventricular regions. The presence of CNPase in all CSF samples was unexpected and indicates a possible novel role for this enzyme in brain development/myelination. Less CNPase in some cases of late-onset hydrocephalus could therefore indicate changes in myelination in these infants. This study demonstrates differential glial damage and loss in the developing human neonatal hydrocephalic brain associated with different aetiologies. Hydrocephalus is a pathological condition characterised by an abnormal production and/or absorption of cerebrospinal fluid (CSF). The aetiology of hydrocephalus is unclear, even with some evidence of genetic predisposition . With a Improvements in prenatal diagnosis and treatment will raise the quality of life for children with hydrocephalus through decreased mortality and morbidity, but this may only be through a better understanding of the underlying aetiology and biological basis of the condition and the development of additional treatments to shunt surgery. The literature in both human and experimental animal studies is not clear with controversy on cause and with much work targeted at understanding the consequences of raised pressure rather than possible earlier events, due in large part to the view of CSF as a mechanical support fluid rather than a fluid with physiological importance. The latter role of CSF is currently under considerable review and renewed research -13 with The severity of hydrocephalus is usually assessed from clinical signs and symptoms. In very young patients an increase in head circumference usually occurs with only minor symptoms due to the compliance of the immature skull. CT-scanning and intracranial pressure-monitoring have proved to be valuable tools in the assessment of the diagnosis, but many patients still do not show satisfactory outcomes after CSF shunt surgery  suggestiThe composition of the CSF should reflect physiological/biochemical changes happening in the brain parenchyma, and more particularly in the periventricular white matter and sub-ventricular zones lining the ventricles ,24 wherePrior to the study, ethical approval was obtained from the UK NHS REC ref: 08/H1015/43), Central Manchester NHS Foundation Trust, Alder Hey Children’s Hospital, Leeds Teaching Hospitals NHS Trust, The University of Manchester, and The Children’s Hospital Lahore. Written informed consent for participation and demographic data including sex, age and ethnicity were obtained from each patient’s parents using their home language with verbal explanation where necessary. This study is based on 25 neonates, 16 males and 9 females varying in age from 1 to 44 weeks  in infants born with classically enlarged heads and associated neurological symptoms (n = 4); 2. Late-onset hydrocephalus (LOH) in infants developing hydrocephalus some days or weeks after birth due to undetermined factors but probably infections (n = 4); 3. Post-haemorrhagic hydrocephalus (PHH) in infants due to an intraventricular or subarachnoid bleed before, during or soon after birth (n = 5); 4. Spina bifida with hydrocephalus (SB/HC) in infants with a primary neural tube defect (n = 4). 5. Control/normal infants with no neurological condition and sera negative for meningitis (n = 8). Sample details are summarised in Table All CSF samples were compared through analysis of equal volumes. This was the most meaningful approach since we expected to see changes in total protein as well as in individual components. The specifications for each antibody on the manufacturer’s web sites also listed previous publications characterising the antibody as well as its specificity and utility for identifying the target protein. All samples were diluted 1:1 by volume in Laemmli sample buffer  containing mercaptoethanol and heated for 7 min at 95°C. Protein standards were included in each gel as well as a molecular weight standard (Biorad161-0375). Rat  astrocyte cell lysates  were used as a positive control for GFAP and vimentin antibodies. The CNPase antibody was well characterised by previous studies of oligodendrocytes and cortical white matter ,34. Furt2 flasks and placed in a humidified incubator for 7–9 days at 37°C with 5% CO2. After 7–9 day incubation, flasks were sealed tight and shaken on an orbital shaker at 230 rpm for 4 hours at 37°C to separate microglial cells from astrocytes. The media was replaced with 10 ml of fresh DMEM and shaken for a further 1 h. The culture was then washed twice with 5 ml of warm PBS with gentle mixing and then the adherent astrocytes lysed using CelLytic M Cell Lysis Reagent (Sigma) for 15 min at room temperature. Cells were scraped off the flask and the whole media centrifuged to remove cell debris. Protein concentration was measured using a Bradford Assay and 5-10 μg total protein loaded on gels as a control for GFAP and vimentin.In order to produce astrocyte cell lysates for positive control, 1-day old Sprague–Dawley rat pups  were killed with an overdose of sodium pentobarbitone given by intraperitoneal injection and then decapitated. 2–4 cerebral hemispheres were dissected under sterile conditions in a laminar flow hood. The cortical hemispheres were placed in ice-cold Dulbecco’s modified medium  and further dissected under a stereo microscope (Leica M3Z). The meninges were removed and the choroid plexus removed from the ventricle. All cleaned hemispheres were then digested with trypsin and gently dissociated through decreasing bore sizes of sterile pipettes. The suspension was centrifuged for 10 min at 1000 rpm, the supernatant discarded and the pellet resuspended in DMEM. After a further centrifugation and resuspension to wash the cells, the pellet was resuspended in 10 ml of DMEM per hemisphere. 10 ml of cell suspension was loaded into poly-D-lysine coated 75 cmp < 0.05 was considered significant. No distribution tests were carried out and the data was assumed to fall within a normal distribution.Films were scanned and the relative density of bands quantified using Image J . Where no bands were obvious a measure of the staining (presumably background) where the band would have been was used for those CSF samples. Data are presented as mean ± SEM. Results of the experiments were entered into Graphpad Prism V software and the data analysed using one way analysis of variance (ANOVA) and post hoc (Tukey) testing to identify significant differences between groups. The value P < 0.05) and showed a non-significant increase in SB/HC CSF when compared with CSF from normal infants  statistical test  increase in CSF of PHH and SB/HC infants compared to normal. The elevation of MBP in PHH and SB/HC infants showed little variation between different samples analysed.A 40 KDa MBP-positive protein band was observed in the CSF from infants with PHH and SB/HC conditions but was below the detection limit in CSF from normal, FOH and LOH infants Figure a. MBP shP < 0.05) although one infant had normal levels. CNPase was reduced but without significance in FOH compared with CSF from normal, PHH and SB/HC infants  early glial progenitors and differentiating astrocytes (vimentin) and oligodendrocytes (MBP) and an enzyme associated with the formation of myelin in oligodendrocytes (CNPase). Their presence in CSF is thus thought to indicate cell compromise, breakdown and release of intracellular components. In this study we found that FOH, the congenital form of hydrocephalus, is not associated with any appearance of cell breakdown products in agreement with previous studies demonstrating absence of cell death  and decret al. reported significantly increased GFAP levels in hydrocephalic rats which decreased after shunting suggesting an effect of fluid accumulation and/or raised intracranial pressure on these cells [GFAP is an intermediate filament expressed in mature astrocytes ,38 and ise cells . Similarse cells . Ependymet al.,[MBP in CSF is an indicator of damage specifically associated with myelination as this protein is only known to be present in oligodendrocytes ,46. MBP et al., reportedet al., giving iVimentin is a cytoskeletal intermediate filament involved in maintaining cell integrity in many different cells types including fibroblasts and endothelial cells  as well et al.[CNPase is a protein accounting for approximately 4% of myelin protein content and is considered an index of myelin formation ,60 whereet al. reportedet al.. CNPase et al., its preThis study has a number of limitations. Firstly, to obtain CSF free of contamination excluded many collected samples so that a much larger study is required to investigate our current findings in more detail. Secondly, it is practically impossible to obtain CSF samples through ventricular catheterisation from healthy neonates due to technical limitations and ethical issues. Similarly, it is unusual to obtain lumbar CSF from hydrocephalic patients. Although some reports indicate that ventricular CSF may be more stable and accurate for measuring pathological changes , others Taken together, the findings of this study indicate that different aetiologies leading to hydrocephalus are associated with different pathophysiological mechanisms affecting different cell types, at least in the initial stages where intracranial pressure may not be pathologically raised. The congenital form of hydrocephalus has no detectable pathological markers in CSF but is known to be affected by a physiological block of cell cycle as well as a folate block ,19. DiffThis study has demonstrates that different aetiologies leading to neonatal hydrocephalus can likely be differentiated by CSF analysis. This presents an important step in understanding this devastating neurological condition. These results suggest that early-stage changes are associated, not with raised pressure, but with currently unknown physiological changes that affect different cell types in different locations within the hydrocephalic brain. Shunting will only address the common end point of these different aetiologies and is unlikely to address the underlying differences in physiology. It is therefore important to understand these physiological processes in order to prevent irreversible cell damage and lasting neurological deficits.CNPase: 2′, 3′-cyclic nucleotide 3′-phosphodiesteras; CSF: Cerebrospinal fluid; FOH: Fetal-onset hydrocephalus; GFAP: Glial fibrillary Acidic protein; H-Tx: Hydrocephalic Texas rat; LOH: Late onset hydrocephalus; MBP: Myelin basic protein; PHH: Post-haemorrhagic hydrocephalus; SB/HC: Spina bifida with hydrocephalus.The authors declare they have no competing interests.JAM and IN conceived and designed the study. IN carried out all of the lab work. KAIW, AWR, SV, CM and JRG were involved in providing clinical samples, discussing the study design and data collected. SNC coordinated the work in Pakistan. All authors contributed to various drafts of the manuscript and have read and approved the final version.


Several studies have reported reduced cerebrospinal fluid (CSF) levels of α-synuclein in synucleinopathy patients and recent data also proposes a significant role of α-synuclein in the pathophysiology of Alzheimer's disease (AD). To investigate potential links between neurosin and its substrate α-synuclein in vivo we used a commercially available sandwich ELISA and an in-house developed direct ELISA to quantify CSF levels of α-synuclein and neurosin in patients diagnosed with DLB, PD and PD dementia (PDD) versus AD patients and non-demented controls. We found that patients with synucleinopathy displayed lower CSF levels of neurosin and α-synuclein compared to controls and AD patients. In contrast, AD patients demonstrated significantly increased CSF α-synuclein but similar neurosin levels compared to non-demented controls. Further, CSF neurosin and α-synuclein concentrations were positively associated in controls, PD and PDD patients and both proteins were highly correlated to CSF levels of phosphorylated tau in all investigated groups. We observed no effect of gender or presence of the apolipoprotein Eε4 allele on neither neurosin or α-synuclein CSF levels. In concordance with the current literature our study demonstrates decreased CSF levels of α-synuclein in synucleinopathy patients versus AD patients and controls. Importantly, decreased α-synuclein levels in patients with synucleinopathy appear linked to low levels of the α-synuclein cleaving enzyme neurosin. In contrast, elevated levels of α-synuclein in AD patients were not related to any altered CSF neurosin levels. Thus, altered CSF levels of α-synuclein and neurosin in patients with synucleinopathy versus AD may not only mirror disease-specific neuropathological mechanisms but may also serve as fit candidates for future biomarker studies aiming at identifying specific markers of synucleinopathy.Neurosin is a protease that  The protein has been found in the pathological senile plaques of AD patients To our knowledge simultaneous quantification of α-synuclein and neurosin in CSF from patients with synucleinopathy versus non-demented controls and AD patients has not yet been performed. In the current study we therefore examined the levels of neurosin and α-synuclein in CSF from patients with AD, DLB, PD and PD with dementia (PDD) versus non-demented controls. We also evaluated potential links between the CSF levels of α-synuclein, neurosin and disease severity.Cerebrospinal fluid samples were obtained from non-demented controls (n = 52) and patients clinically diagnosed with AD (n = 46), PD (n = 38), PDD (n = 22) and DLB (n = 33) at the Neurology and Memory Clinics at Skåne University Hospital (Sweden). The patients diagnosed with PD met the NINDS Diagnostic Criteria for Parkinson Disease Lumbar puncture was performed in the L3/L4 or L4/L5 intervertebral space and CSF was collected passively by gravity flow in polypropylene tubes. Within 30 minutes the CSF was centrifuged at 4°C at 2000×g for 10 min. The supernatant was collected, gently mixed, aliquoted in polypropylene tubes and frozen within 2 h at −80°C pending biochemical analysis. The procedure followed the Alzheimer's Association Flow Chart for LP and CSF sample processing The specificity of the biotinylated polyclonal goat anti-human neurosin antibody (R&D Systems) against human neurosin (amino acids 22–244), was assessed by western blotting (WB). The same antibody was used to immunoprecipitate neurosin from pooled human CSF using a Protein G Immunoprecipitation Kit (Sigma Aldrich) according to the recommendations by the supplier. Neurosin was eluted in 0.1 M glycine buffer pH 2.5 and immediately neutralized with 1 M Tris pH 9.5. Recombinant human neurosin with a C-terminal 10 His-tag, expected to run at 35 kDa (R&D Systems), was separated along with the immunoprecipitated CSF neurosin by use of a 12.5% SDS-PAGE under reducing conditions. The separated proteins were transferred to a PVDF membrane (Millipore) by use of the trans-blot semi-dry transfer cell (Bio-Rad) where-after the membrane was blocked at 4°C over night (o/n) in phosphate buffered saline (PBS) with 0.5% skimmed milk  and 0.1% Tween20 (Merck). For the WB analysis the membrane was probed with the goat anti-human neurosin antibody (diluted in PBS with 0.1% Tween20), rinsed and then further incubated with a donkey anti-goat IRDye secondary antibody (Li-Cor Biosciences). Immunolabeled proteins were visualized using the Odyssey Infrared Imaging System (Li-Cor Biosciences).2SO4. The optical density at 450/540 nm was determined using a microplate reader (Labsystems iEMS) with background correction. Readings of the standards and CSF samples were averaged and neurosin concentrations determined by use of a 4 parametric curve fit. The intra- and inter-assay variations coefficients (CV%) throughout the standard range were <10 and <15 and recovery of spiked standards (full standard curve range) into appropriately diluted CSF samples was between 95–110%. To limit the effect of inter-assay variations the results of the CSF samples were corrected against the results of repeated analyses of two control samples (pooled CSF).A direct neurosin enzyme-linked immunosorbent assay (ELISA) was used for the quantification of neurosin in CSF. Recombinant human neurosin (R&D Systems) (standard concentration range 1.25–80 ng/mL) and CSF samples were diluted in PBS and added in duplicate (100 µL) to the wells of a MaxiSorb 96-well polystyrene plate (Nunc A/S). Standards and samples were incubated o/n at 4°C after which they were discarded and the plate rinsed 3× with wash-buffer (PBS with 0.05% Tween20). After rinsing the wells were blocked with blocking buffer  for 1 h at room temperature (RT) followed by rinsing (see above) and incubation with detection antibody , diluted in blocking buffer, for 2 h at RT. After another rinse step HRP-streptavidin (R&D Systems) solution was added to the wells and the plate incubated for 20 min at RT. For the colorimetric reaction to occur peroxidase substrate (tetramethylbenzidine) (KBL) was added to the wells and after 20 min incubation in the dark the reaction was stopped with 1 M HConcentrations of α-synuclein were determined by use of a sandwich ELISA (Invitrogen) previously described For the statistical analyses the PASW Statistics 18 (SPSS Inc) software was used. The α-synuclein and neurosin data was normally distributed, assessed by the Kolmogorov-Smirnov test, therefore parametric tests were used throughout the analyses. The independent t-test was used to compare results between two groups  carriers and non-carriers) whereas the one-way ANOVA or ANCOVA, with correction for age, was used to compare results between more than two groups with adequate correction for multiple comparisons (Bonferroni). To analyze frequency differences of dichotomous variables the chi-square test was used. The Pearson correlation test was used to assess correlations between variables. Levels of neurosin, but not α-synuclein, were related to age and all analyses including neurosin were therefore adjusted for age. The data is presented as mean ± SD and p<0.05 was considered significant.The demographic data of the study participants is summarized in For the quantification of neurosin in CSF we developed and validated an in-house direct ELISA assay. The biotinylated goat anti-human neurosin antibody used in this assay detected the recombinant human neurosin  at the expected migration position of ≈35 kDa (used as standard in our assay). The antibody also detected neurosin immunoprecipitated from pooled human CSF as a double band at approximately 26–30 kDa, as assessed by western blotting . DetermiIn order to determine the relationship between CSF neurosin and α-synuclein levels we determined the concentrations of the latter marker, by use of an assay previously employed in another CSF study Our previously presented results suggested gender-specific variations in α-synuclein levels between AD and DLB patients versus controls By use of correlations analysis we evaluated potential links between the CSF levels of both neurosin and α-synuclein, age, cognitive function  and PD severity . Earlier reports have described a link between age and CSF levels of neurosin In order to evaluate potential associations between neurosin, α-synuclein and the established AD biomarkers, as additional indicators of disease severity, we compiled AD biomarker data from a subset of the individuals included in our study and of whom the CSF had undergone routine analysis for Aβ42, t-tau and p-tau. Few of the PD and PDD patients had undergone routine screening for CSF AD biomarkers and these individuals were pooled into one group (PD/PDD n = 11 since PD n = 2 and PDD n = 9). The AD biomarker data is presented in Next we analyzed potential links between neurosin, α-synuclein and CSF AD biomarkers in controls, AD, DLB and the PD/PDD group. In the current study we elucidated whether CSF levels of neurosin and its substrate α-synuclein differ between patients with synucleinopathy, DLB, PD, PDD, and patients with AD, versus non-demented controls. Our results revealed that all three disease groups with synucleinopathy exhibited similar neurosin levels. When pooled, the CSF neurosin concentrations in the combined group of total synucleinopathies were significantly lower compared to neurosin found in both AD patients and non-demented controls. Interestingly, the lowest neurosin concentrations were found in the DLB group, which differed significantly from AD patients and controls. In agreement with recent studies we also show 14% higher levels of α-synuclein in AD patients versus controls however in the absence of altered neurosin levels.To our knowledge we are first to describe decreased neurosin levels in CSF from patients with synucleinopathy disorders versus controls and AD patients. In support, we have found similar results in a previous study on CSF samples from a different cohort  Neurosin levels in CSF from AD patients and controls have previously been investigated in two different studies. These two studies employed different ELISA assays In support of the earlier proposed link between AD and neurosin, we here described a correlation between neurosin and Aβ42 levels in CSF from controls. This association supports a role for neurosin in the Aβ pathway, at least in non-demented controls, which however might be rendered upon disease, as earlier suggested by experimental data The number of studies reporting CSF concentrations of α-synuclein in patients with synucleinopathy is rapidly increasing. Whereas few studies have reported unaltered levels of α-synuclein in these patients in vivo mirror increased amyloid deposition in the brain The results presented in the current study are the first to demonstrate that decreased CSF neurosin concentrations are significantly associated with decreased α-synuclein concentrations in the CSF of controls and patients with PD and PDD but not AD and DLB. These findings are to date difficult to interpret since little is known about the relationship between the extracellular and intracellular pool of neurosin and α-synuclein as well as the association between the brain tissue concentrations and quantities of these proteins in the CSF. As suggested in the recent report by Larson and colleagues the pool of soluble α-synuclein in the brain tissue of AD patients may in fact be significantly increased Correlations between α-synuclein and the AD biomarkers Aβ42, t-tau and p-tau in synucleinopathy patients have lately been investigated and the combination of CSF α-synuclein, tau and Aβ1–42 have shown promising results for facilitated discrimination of synucleinopathy We recently demonstrated significantly lower α-synuclein in female, but not male patients with DLB versus AD patients and controls To summarize, data from recent reports in combination with our current findings of lower versus elevated CSF levels of α-synuclein in patients with synucleinopathy versus AD respectively and decreased levels of neurosin in the former but not the latter group, may point to different disease-specific neuropathological mechanisms in these disease groups. Alterations in neurosin production or activity might hamper clearance of specific α-synuclein forms leading to Lewy pathology in patients with synucleinopathy but not AD. Last, we propose neurosin as a potential marker of synucleinopathy and a natural candidate in studies elucidating whether a specific panel of CSF proteins will aid the identification, discrimination and differential diagnosis of various synucleinopathies versus AD.


The dynamic and molecular composition of the cerebrospinal fluid (CSF) and, consequently, the CSF physiology is much more complex and fascinating than the simplistic view held for decades. Signal molecules either transported from blood to CSF or secreted into the CSF by circumventricular organs and CSF-contacting neurons, use the CSF to reach their targets in the brain, including the pre- and postnatal neurogenic niche. The subcommissural organ (SCO), a highly conserved brain gland present throughout the vertebrate phylum, is one of the sources for signals, as well as the choroid plexus, tanycytes and CSF-contacting neurons. The SCO secretes into the fetal and adult CSF SCO-spondin, transthyretin, and basic fibroblast growth factor. These proteins participate in certain aspects of neurogenesis, such as cell cycle of neural stem cells, neuronal differentiation, and axon pathfinding. Through the CSF, the SCO-secretory proteins may reach virtually any target in the embryonic and adult central nervous system. Since the SCO continues to secrete throughout life span, it seems likely that the neurogenetic property of the SCO compounds would be targeted to the niches where neurogenesis continues in adulthood. This review is aimed to bring into discussion early and new evidence concerning the role(s) of the SCO, and the probable mechanisms by which SCO compounds can readily reach the neurogenic niche of the subventricular zone flowing with the CSF to participate in the regulation of the neurogenic niche. As we unfold the multiples trans-fluid talks between discrete brain domains we will have more tools to influence such talks. The identification of neural stem cells (NSCs) in the adult central nervous system closed down a long-held dogma that neurons are formed exclusively during brain development. The mammalian brain retains the capacity to generate new neurons throughout life in two main locations, the subventricular zone (SVZ) of the lateral ventricles and the hippocampal dentate gyrus .The cellular and molecular mechanisms that guide the progression from a dividing NSCs to a functional neuron are far from being understood. A series of components of the neurogenic niche has been identified, including cell–cell interactions, secretory factors, vascular requirements, and specific innervation . HoweverThe design of the CSF-neurogenic niche interphase, i.e., NSC projecting a process to the CSF and bearing a 9+0 cilium, neighboring bi-ciliated and multiciliated cells organized as spatial units around the NSC process , and theThe CSF results from the secretion by the choroid plexuses and the bulk flow of the interstitial fluid of brain parenchyma to the ventricles and to the subarachnoid space. In humans, approximately 600 ml of CSF is produced each day. The rate of CSF production displays circadian variations, with lowest levels around 06:00 PM and a nightly peak at about 02:00 AM . The CSFCerebrospinal fluid proteomics is showing a wealth of over 200 proteins . A long The CSF is a heterogeneous and highly dynamic compartment that changes its molecular composition as it unidirectionally moves through the various ventricular and subarachnoidal compartments. The choroid plexus of the lateral ventricles, the interstitial fluid of the parenchyma surrounding these ventricles and axons endings secreting into these cavities are the source of molecules forming this “first” fluid. At the third ventricle new compounds are added to the CSF by hypothalamic neurons, the pineal gland and the local choroid plexus . When enFigure 1). (3) Selective and circadianly regulated secretion by CSF-contacting neurons of serotonin and neuropeptides such as vasopressin, oxytocin, and somatostatin . Furthermore, recent findings indicate that cells forming the ventricular walls release into the CSF microvesicles containing signaling and intracellular proteins  Neurotransmitters and their metabolites reach the CSF via the bulk flow of parenchymal fluid. (2) Regulated secretion into the CSF of biologically active compounds by the circumventricular organs , such as SCO-spondin, basic FGF, melatonin, TTR, TTR-T4 complex, TTR-T3 complex, nerve growth factor (NGF), transforming growth factor-β (TGFβ), vascular endothelial growth factor (VEGF), transferrin, and vasopressin (tostatin . (4) Traproteins .Figure 4D).Thus, the early view that the CSF is a medium carrying brain-borne and blood-borne signals to distant targets within the brain  has largFigures 2A–E). The astonishing amphioxus, an evolutionary leap made at the bottom of the ocean over 500 million years ago, already has a small group of cells secreting a very thin Reissner fiber (RF)  that immunoreacts with antibodies against mammalian SCO-spondin . SCO-spondin could be considered a member of an exclusive group of proteins accompanying the brain through its long lasting evolution what, in turn, highlight the functional significance of this molecule.The SCO is an ancient and highly conserved brain gland present throughout evolution of chordates, from amphioxus  to man . In the human, the SCO can be morphologically distinguished in 7-weeks-old embryos. By the 13th gestational week , the SCO is a fully differentiated gland that remains secretory active throughout the fetal life, releasing CSF-soluble proteins .In ontogeny, the SCO is one of the first brain structure to differentiate . The secretory cells of the SCO are arranged into two different layers, the ependyma and the hypendyma.The SCO is located in the dorsocaudal region of the third ventricle, at the entrance of the Sylvian aqueduct . The cell body presents a clear zonation, which has facilitated the investigation of the secretory process. Different phases of this process occur in discrete but separate areas of the cell, namely, (1) synthesis in the perinuclear and intermediate regions, (2) storage of precursor forms in big RER cisternae located in the subnuclear region, (3) processing and packaging in the intermediate region, (4) transport in the subapical region, (5) storage of processed forms and release in the apical cell pole . Further, the SCO offers a unique feature: the secretory material upon release condenses, first as a film on the surface of the organ and then, after further packaging, into RF . Most of the ultrastructural characteristics of the hypendymal cells are similar to those described for the ependymal cells.The ependymal cells of the SCO are bipolar, with and apical pole contacting the ventricular CSF and a basal process projecting to local capillaries and to the subarachnoid space . Their terminals are loaded with secretory granules. The most likely fate of this secretion is the local leptomeningeal cistern . In mammals, the basal processes of the SCO cells containing secretory granules either project to the subarachnoid space or to the subependymal capillaries. Here, the processes end on a network of extensions of the perivascular basement membrane formed by long-spacing collagen, a unique arrangement and a landmark of the SCO .In non-mammalian species all ependymal cells of the SCO display long and slender processes that traverse the posterior commissure and end on the external basement membrane of the brain  they secrete compounds to the ventricular CSF, the subarachnoidal CSF and probably to blood; (ii) this secretory activity is under neural control. The nature of the compounds secreted into ventricular CSF is only partially known , whilst that of the compounds contained in the secretory granules stored at the perivascular and subarachnoidal ependymal terminals is unknown.island within the brain .The SCO secretes into the ventricular CSF two classes of proteins, the ones that remain soluble in the CSF and that, consequently, go with the flow and those that aggregate to form an insoluble, ever-growing structure, the RF . RF material continuously arrives at the dilated caudal end of the central canal, known as the terminal ventricle or ampulla, where RF-glycoproteins undergo chemical modifications , disaggregate and then escape through openings in the dorsal wall of the ampulla to finally reach local vessels . At variance with SCO-spondin forming RF, there are compounds of 200, 63, 50, and 25 kDa molecular mass that are consistently found in the CSF of rodents . These compounds react with specific antibodies against SCO-spondin and most likely result from a further processing of SCO-spondin. We regard these proteins as CSF-soluble SCO-spondin-derived compounds. In adulthood, the CSF contains both RF-SCO-spondin and the soluble SCO-spondin related compounds , including the human , secretes CSF-soluble SCO-related proteins while RF is missing  secreted by the SCO into the ventricular CSF, where it contributes to form the RF . The floor plate, a key structure in brain development, participates in the neural patterning and axon guidance of the ventral neural tube.At early developmental stages SCO-spondin is also expressed by the floor plate cells that release it into the fetal CSF and also transport it along their basal processes . TTR was detected in the SCO of bovine embryos and human embryos  suggesting that this ependymal gland is a source of TTR during brain development SCO  obtained from the CSF of hydrocephalic children react with the human and rat SCO . ImmunorFigure 3A). Because both CSF compartments are in open communication with the brain tissue, the SCO-soluble secretion could reach any region of the central nervous system, with the exception of the other circumventricular organs that have a tight barrier with the CSF.The detection in the CSF of the lateral ventricle and cisterna magna of CSF-soluble compounds secreted by the SCO  indicateFigure 3D). SCO-cells also express receptors for angiotensin II  and the subgranular zone (SGZ) of the hippocampal dentate gyrus  of the developing brain. After birth, and during life span, neurogenesis continues at specific brain areas, known as neurogenic niches. Adult neurogenesis is mostly confined to two brain regions, the SVZ of the lateral ventricles ; extrinsic factors include signals generated in the vicinity of the niche as well as blood-borne and CSF-borne compounds .The molecular mechanisms that control neurogenesis are being extensively studied . Interestingly, primary cilia ablation leads to disruption of hedgehog signaling which plays key roles in brain development and in adult neurogenesis . The design of the CSF-neurogenic niche interphase and the numerous neurotropic factors secreted into the CSF, point to the CSF as a key milieu for the SVZ niche. A further thought concerns the properties of the CSF–SVZ barrier. Neither the cell junction complexes between the different component of the ependymal component of the niche  nor the barrier properties of this cell layer have been properly investigated. This information is required for a better understanding of the relationships between the processes taking place in the SVZ and, via the CSF, in other brain regions.Cerebrospinal fluid-long-distance cues may act directly on NSC and progenitor cells to regulate neurogenesis . Many ofThe fetal CSF may be regarded as the main component of the milieu of stem cells and progenitor cells of the germinal zone providing signals participating in embryonic brain growth and differentiation . QualityFigure 2I). There is evidence that SCO-spondin is released from these processes becoming part of the ECM , the SCO remains highly differentiated and secretory active through life span . During in vitro and in vivo . Within the choroidal cells, TTR binds thyroxin (T4) that has entered these cells either by passive diffusion or by specific transporters .Transthyretin synthetized by the choroid plexus and the SCO is secreted into the CSF. A marked difference between these two sources of TTR is that the SCO cells, at variance with the choroidal cells, are not open to the blood stream and their secretory activity is under the control of a complex neural input .T4 is the predominant iodothyronine in plasma. However, T3 is the major receptor-active form of thyroid hormones. Consequently, T4 has to be converted by the effect of diodinase 2 into T3. The conversion of thyroxin present in the CSF into T3 takes place, exclusively, in the tanycytes located in the hypothalamus , 2010. TFigure 1).These findings point to a functional relationship, via the CSF, between three different types of ependymal cells, namely, the ependymocytes of the SCO, the choroidal cells of the choroid plexus and tanycytes. The outcome of such an association is to provide signals to the neurogenic niche . Due to the large number of TSR, SCO-spondin is regarded as an extra cellular matrix-like protein belonging to the TSR superfamily. It is involved in multiple functions including cell attachment, motility, proliferation, cell–cell contact, cell aggregation and angiogenesis, all of which are thought to contribute to vascular homeostasis and brain functions . This interaction may be essential for the neurite outgrowth induced by SCO-spondin in vitro  by which SCO-spondin and SCO-spondin-derived compounds would promote neurogenesis in the adult SVZ niche. This protein displays a unique arrangement of several conserved domains, including 26 thrombospondin type 1 repeats (TSRs), 9 low density lipoprotein receptor (LDLr) type A domains, 2 epidermal growth factor (EGF) like domains, and NH2 and COOH von Willebrand cysteine-rich domains vWD; . All theunctions . This isunctions , and theunctions . Interesin vitro  and for . Interestingly, bFGF, also secreted by SCO-cells, increases the expression of β1-integrin . Explants synthetize  and secrete SCO-spondin and TTR into the culture medium . The basal lamina of the newly formed capillaries, but not the capillaries of the neighboring brain parenchyma, contains long spacing collagen, indicating that the expression of this special type of collagen is triggered by signals of the grafted SCO cells .Rat SCO explants grafted into a lateral ventricle of normal adult rats become re-vascularized and secrete RF-glycoproteins into the CSF forming a RF, now located in the lateral ventricle (CO cells . XenograA good body of evidence is revealing that the dynamic and molecular composition of the CSF and, consequently, the CSF physiology is much more complex and fascinating than the simplistic view held for decades. Signal molecules either specifically transported from blood to CSF or secreted into the CSF by a series of periventricular structures, use the CSF to reach their targets in the brain. This allows a cross talk between brain regions located beyond the blood-brain-barrier, thus keeping the brain milieu private. One of these brain target is the neurogenic niche, and the SCO, choroid plexus, and tanycytes are some of the sources of signals that reach this target via the CSF. Thus, the CSF path has made it possible for these four brain structures to become good functional partners.As we unfold the multiples trans-fluid talks between discrete brain domains we will have more tools to influence, in one way or another, such talks. The CSF may become an appropriate medium to deliver foreign molecules or to host cell grafts.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.


Background. Neoangiogenesis is crucial in plaque progression and instability. Previous data from our group showed that Nestin-positive intraplaque neovessels correlated with histological complications. The aim of the present work is to evaluate the relationship between neoangiogenesis, plaque morphology, and clinical instability of the plaque. Materials and Methods. Seventy-three patients  were consecutively enrolled. Clinical data and 14 histological variables, including intraplaque hemorrhage and calcifications, were collected. Immunohistochemistry for CD34 and Nestin was performed. RT-PCR was performed to evaluate Nestin mRNA . Results. Diffusely calcified plaques (13/73) were found predominantly in females (P = 0.017), with a significantly lower incidence of symptoms (TIA/stroke (P = 0.019) than noncalcified plaques but with the same incidence of histological complications (P = 0.156)). Accordingly, calcified and noncalcified plaques showed similar mean densities of positivity for CD34 and Nestin. Nestin density, but not CD34, correlated with the occurrence of intraplaque hemorrhage. Conclusions. Plaques with massive calcifications show the same incidence of histological complications but without influencing symptomatology, especially in female patients, and regardless of the amount of neoangiogenesis. These results can be applied in a future presurgical identification of patients at major risk of developing symptoms. RT-PCR data were analyzed assuming the null hypothesis that the CT differences between target and reference genes will be the same in type V tissue versus type VI tissue. If the null hypothesis is not rejected, then the ΔΔCT would not be significantly different from 0. Analyses of differences between the three groups  were performed with one-way ANOVA test, followed by Tukey's test. All the P values are derived from testing the null hypothesis that ΔΔCT are equal to 0 (at P = 0.05). SEM, SD, and the confidence interval (CI) of 2−(ΔΔCT) are all derived from the SEM, SD, and CI of ΔΔCT as explained by Yuan et al. [All statistical analyses were carried out with SPSS software for Windows, version 20. All continuous variables are expressed as means, standard deviations, and ranges; all categorical variables  are expressed as number of cases and percentages. The Spearman test, the chi-square test, the Mann-Whitney n et al. .Seventy-three patients were finally enrolled, 53 (72.6%) males and 20 (27.4%) females, with a mean age at the time of endarterectomy of 70.8 ± 8.7 years (range 42–86 years). The clinical characteristics of the patients, including ongoing therapy, are summarized in μm (range 120–2500 μm) and mean minimum cap size was 284.3 ± 199.7 μm (range 40–1125 μm); the lipid core was not evident in 8 (11.0%) cases, 1/4 in 16 (21.9%), 2/4 in 18 (24.7%), 3/4 in 23 (31.4%), and 4/4 in 8 (11.0%); the inflammatory infiltrate was mild/focal or absent in 14 (19.2%) cases, moderate in 20 (27.4%) cases, and severe/diffuse in 39 (53.4%) cases. Fifty-two (71.2%) plaques were classified as AHA type VI [At histopathological analysis, mean maximum cap size was 1132.8 ± 485.6  type VI : the mos type VI .Intraplaque calcifications were graded 0 in 11 (15.1%) cases, 1+ in 21 (28.8%), 2+ in 20 (27.4%), 3+ in 16 (21.9%), and 4+ in 5 (6.8%). According to this semiquantitative assessment of the calcification extent, low-grade calcifications (up to 2+) were recorded in 52 (71.2%) plaques, and high-grade calcification (3+ and 4+) was recorded in 21 (28.8%) plaques .N = 39), calcified and complicated plaques (type VI-c N = 13), calcified noncomplicated plaques , and noncalcified noncomplicated plaques . No correlations were found between plaque morphological criteria used in the present study  and the occurrence of chronic ischemic cardiopathy, chronic obstructive bronchopneumonia, chronic renal failure, smoke, diabetes, hypertension, dyslipidemia, or therapy with acetylsalicylic acid or statins .Finally, according to the occurrence of intraplaque complications and/or calcifications, our cases were sorted in noncalcified complicated plaques . The mean density of positivity for Nestin, evaluated in the same ROI, was 6.8 ± 3.7 structures/field (range 1.4–18.5/field). The mean Nestin/CD34 ratio was 0.7 ± 0.2, which means that 70% of the CD34-positive neovessels examined coexpressed Nestin. This result is in line with what is previously described [P = 0.111, Mann-Whitney U test), while the density of Nestin-positive neovessels was significantly higher in type VI  showed overall less neoangiogenesis, measured with both CD34 and Nestin, than noncalcified plaques (P = 0.0001); thus the null hypothesis was rejected, which indicated a change in Nestin gene expression among healthy, type V and type VI plaques. In type V and type VI plaques, the mean ΔΔCT Nestin was, respectively, 4.25 and 3.25; this corresponds to 2−(ΔΔCT) of 0.05 for Nestin gene expression in type V plaques and 0.11 in type VI plaques. The type VI plaques showed a 2-fold expression increase for Nestin gene compared to type V plaques, as a confirmation of the IHC results  and the mRNA level (RT-PCR), plays a key role in the development of intraplaque complications and that plaques with massive calcifications generally have a minor density of neoangiogenesis compared to other plaques.P = 0.167, chi-square test; Despite the differences in neoangiogenesis, the incidence of histological complications did not differ significantly between calcified and noncalcified plaques . Interestingly, among the 21 patients with calcified plaques, only 4 (19.0%) had symptoms at the onset, regardless of the occurrence of histological complications; conversely 25 out of 52 (48.1%) patients with noncalcified plaques were symptomatic .As for the clinical presentation, unsurprisingly, 25 out of 29 (86.2%) symptomatic patients  had a type VI plaque, versus 27 out of 44 (61.4%) asymptomatic patients . All three symptomatic female patients had a type VI-nc atheromatous lesion. Notably, a higher incidence of calcified plaques was observed in female patients (P = 0.017): 10 (50.0%) females had calcified plaques (5 type VII and 5 type VI-c) versus only 11 (20.8%) males (3 type VII and 8 type VI-c).The patients' gender was correlated with the plaque morphology and instability as well; first of all only 3 (15.0%) of the 20 female patients in our study were symptomatic versus 26 (49.1%) out of 53 males , and the clinical plaque instability. For these purposes, neoangiogenesis was evaluated and semiquantified by CD34 and Nestin IHC, followed by RT-PCR. The majority of tissue obtained from endarterectomy was used for the routine histological diagnosis of surgical specimens. The remaining tissue had to be fully processed for RT-PCR and Western blot analysis was not included in our protocol. However, in our experience , 9 we obAccording to our data calcified plaques show less inflammatory infiltrate, a smaller lipid core, and less neoangiogenesis than other plaques, but with the same incidence of hemorrhage, thrombosis, and surface defects, which define the plaque as histologically complicated . Yet, inThe reason why in the massively calcified plaques the neoangiogenesis and the histological complications do not affect symptomatology remains unclear, but it is possible that the hemorrhages and erosions found in these plaques might have a different pathogenesis. For example, it is possible that they can be due directly by the mechanical stresses of the calcified mass and not by immature neoangiogenesis and endothelial damage. Another possible explanation is that the “calcified type VI” plaques can represent an early stage of type VII plaques, in which the regressive process is more recent, and the complications have not disappeared yet .The last result that emerged from our data is that 50% of the female patients had calcified plaques, showing a significantly lower incidence of symptoms and type VI plaques than the male patients. Ten years ago, Allison et al. have found 53% and 30% prevalence of “zero calcification” in female and male patients, respectively, before age of 50; after that age the prevalence of a diffuse vessel calcification increases, in a linear fashion in males and exponentially in females . ActuallStudy Limitations. A limitation of our study is represented by heterogeneity in the sample size of each plaque group . However, this is a monocentric perspective study, and our series reflects the incidence of the different plaque types in the general population. Moreover, the patients submitted to surgery generally have advanced plaques, and complicated type VI plaques are the most represented. Furthermore, there is a discrepancy between the age of the patients with plaques and the controls, due to obvious differences in the populations of multiorgan donors and atheromatous patients. de facto a sort of protection compared to male patients.In conclusion, this study confirms that the Nestin+/WT1− phenotype characterizes the plaques with morphological features of instability, regardless of the actual amount of the neoangiogenesis (expressed as CD34-positive vessels). The plaques with massive calcifications show the same incidence of histological complications but with a lower incidence of neurological symptoms. Female patients show a much higher incidence of noncomplicated or calcified plaques, receivingA possible indication emerging from these findings could be a comparison between the plaque dynamic imaging and the histological assessment of calcification, to evaluate the possibility of a presurgical risk stratification of patients, based on their sex, risk factors, and intraplaque calcification. The presurgical identification of those patients at major risk of developing stroke or brain lesions is likely to make the priority for endarterectomy more rational.


P = 0.045).Further analysis showed that anti-SS-A in CSF specimens was related to neuropsychiatric syndromes of the central nervous system in SLE (P = 0.009). Analysis with Spearman’s rank correlation coefficient indicated that the titers of anti-RPLP2 and anti-SS-A in paired CSF and serum specimens significantly correlated. Human proteome microarrays offer a powerful platform to discover novel autoantibodies in CSF samples. Anti-SS-A autoantibodies may be potential CSF markers for NPSLE.Autoantibodies in cerebrospinal fluid (CSF) from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) may be potential biomarkers for prediction, diagnosis, or prognosis of NPSLE. We used a human proteome microarray with~17,000 unique full-length human proteins to investigate autoantibodies associated with NPSLE. Twenty-nine CSF specimens from 12 NPSLE, 7 non-NPSLE, and 10 control (non-systemic lupus erythematosus)patients were screened for NPSLE-associated autoantibodies with proteome microarrays. A focused autoantigen microarray of candidate NPSLE autoantigens was applied to profile a larger cohort of CSF with patient-matched sera. We identified 137 autoantigens associated with NPSLE. Ingenuity Pathway Analysis revealed that these autoantigens were enriched for functions involved in neurological diseases (score = 43).Anti-proliferating cell nuclear antigen (PCNA) was found in the CSF of NPSLE and non-NPSLE patients. The positive rates of 4 autoantibodies in CSF specimens were significantly different between the SLE  and control groups: anti-ribosomal protein RPLP0, anti-RPLP1, anti-RPLP2, and anti-TROVE2 . The positive rate for anti-SS-A associated with NPSLE was higher than that for non-NPSLE (31.11% cf. 10.71%;  Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by production of pathogenic autoantibodies and multiple organ and tissue damage. InvolveCerebrospinal fluid (CSF)contains metabolic products of the central nervous system, and pathology in the central nervous system can be reflected in the traits and composition of the CSF. For example, a variety of autoantibodies have been detected in the CSF of SLE patients, including anti-glutamate receptor ɛ2 subunit (GluRɛ2), anti-neIn a normal adult the CSF volume is only 125–150 mL, and the amount of sample that can be collected from one person is very limited. In addition, the concentration of immunoglobulin G (IgG) in CSF is~42 ± 21mg/L, much leDuring the past decade, functional protein microarrays have become a powerful proteomics tool enabling novel discoveries in many biological fields–19.WithiIn the present study, we used a human proteome microarray to profile disease-related autoantibodies in the CSF of SLE patients with neuropsychiatric syndromes.The study was approved by the Ethics Committee of Peking Union Medical College Hospital. All patients provided written consent.Inpatients and outpatients of Peking Union Medical College Hospital during 2004–2011 were enrolled. SLE patients received diagnoses based on the criteria established by the American College of Rheumatology (ACR) in 1982,which was revised in 1997; NPSLE pTwo cohorts of samples were used in the study. The first cohort was used to screen candidate autoantigens with the human proteome microarray, including CSF specimens of 12 NPSLE, 7 non-NPSLE, and 10 non-SLE(5 meningitis and 5 scoliosis) patients. The second cohort was for validation with a focused autoantigen microarray, including CSF specimens of 51 non-SLE, 45 NPSLE, and 28 non-NPSLE patients, and 41 matched serum specimens of 5 non-SLE, 26 NPSLE, and 10 non-NPSLE patients.2 was calculated to evaluate the consistency of duplicate spots.The human proteome microarrays contained about 17000 unique human full-length proteins with an N-terminal GST (glutathione S-transferase) tag and some protein controls in duplicates. These pThe procedures for the anti-GST and specimen assay with the human proteome microarray were performed as described previously, with miIjdenoted the normalized intensity of spot j on the protein microarray. All Ij(control spots omitted) on each protein microarray were used to construct an intensity distribution curve. In general, if there is no positive hit on the protein microarray, the intensity distribution curve for the protein microarray is bilaterally symmetrical with the line x = 1 as the axis of symmetry. Here, positive hits resulted in an intensity distribution with a positively skewed distribution. Therefore, we inverted (mirrored) the actual intensity distribution curve on the left of line x = 1 to the right side, to fabricate a bilaterally symmetrical curve, and calculated the standard deviation (SD) for this new curve. We used meani to denote the average of Ij(control spots omitted) on protein microarrayi. The cutoff of the meani+6·SD was applied to determine a positive hit on protein microarrayi. Human proteins and various controls were considered as a positive feature only when both of their duplicate spots were simultaneously judged as a positive hit.GenePix 5.1 was used to obtain microarray signal intensity. Fmedian/Bmedian was used to execute background correction and to normalize the intra-array signal intensity. Twelve candidate autoantigens resulted from screening with the human proteome microarray, and 8autoantigen control proteins were used to construct a focused autoantigen microarray. Plasmid pEGH-bearing coding sequences of autoantigens were transformed into yeast strain Y258 and the proteins were expressed as GST-fusion proteins and purified by affinity chromatography. Spotting buffer, bovine serum albumin (BSA), GST, H5N1, human IgG, and mouse IgG were used as controls on the focused autoantigen microarray. GST protein was expressed from an empty pEGH vector.BSA was purchased from Sigma-Aldrich. H5N1 protein, mouse IgG, and human IgG were supplied by Beijing Protein Innovation (Beijing). The Bradford method was applied to determine the concentration of proteins. The layout of the focused autoantigen microarray is displayed in One hundred and twenty-four CSF specimens (1:20 diluted) and 41 matched serum specimens (1:500 diluted) were assayed using the focused autoantigen microarray. Anti-GST mAb (1:2000) was used to measure the quality of the customized microarray. A SmartGrid  was fixed to the slide surface to form 12 identical chambers, and each chamber was blocked with3% BSA in PBST at 37°C for 1h. CSF or serum was diluted using3% BSA in phosphate-buffered saline with Tween 20 (PBST). Each diluted specimen was randomly pipetted into 2 different chambers and incubated at 37°Cfor 1h. Specimens were removed by centrifuging for 2 min at 2000 rpm in a 50-mL centrifuge tube, and PBST, preheated to 37°C,was used to wash the slide 3times for 10 min each.After removing the PBST, Cy5-conjugated anti-human IgG or anti-mouse IgG was used to probe the primary antibodies bound to the slide. Secondary antibodies and the SmartGrid were removed from the slide. The slide was consecutively washed with preheated PBST and Milli-Q water 3 times for 10 min each. After removing the Milli-Q water and drying, the slides were scanned using theLuxScan-10K/A scanner at optimal settings  for Cy5.The signal intensity of the spots on the microarray was extracted using Genepix 5.1. Because the autoantigens on the microarray were GST-fused proteins, the signals measured may come partially from the GST-tag, or other non-target-coding parts of the fusion proteins. To eliminate these potential interferences, we developed the following method to differentiate the positive feature on the focused autoantigen microarray.Ip, could be deduced from the fitted unary linear regression equation by the molecular amount of autoantigen.The average mean intensity of the foreground pixels from duplicate spots for each autoantigen or control was characterized as the actual intensity (Ia). For each autoantigen or control, we obtained a ratio for Ia-to-Ip. In CSF, we considered an autoantigen or control with a ratio ≥2, simultaneously in 2different assays, as positive spots. In the serum group, the ratio was set at ≥3.GST proteins, expressed and purified from the empty pEGH vector, were spotted onto each block at 4concentrations. In each block, the signal intensity and natural logarithm of the molecular amount of GST proteins from the 4concentrations fit a unary linear regression equation. We assumed that proteins with the same molecular amount would have the same interference signal intensity. With this assumption, the interference signal intensity for a given autoantigen, denoted Five autoantigens were expressed: RPLP0, RPLP1, RPLP2, SS-A, and PCNA. The GST-tag was removed from the GST-fusion protein as previously described. The recP<0.05 was considered statistically significant.Fisher’s exact test, the chi-squared test, the Kruskal-Wallis test, and Spearman’s rank correlation coefficient analysis were used in our study using the R software package. 2 of 0.98 was obtained, indicating that this batch of microarrays were of high quality .To identify NPSLE-specific CSF autoantibodies, we profiled the autoimmune reactivity by incubating the human proteome microarrays with 29 individual CSF samples from 12 NPSLE, 7 non-NPSLE, and 10 non-SLE patients, and then performed signal detection with Cy5-labeled anti-human IgG antibodies. Each of the 29 CSF specimens was incubated on a randomly selected human proteome microarray of the same printing batch. Human proteins that showed strong autoimmunogenicity could be readily detected in the CSF specimens .Using a To analyze the autoantibodies specifically present in the CSF of SLE patients, especially in the NPSLE patients, we first excluded the autoantigens that showed positive signals observed in the non-SLE control samples. By using a positive rate of 15% (ratio≥2) within the group as a cutoff, we found 159 and 31 autoantigens associated with the NPSLE and non-NPSLE patients, respectively; 22 were shared. The assP-value, using the right-tailed Fisher’s exact test. The 137 autoantigens specifically associated only with NPSLE specimens were found significantly enriched in neurological disease (with a score of 43). Autoantigens common in NPSLE and non-NPSLE were prone to participate in an inflammatory response (with a score of 29).Ingenuity Pathway Analysis(IPA), a web-based functional analysis tool for comprehensive omic data, was used to identify potential biological function enrichment among those autoantigens associated with CSF obtained from NPSLE and non-NPSLE patients  Tables. One major concern with biomarker identification is statistical overfitting, caused by a relatively small cohort size or complex factors or variables involved with a particular disease. To minimize overfitting, we recruited a much larger cohort of CSF specimens to validate those autoantigen candidates identified on the human proteome microarrays. Using our previously established two-phase approach, we fabrAs described previously, purified candidate autoantigen proteins, as well as positive and negative controls, were spotted in duplicate in a 2×6 format to form 12 identical blocks on each glass slide. Each CSP = 0.045). All the autoantigens are listed according to the positive rates, in decreasing order, in the NPSLE group.The consistency of discriminations in2 independent assays reached 95.8% among all CSF specimens tested. The positive rates of autoantibodies against RPLP0, RPLP1, RPLP2, and SS-A were significantly higher in the NPSLE group than in then on-NPSLE or non-SLE groups. Further+ and anti-SS-A—groups. In each group, neuropsychiatric syndromes were recorded and P-values were calculated using Fisher’s exact test or the chi-squared test (P = 0.009).Since the prevalence of anti-SS-A in CSF specimens was significantly different between the NPSLE and non-NPSLE groups, we wanted to determine further whether it had any association with particular neuropsychiatric syndromes. Seventy-three CSF specimens from SLE patients were divided into anti-SS-Ared test . We founAlthough autoantibodies were previously reported detectable in both CSF and serum samples in SLE patients, a potential correlation between CSF- and serum-based autoantibodies has not been extensively investigated. TherefoP-values between the CSF and serum samples .Interestingly, the correlation coefficient should be even higher for those matched samples with Z-scores>2 . Therefore, autoantibody titers of the CSF and serum samples strongly correlated, indicating that autoantibodies found in the CSF were probably due to leakage through the compromised blood-brain barriers from the blood.The Z-scores between the CSF and serum samples showed rather high correlations for both autoantigens, with correlation coefficients of 0.71 and 0.75 for RPLP2 and SS-A, respectively. Statistical analyses also revealed significant Because of the limited availability of the CSF specimens, we only employed 15 CSF specimens in the validation assay using the standard immunoblotting assays. Previously, anti-RPLP0, anti-RPLP1, and anti-RPLP2 autoantibodies had been widely reported in CSF specimens of SLE patients, 32. TheAutoantibodies against both SS-A and PCNA were readily detected in both CSF and serum samples .As expecUnder normal conditions, the integrity of the blood-brain barrier helps maintain the homeostasis of the central nervous system. When the integrity is undermined under pathological conditions, such as NPSLE, substances in the blood will leak into the central nervous system, resulting in changes in the properties and composition of the CSF. Based on this notion, laboratory results from CSF are often used to assist clinical diagnosis. Indeed, autoantibodies have been detected in the CSF of SLE patients; some of them are correlated with neuropsychiatric syndromes, 7, 32.In this study, we chose to use a human proteome microarray, comprising17000 full-length individually purified human proteins as an unbiased platform, to survey for novel NPSLE-associated autoantibodies in the CSF of SLE patients. We found that the prevalence of disease-associated autoantibodies in the CSF of SLE patients was low , <45% inAlthough the pathology of NPSLE and non-NPSLE is quite different, our unbiased survey on the human proteome microarray led to the identification of 22 shared autoantigens between these2 subtypes of SLE. Interestingly, bioinformatics analyses of these 22 autoantigens revealed a significant enrichment in the inflammatory response (score = 29). Because SLE is a prototype autoimmune disease, and because activation of autoreactive B or T cells leads to chronic tissue inflammation and often irreversible structural and functional damages, our anaIn NPSLE, autoantibodies directly cause damage to the nervous system. In adult mouse models, when the blood-brain barrier is compromised, anti-NMDA receptor autoantibodies can access the brain and elicit neuronal death with ensuing cognitive dysfunction and emotional disturbance. InjectiP<0.01). This indicated that autoantibodies in the CSF of SLE patients were mainly from the leakage of autoantibodies through the compromised blood-brain barrier.Autoantibodies in CSF may come from intrathecal antibody synthesis or leakaP = 0.045) in the NPSLE group (31.11%) than in the non-NPSLE group (10.71%). Further analysis showed that anti-SS-A in CSF specimens was associated with neurological syndromes of the central nervous system in SLE patients (P = 0.009). SS-A is an RNA-binding protein that binds to misfolded non-coding RNAs, pre-5S rRNA, and several small cytoplasmic RNA molecules known as Y RNAs. It may stabilize some of these RNAs and protect them from degradation. It is not clear whether anti-SS-A antibodies participate in the etiology of neurological syndromes.In this study, we detected and validated that anti-SS-A autoantibodies are significantly associated with NPSLE, based on both protein microarray and standard immunoblot assays. To our knowledge, this is the first report to firmly connect anti-SS-A autoantibodies with neuropsychiatric symptoms in SLE, although anti-SS-A was previously detected in the CSF and sera of NPSLE patients. ImportaPreviously, anti-PCNA autoantibodies had only been reported in<5% of the serum samples of SLE patients. In thisS1 FigRandomly selected proteome microarray was hybridized with anti-GST mAb, and the scanning result is displayed in S1A Fig. Intensity of duplicate spots was extracted to draw scatter plot and calculate intensity consistency. Linear regression analysis was applied to quantify the consistency of signal intensity of duplicate spots in S1B Fig.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(XLS)Click here for additional data file.


Background. The two most frequent types of microcytic anemia are beta thalassemia trait (β-TT) and iron deficiency anemia (IDA). We retrospectively evaluated the reliability of various indices for differential diagnosis of microcytosis and β-TT in the same patient groups.  Methods. A total of 290 carefully selected children aged 1.1–16 years were evaluated. We calculated 12 discrimination indices in all patients with hemoglobin (Hb) values of 8.7–11.4 g/dL. None of the subjects had a combined case of IDA and β-TT. All children with IDA received oral iron for 16 weeks, and HbA2 screening was performed after iron therapy. The patient groups were evaluated according to red blood cell (RBC) count; red blood distribution width index; the Mentzer, Shine and Lal, England and Fraser, Srivastava and Bevington, Green and King, Ricerca, Sirdah, and Ehsani indices; mean density of hemoglobin/liter of blood; and mean cell density of hemoglobin.  Results. The Mentzer index was the most reliable index, as it had the highest sensitivity (98.7%), specificity (82.3%), and Youden's index (81%) for detecting β-TT; this was followed by the Ehsani index  and RBC count .  Conclusion. The Mentzer index provided the highest reliabilities for differentiating β-TT from IDA. It is estimated that about 50% of the world's population with β-TT are in Southeast Asia; it is also common in the Mediterranean region, the Middle East, Southeast Asia, Southwest Europe, and Central Africa [β-TT is found in people with no obvious ethnic connection to the disorder.Anemia resulting from lack of sufficient iron to synthesize hemoglobin is the most common hematological disease in infants and children. It has been estimated that 30% of the global population suffers from iron deficiency anemia (IDA), and most of those affected live in the developing countries. Microcytic anemia in a case of thalassemia results from impaired globin chain synthesis and decreased hemoglobin (Hb) synthesis, resulting in microcytosis and hypochromia; 1.5% of the world's population carries genes for l Africa . Due to β-TT and IDA is based on the result of HbA2 electrophoresis, serum iron levels, and a ferritin calculation [β-TT. The purpose of using indices to discriminate anemia is to detect subjects who have a high probability of requiring appropriate follow-up and to reduce unnecessary investigative costs. Since 1970, a number of complete blood count indices have been proposed as simple and inexpensive tools to determine whether a blood sample is more suggestive of β-TT or IDA [β-TT (high sensitivity) while eliminating patients with IDA (high specificity). In this study, we compared the ability of different 12 indices to distinguish β-TT from IDA by calculating their sensitivity, specificity, and Youden's index values.A definitive differential diagnosis between culation . ElectroT or IDA –12. Most2 values were determined in all children. HbA2 was detected by high-performance liquid chromatography (Shimadzu LC-MS). SI and total iron binding capacity (TIBC) were determined calorimetrically , and ferritin was measured by immunoassay using a Siemens Advia XP Analyzer. Transferrin saturation was calculated as the ratio of SI to TIBC. The hematological and biochemical data of the groups are evaluated before iron replacement regimen. Those are shown in 2 synthesis, resulting in reduced HbA2 levels in patients with IDA [2 levels (>3.5%) is the most significant parameter for identifying beta thalassemia carriers. Patients with β-TT and concomitant iron deficiency may show normal HbA2 levels. Therefore, none of the subjects in the present study had both IDA and β-TT. All children with IDA received oral iron (3–5 mg/kg/day) for 16 weeks. HbA2 screening was performed after completion of the 16-week iron replacement regimen.We retrospectively analyzed 290 children with microcytic anemia . Samples were obtained from 121 boys and 169 girls with no clinical symptoms of acute or chronic inflammation or infectious diseases. None of them had received a transfusion or had an acute bleeding episode in the previous month. The samples were obtained during the course of routine analysis and collected in EDTA anticoagulant tubes. Red blood cell (RBC) count and red blood cell distribution width (RDW) were assessed on a Siemens Advia 2120 Hematology Analyzer. Serum iron (SI), serum iron binding capacity, serum ferritin, and HbAwith IDA , 14. Theβ-TT. The β-TT group consisted of children with Hb levels of 9–11.46 g/dL, mean corpuscular volume (MCV) < 80 fL at age > 6 years or MCV < 70 fL at age < 6 years, serum iron level > 30 μg/dL, transferrin saturation > 16%, serum ferritin level > 12 ng/dL, and HbA2 > 3.5%. A total of 136 children were confirmed to have IDA with serum ferritin levels ≤ 12 ng/dL. The IDA group consisted of children with Hb levels of 8.7–11.4 g/dL, mean corpuscular volume (MCV) < 80 fL at age > 6 years or MCV < 70 fL at age < 6 years, serum iron level < 30 μg/dL, transferrin saturation < 16%, and serum ferritin level ≤ 12 ng/dL. Children with Hb levels < 8.7 g/dL were excluded because these cases of severe anemia are not confused with β-TT in daily practice. None of the subjects of the present study had a combined case of β-TT and IDA. The combined cases were excluded from the early stages of the evaluation study.A total of 154 children were confirmed to have The 12 discrimination indices used in the evaluation were calculated and are summarized in t-test was performed to detect differences between the two groups of anemic children. P values < 0.05 were considered significant. The differential values for each discrimination index were applied as defined in the original published reports: red blood distribution width index (RDWI), Mentzer index [β-TT and IDA and the number and proportion of correctly identified patients (true positives) calculated using these indices are shown in β-TT and IDA are shown in Data were analyzed with computerized statistical package for social sciences (SPSS) version 15.0. An independent sample er index , the Shier index , the Srier index , the Greer index , the Ricer index , and theer index . The Ehser index , mean deer index , and RBCβ-TT group were 10.39 ± 0.69, and those in the IDA group were 10.23 ± 0.95 (P > 0.05). MCV was 60.11 ± 3.49 and MCHD was 18.9 ± 1.37 in the β-TT group, and these values were lower than those in the IDA group . The RDWI values were increased in both groups: 17.4 ± 3.48 in the IDA group and 16.76 ± 1.83 in patients with β-TT (P > 0.05). Red cell values at various ages of study groups are shown in Hb values in the β-TT (5.56 ± 0.4) group than that in the IDA . A high erythrocyte count (RBC > 5.0 × 106/μL) is a common feature of IDA and β-TT. The RBC count is one of the most accurate indices available. The RBC count provided the best sensitivity (94.8%) but had low specificity (70.5%), and Youden's index was 65.3%.RBC count was higher in the β-TT. The Ricerca et al. and the Shine and Lal indices demonstrated the highest sensitivity (100%) but had low specificities for correctly identifying IDA (14.7%) and β-TT (10.2%). Therefore, according to our results, these indices cannot be used as screening tools for β-TT, as using them could result in a significant number of false-negative results. The England and Fraser index had the lowest sensitivity of 66.2%, and identification was wrong in about 28% of β-TT cases. The England and Fraser and the Mentzer indices demonstrated the highest specificities at 85.3% and 82.3%, respectively. Furthermore, β-TT and IDA.As indicated in β-TT and IDA: Mentzer index > Ehsani et al. index > RBC count > Sirdah et al. index > RDWI > Srivastava index > Green and King index > England and Fraser index > MDHL > Ricerca et al. index > Shine and Lal index > MCHD. The difference between the results of all of these indices and the gold standard (HbA2) was significant (P < 0.001).The Mentzer index showed good sensitivity, specificity, and Youden's index values of 98.7%, 82.3%, and 81%, respectively. When the Mentzer index was calculated, 264 children with microcytic anemia (91%) were correctly diagnosed. Youden's index showed the following ranking with respect to the indices' ability to distinguish between β-TT and IDA are among the most common types of microcytic anemia encountered by pediatricians. Distinguishing β-TT from IDA has important clinical implications because each disease has an entirely different cause, prognosis, and treatment. Thalassemia is endemic in Turkey. Misdiagnosis of β-TT has consequences for potential homozygous offspring. Up to now, many investigators have used different mathematical indices to distinguish β-TT from IDA using only a complete blood count. This process helps to select appropriate individuals for a more detailed examination; however, no study has found 100% specificity or sensitivity for any of these RBC indices. Our data  at the time of diagnosis. However, the frequency of high RBC count was 29.4% in children with IDA. It seems that RBC alone was not a reliable tool for distinguishing β-TT from IDA. Elevated RBC count might be associated with erythrocytosis. We observed that the RBC count increased at the initiation of iron therapy in patients with IDA and decreased by the end of therapy. A similar observation was made by Aslan and Altay, who reported an elevated RBC count in 61% of cases with mild anemia [Our data  showed sle index , but we d anemia .In a 2010 study, Ferrara et al. demonstrated that RDWI had the highest sensitivity (78.9%), that the England and Fraser index had the highest specificity and highest Youden's index , and that the Green and King index had the highest efficiency (80.2%) in 458 children with mild microcytic anemia aged 1.8–7.5 years .β-TT and IDA, whereas the Shine and Lal index was ineffective for differentiating microcytic anemia [β-TT and IDA at 81%. When the Mentzer index was calculated, 91% of children with microcytic anemia were correctly diagnosed. The England and Fraser and the Shine and Lal indices had the lowest Youden's index values of 51.4% and 10.2%, respectively.AlFadhli et al. compared nine discriminant functions in patients with microcytic anemia and measured validity using Youden's index. Youden's index considers both sensitivity and specificity and provides an appropriate measure of validity for a particular question or technique. They showed that the England and Fraser index had the highest Youden's index value (98.2%) for correctly differentiating c anemia . AccordiIn 2009, Ehsani et al. showed that the best discrimination index according to Youden's criteria was the Mentzer index (90.1%), followed by the Ehsani et al. index (85.5%). In their study, the Mentzer and Ehsani et al. indices were able to correctly diagnose 94.7% and 92.9% of cases, respectively, and both are easy to calculate . Similarβ-TT and 170 patients with IDA. According to Youden's index, the Shine and Lal index and RBC count showed the greatest diagnostic value in patients < 10 years . They found that the Mentzer index had 85% sensitivity, 93% specificity, and 79% Youden's index [Rahim and Keikhaei examined the diagnostic accuracy of 10 indices in 153 patients with 's index .β-TT [β-TT from IDA could be attributed to differences in the mutation spectrum of the thalassemia disease in different populations [In 2007, Ntaios et al. reported that the Green and King index was the most reliable index, as it had the highest sensitivity (75.06%), efficiency (80.12%), and Youden's index (70.86%) for detecting β-TT . A similulations .β-TT involves measuring the HbA2 concentration of lysed RBCs via HPLC. The HbA2 analysis is considered the gold standard for diagnosing thalassemia. Several studies have shown that iron deficiency directly affects the rates of HbA2 synthesis in bone marrow; therefore, 16–20 weeks of iron therapy should be instituted, after which a repeat serum iron with electrophoresis is done to confirm improvement in the HbA2 levels [The diagnosis of 2 levels .β-TT. According to our results, the percentage of correctly diagnosed patients was the highest with the Mentzer index (91%) followed by the Ehsani et al. index (84.8%). The third highest one was RBC count (83.4%). Cell-count-based parameters and formulas, particularly the MCV and RBC counts and their related indices , have good discrimination ability in diagnosing β-TT.In conclusion, the cell-count-based indices, particularly the Mentzer index, are easily available and reliable methods for detecting