Ceramides are sphingolipids with defined acyl chain lengths, which are produced by corresponding ceramide synthases (CerS1-6). In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), the ablation of CerS2 suppresses EAE-pathology by reducing neutrophil migration into the central nervous system. This migration is induced by granulocyte-colony stimulating factor (G-CSF) signaling. G-CSF signaling leads to a signal cascade including the phosphorylation of Lyn kinase and STAT3. This in turn regulates expression of the neutrophil surface receptor chemokine receptor 2 (CXCR2) and causes translocation of the receptor into detergent-resistant membranes (DRMs). In this study we investigated the role of ceramides in G-CSF signaling. We found, that G-CSF treatment of wild type bone marrow cells (BMCs) leads to translocation of G-CSF-receptor (G-CSF-R) into DRMs. G-CSF also induces downregulation of ceramides in WT and CerS2 null BMCs, as well as upregulation of very long chain lactosylceramides. However, in CerS2 null BMCs, G-CSF failed to induce translocation of G-CSF-R into DRMs, leading to reduced phosphorylation of Lyn and reduced CXCR2 expression. Interestingly, G-CSF signaling in CerS6 null BMCs was not affected. In conclusion, very long chain ceramides are important for G-CSF signaling and translocation of G-CSF-R into DRMs. Among other immune cells involved in the pathogenesis of EAE, neutrophils are the first to infiltrate into the CNS4. Migration into the CNS occurs after neutrophil expansion in the bone marrow and accumulation in the peripheral vascular system3.Multiple sclerosis (MS) is a neurodegenerative autoimmune disease characterized by the infiltration of immune cells into the central nervous system (CNS). The infiltrating immune cells release chemokines to recruit further immune cells and cytokines/inflammatory mediators to promote the inflammatory process, leading to the death of oligodendrocytes and subsequently to demyelination and neurodegeneration. A commonly used animal model for MS is experimental autoimmune encephalomyelitis (EAE), which has been used successfully for translation of drug candidates, including mitoxantrone, glatiramer acetate and natalizumab, into human therapeutic approaches7. This signaling pathway is also regulated by suppressor of cytokine signaling 3 (SOCS3) which is induced by STAT3 and inhibits the catalytic activity of Jak and thereby, the expression of STAT3. This results in negative feedback regulation9. Activated STAT3 translocates into the nucleus and induces the expression of several genes such as CXCR210.Granulocyte colony-stimulating factor (G-CSF) induces migration of neutrophils by upregulation of the chemokine receptor CXCR2. The binding of G-CSF to its receptor (G-CSF-R) leads to the dimerization of the extracellular domain of the receptor that in turn activates Lyn kinase and Janus kinases (Jak). These kinases phosphorylate one or more tyrosine residues in the C-terminal region of the G-CSF-R leading to the activation of multiple intracellular signaling proteins, including the signal transducers and activators of transcription (STAT) and mitogen-activated protein (MAP) kinases11. Binding of the ligand G-CSF to the G-CSF-R activates neutrophils and promotes their adhesion to the endothelium and subsequently, the migration of the cells into tissues12. The CXCR2 receptor is the primary mediator of CXCL2 chemokine signaling11 and antagonism of CXCR2 ameliorates clinical symptoms in EAE mice13. Recently, we demonstrated that expression of CXCR2 in neutrophils induced by G-CSF is regulated by ceramide synthases (CerS2 and CerS6). Furthermore, ablation of CerS2 or CerS6 leads to an amelioration or worsening of EAE pathology, respectively, and is linked to CXCR2 expression and neutrophil infiltration14.The G-CSF-R is expressed on mature neutrophils in bone marrow and blood15. CerS2 synthesizes predominantly C22-C24-ceramides (very long chain ceramides), whereas CerS6 produces mainly C16-ceramides (long chain ceramides)17. The ratio between long chain ceramides and very long chain ceramides may influence the fluidity of the membrane and therefore the activation potential of membrane receptors18.The six known ceramide synthases are expressed in a tissue-specific manner and synthesize ceramides (Cer) by catalyzing the N-acylation of a long chain base such as sphingosine to fatty acyl chains of specific lengths. Ceramides are sphingolipids, which have emerged as interesting targets for MS therapy since FTY720 (fingolimod), a sphingosine analogue, has been used successfully in MS treatment, acting to confine lymphocytes to the lymphoid organs19. For lipid raft characterization cell membranes can be divided into biochemical fractions, which are non-soluble in detergents such as Triton X-100. Therefore, these fractions are called detergent-resistant membranes (DRMs). DRM isolation is a useful tool to identify proteins, which are situated in lipid rafts microdomains20. Previously it was shown that the sphingolipid composition of the membrane can influence the localization of membrane proteins such as CD36 or connexin 3221. It is still unclear whether this is due to the lack or elevation of specific sphingolipids or the fluidity of the membrane.Within the outer leaflet of the cell membrane bilayer complex sphingolipids such as glycosphingolipids and cholesterol cluster together in distinct domains, the so called lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins14. The mechanisms by which G-CSF induces the expression of CerS2 and CerS6, and how CerS2 and CerS6 mediate the regulation of CXCR2 are currently unknown. In this study, we investigate the hypothesis that a deficiency in ceramides with specific acyl chain lengths alters membrane properties and thereby prevents or promotes G-CSF signaling.Our previous results indicate that G-CSF regulates CerS2 and CerS6 expression, which in turn mediates CXCR2 expression2. The impaired neutrophil migration was linked to reduced CXCR2 expression on neutrophils in the preclinical phase2. Because G-CSF induces CXCR2 expression, we suggested that CerS2 is required for proper activation of the G-CSF-R and thereby promotes the expression of CXCR2 and the neutrophil migration potential. To exclude the possibility that higher CXCR2 expression in EAE WT mice compared to EAE CerS2 null mice2 is due to increased basal levels of G-CSF or G-CSF-R, G-CSF levels in plasma and G-CSF-R mRNA levels in white blood cells (WBCs) of CerS2 null and WT EAE mice were measured. As expected, G-CSF levels increased in WT EAE mice at four days post immunization  by determination of sphingolipid and cholesterol content. The DRMs from BMCs were isolated using sucrose gradient centrifugation. The DRMs were located predominantly in fraction two, as determined by the cholesterol content in BMCs from WT mice  is CerS2 or CerS6 dependently regulated. SOCS3 is a key negative regulator for G-CSF signalingMCs Fig. . CerS6 WMCs Fig. .10. However, to our knowledge it is unknown if Lyn kinase signaling plays a role in CXCR2 expression in neutrophils. Therefore, we investigated whether Lyn kinase also influences CXCR2 expression. We also checked whether an inhibitor of the Lyn kinase (PP1) can prevent the upregulation of CXCR2 on neutrophils isolated from BMCs. The cells were treated with G-CSF for 24 h or 46 h in the presence or absence of the inhibitor. G-CSF induced upregulation of CXCR2, whereas PP1 prevents the increase of CXCR2 expression after 24 h . In unstimulated BMCs, the CXCR2 was primarily localized in fraction 10, whereas the activation with G-CSF led to an increase of CXCR2 in fraction 6 after 24 h. This was followed by an increase of CXCR2 in the next 22 h, in fractions 2 and 3, accompanied by a decrease in CXCR2 in fraction 6, indicating that G-CSF induces the expression of CXCR2 and leads to its translocation into DRM fractions 2 and 3 Fig. . Therefo14. Therefore, we investigated whether this translates into an altered ceramide or cholesterol content of the DRMs. After 46 h G-CSF treatment cholesterol was found in fraction 2 or 3 in WT and CerS6 null cells, whereas in CerS2 null cells cholesterol was found in each fraction , located upstream of the start codon of the murine CerS6  of different chain lengths. In CerS2 null BMCs, DRMs also consist of ceramides and glucosylceramides but with almost no contribution of sphingolipids with a C24- and C24:1-Cer backbone. The lack of very long chain ceramides does not prevent the formation of DRMs but alters the composition of the DRM and the fraction location (at least in hepatocytes21) and thereby the biophysical properties of the membrane. The lack of very long chain ceramides in CerS2 null cells leads to a more fluid26 membrane and might influence cell signaling. For example the incorporation of 2-hydroxylated fatty acids (2OHFAs) into the cell membrane results in decreased lipid order and dipole potential as well as less rigid packing of acyl chains and hence, modulates membrane proteins by microdomain reorganization and membrane fluidity27. Furthermore, activation with G-CSF and thereby the switch to neutrophils leads to an increase in C24-LacCer in WT cells, whereas in CerS2 null cells, C16-LacCer and C24-LacCer were increased. This unexpected C24-LacCer increase in CerS2 null BMCs might be produced by CerS3 or CerS4, which are also capable of very long chain ceramide synthesis28. Also, in the dextran sodium salt (DSS) evoked model of ulcerative colitis, an increase in very long chain ceramides in CerS2 null mice was shown, accompanied by an increase in CerS3 mRNA expression29. The observed decrease in ceramides after G-CSF induction in CerS2 WT and null BMCs is possibly due to the fact that ceramides are precursors for the glucosylceramides. Additionally, the G-CSF induced altered membrane composition prevented the formation of DRMs in CerS2 null cells, as indicated by the lack of cholesterol accumulation in DRM-fractions  and experimentally resist extraction in cold detergent (detergent-resistant membrane)30. The gap junction protein connexin 32 displays a mislocalization in CerS2 null hepatocytes due to disruption of DRMs31. For FcγRIIB, it was shown that exclusion from DRMs prevents interaction with signaling molecules33 and therefore, possibly their inhibitory potential in immune regulation. In CerS2 null hepatocytes, TNFR1 is not internalized and inhibits selective pro-apoptotic downstream signaling for apoptosis34.The altered sphingolipid composition, the more fluid membrane and the lack of DRM in G-CSF-activated cells are possibly responsible for the lack of G-CSF-R translocation and the impaired G-CSF signaling, such as reduced Lyn phosphorylation and reduced CXCR2 expression. The importance of sphingolipid composition and altered membrane properties for the function of membrane receptors has already been observed by others. CD36 which is responsible for the uptake of triglycerides and the insulin receptor did not translocate into DRMs in CerS2 null hepatocytes and consequently loose their functions35. The existence of cholesterol and sphingolipid rich microdomains is a prerequisite for this activation. It is also possible that ceramides directly interact with proteins and thereby, induce dephosphorylation of Jak2 or Lyn kinase. For example, C18 ceramide activates protein phosphatase-2A36 which in turn, can dephosphorylate Jak237.We cannot exclude that ceramides synthesized by sphingomyelinase (SMase) also contribute to the observed effects, since ceramides generated by SMase enhance Tyr-phosphorylation of Jak2 and subsequently STAT3 phosphorylation in fibroblasts39. G-CSF also induced CerS2 promotor activity. To identify which transcription factor, activated by G-CSF signaling, was responsible for CerS2 promotor activity, we analyzed five different upstream regions of the start codon of CerS2. Promotor construct prom2_III was able to function as a promotor. Prom2_III comprises exon 1 and intron 1. To narrow down the putative binding site, we took into account that construct prom2_IV lacks the first 643 Bp of intron 1 and has no promotor activity. These data suggest that the transcription factor binds to intron 1 at the first 643 Bp and/or to exon 1. This is in line with the findings of Gong et al. who identified exon 1 as a regulatory region for gene transcription in human CerS2 and as binding site of the transcription factor SP1, which interacts with KLF640. Analysis of the putative promotor region DNA fragment of CerS2 including exon 1 and 700 Bp from intron 1 by the “Promo program” (maximum matrix dissimilarity rate: 5%)42 suggests several putative transcription factor binding sites. This includes CCAAT/enhancer-binding protein C/EBPα, C/EBPβ, IRF-1 and STAT5 which are induced by G-CSF or belong to G-CSF-responsive genes22, but no STAT3 binding site. These data indicate that CerS2 mRNA expression is not induced via STAT3 but via a yet unknown transcription factor.As it has already been shown by others, our data confirm that G-CSF induces the Lyn signaling cascade and leads to activation of STAT3Previously, we demonstrated that the genetic deletion of CerS2 ameliorates clinical symptoms in EAE mice. We showed that the ameliorated clinical symptoms in CerS2 null mice were due to a reduced potential for migration of neutrophils that express reduced CXCR2 protein levels. In this study, we showed that in CerS2 null cells, translocation of the G-CSF-R is prevented leading to impaired receptor function and subsequently, to reduced expression of CXCR2. We assume that besides the G-CSF-R, other DRM-located proteins that regulate the immune response or inflammatory processes can be influenced by the alteration of DRM properties in CerS2 null mice. Further insight into the influence of chain-length specific ceramides on cell signaling is therefore important to better understand their involvement in diseases such as cancer and autoimmune diseases and to pinpoint possible therapeutic targets.2. G-CSF was purchased from BioLegend .BMCs were cultured and stimulated in RPMI 1640 GlutaMAX medium containing 2.5% hormone-free fetal calf serum (FCS) and 1% penicillin/streptomycin. RAW247 macrophages were cultured in RPMI 1640 medium – GlutaMAX containing 10% FCS. For the reporter gene assay, RAW247 macrophages were incubated in RPMI 1640 medium – GlutaMAX containing 2.5% FCS . Cells were cultured at 37 °C in an atmosphere containing 5% COIn all experiments, the ethics guidelines for investigations in conscious animals were followed and the experiments were approved by the local Ethics Committee for Animal Research . Unless otherwise stated, mice for all experiments were female, between 8–14 weeks of age.2. Untreated mice were used as the control group.10- to 13-week-old female 129S4/SvJae × C57BL/6 mice (F1 WT or F1 CerS2 null) were used for induction of EAE. The induction was achieved as recommended by the supplier using an EAE kit of Hooke Laboratories . The induction of the EAE model has been described previously2O. The absorbance was determined using an Infinite 200 PRO Reader .The concentration of G-CSF in blood plasma was determined using a mouse G-CSF enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions. Blood plasma was isolated by centrifugation , stored at −80 °C and diluted 1:50 in VE-H4Cl, 100 mM NaHCO3, 0.1 mM Na-EDTA pH 7.4) and incubated for 5 min before adding 5 ml of cold PBS. Cells were centrifuged, washed with PBS, and counted using a hemocytometer. For the experiments BMCs from 2 mice were pooled.Animals were killed and their femurs and tibias removed aseptically. Bone marrow cavities were flushed with phosphate buffered saline (PBS) using a 27G1/2 needle. Single cell suspensions were prepared by repeated pipetting and passing through a 70 μm nylon filter. After centrifugation at 4 °C and 400 g for 3 min, cells were resuspended in 0.5 ml of erythrocyte lysing solution (150 mM NH7 cells/ml) were stimulated with 10 ng/ml G-CSF for 5 min or 46 h in RPMI 1640 GlutaMAX medium. 5 min G-CSF stimulation was performed for determination of G-CSF-R levels by ELISA, since the activation of the receptor occurs at the beginning of the signaling cascade. Cellular processes with longer time spans, such as CXCR2 activation/translocation and production of ceramides were measured after 46 h. Cells were scraped off, washed with PBS and stored at −20 °C. 67.5%, 35% and 5% sucrose was freshly prepared and dissolved in water. The BMCs were thawed on ice and resuspended in 566 μl MES-buffer ). Cells were homogenized and sonicated 5 times for 20 s at level 3 with constant ultrasonic frequency . BMCs were kept on ice and resolved thoroughly in 1.13 ml 67.5% sucrose and filled into a 4.0 ml centrifugation tube . Thereafter, 1.7 ml 35% sucrose was layered on top, followed by 0.85 ml 5% sucrose. The sucrose gradient was centrifuged in the ultracentrifuge  for 20 h at 32,000 rpm  and 4 °C with slow deceleration. 9 DRM fractions were isolated (counting from the top of the sucrose gradient) at a volume of 450 μl with the second and third fraction containing the DRMs (shown by cholesterol and sphingolipid content). The pellet was resuspended in 450 µl MES-buffer and referred to as fraction 10. The samples were stored at −80 °C. Protein levels were determined using the Bicinchoninic acid (BCA) method.BMCs from two CerS2/6 null and two WT animals  or RNA Mini Kit  according to the manufacturer’s instructions. The cDNA synthesis was performed using a First Strand cDNA-Synthesis kit  including random hexamers. The expression levels of CXCR2, G-CSF-R, SOCS3 and peptidyl prolyl isomerase A (PPIA) were determined using Maxima SYBR Green  with an ABI Prism 7500 Sequence Detection System . Relative mRNA expression was determined using the comparative CT (cycle threshold) method, normalizing relative values to the expression level of murine PPIA. The sequences for the primer sets of CXCR2 and PPIA are publishedFor quantification of sphingolipids, high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) was used. 50 µl sample were mixed with 150 µl water, 150 µl extraction buffer  and 20 µl of the internal standard solution containing C17:0-Cer, C16:0-Cer-d31, C18:0 Cer-d3, C17:0-LacCer C16:0-LacCer-d3  and C24:0-Cer-d4  (400 ng/ml each). The mixture was extracted once with methanol/chloroform/hydrochloric acid . The organic phase was evaporated at 45 °C under a gentle stream of nitrogen and reconstituted in 200 µl of tetrahydrofuran/water  with 0.2% formic acid and 10 mM ammonium formate. Afterwards, 10 µL of the reconstituted solution were analyzed using a Zorbax EclipsePlus C18 column  and water with 0.2% formic acid and 2 mM ammonium formate and acetonitrile/isopropanol/acetone  with 0.2% formic acid as mobile phases run in gradient elution mode. Total running time was 19.5 min. MS/MS analyses were performed using a mass spectrometer 5500QTRAP , operating as triple quadrupole, in positive electrospray ionization mode and Multiple Reaction Monitoring (MRM) mode.Data Acquisition was done using Analyst Software V 1.6 and quantification was performed with MultiQuant Software V 3.0 , employing the internal standard method (isotope dilution mass spectrometry). Calibration ranges were ranged from 0.05 to 5 ng/sample, 0.2 to 20 ng/sample and 2 to 200 ng/sample depending on the analyte.20 and the concentrations of G-CSF-R and CXCR2 were determined according to the manufacturer’s instructions using an Infinite 200 PRO Reader .The G-CSF-R ELISA kit was purchased from MyBioSource  and the CXCR2 ELISA kit was from Cloud-Clone Corp. . The DRM fractions were diluted 1:10 in VE- HBMCs of WT, CerS2 null and CerS6 null mice were incubated for 5, 10, 20 and 40 min with 10 ng/ml recombinant mouse G-CSF. Cell pellets were homogenized and sonicated in PhosphoSafe Extraction Reagent  and supplemented with 1:10 Pefabloc . The homogenate was centrifuged, the supernatants were collected and stored at −80 °C. Protein concentrations were assessed using the Bradford method.Samples of 30–60 µg of total protein extract were separated electrophoretically by 8% SDS-PAGE and electroblotted onto nitrocellulose membranes . Membranes were blocked in 5% bovine serum albumin (BSA) in PBS and incubated with the respective primary antibody directed against P-Src , Lyn (1:100), P-STAT3 (1:100) and STAT3 (1:100). All antibodies were diluted in 5% BSA in 0.1% Tween 20 in PBS. Membranes were washed three times with 0.1% Tween 20 in PBS and then incubated with an IRDye800 or IRDye700 conjugated secondary antibody  in Odyssey Blocking Buffer: 0.1% Tween 20 in PBS (1:1) for 2 h at RT. Membranes were analysed on the Odyssey infrared scanner from LI-COR . Anti-P-Src, anti-P-STAT3 and anti-STAT3 antibodies were purchased from Cell Signaling  and anti-Lyn was purchased from Biolegend .BMCs were treated with 10 ng/ml recombinant mouse G-CSF for 24 h and 46 h to check for CXCR2 surface expression on neutrophils. One hour before G-CSF treatment, the cells were incubated with 10 µM Lyn inhibitor PP1  and then with an antibody cocktail consisting of CD11b-eFluor450 , Ly6G-APC-Cy7  and CXCR2-APC  for 15 min at room temperature. Samples were acquired with a MACSQuant Analyzer 10 flow cytometer  and analyzed using FlowJo software v10 . All antibodies were previously titrated to determine optimal concentrations. Antibody-capturing CompBeads  were used for single-color compensation to create multi-color compensation matrices.The putative promoter region of murine CerS2 was assumed to lie upstream of exon 1, in exon 1 or in intron 1, because the ATG lies in exon 2  and 12.5 ng of the Renilla luciferase control reporter vector  using 0.5 μl MACSfectin reagent. One day after transfection, the cells were incubated for 46 h with or without 10 ng/ml G-CSF. The luciferase assay was performed with luciferase substrate buffer 4Mg(OH)2); 1.07 mM MgSO4; 100 μM ethylenediamine tetraacetic acid (EDTA); 0.33 mg/ml 3.3′-diethylthiatricarbocyanine iodide (DTTC); 0.21 mg/ml co-enzyme A; 470 mM d-luciferin) in a luminometer  according to the manufacturer’s instructions. The activity of firefly luciferase was normalized to the RLU of renilla luciferase .Transient transfection was performed with MACSfectin  according to the manufacturer’s protocol. 1.2 × 10Results are presented as means ± SEM (standard error of the mean) or ± SD (standard deviation). Significant differences in multiple data sets were analyzed by Two-way ANOVA with Bonferroni post hoc-test or alternatively with Tukey’s or Sidak’s multiple comparisons test. Significant differences between two groups were analyzed by t-test (Graphpad Prism 6 software). The level of significance was set at p < 0.05.Supplemental Information


In vitro and in vivo studies have demonstrated that antimicrobial proteins and peptides (AMPs) are bactericidal against otopathogens, indicating potential therapeutic value for recalcitrant OM. We measured concentrations of 6 AMPs and 14 cytokines in middle ear effusion (MEE) from 67 children undergoing ventilation tube insertion for recurrent acute OM. Sixty one percent of children had bacterial otopathogens detected in their MEE, 39% by PCR and 22% by PCR and culture. Groups were defined as: PCR-negative/culture-negative , n = 26; PCR-positive/culture-negative , n = 26; PCR-positive/culture-positive , n = 15. Age, antibiotic usage, day-care attendance, presence of respiratory viruses in MEE and number of AOM episodes were similar between groups. AMP and cytokine concentrations were higher in children with bacterial otopathogens in their MEE compared to those with no bacterial otopathogens. Median concentrations of AMPs (except HBD2) were 3 to 56-fold higher in MEE from children with bacterial otopathogens detected in their MEE (P ≤ 0.01). Similarly, median cytokine concentrations (except TGFβ) were >16-fold higher in MEE with bacterial otopathogens detected (P ≤ 0.001). This is the first study to measure AMPs in MEE and together with the cytokine data, results suggest that elevated AMPs and cytokines in MEE are a marker of inflammation and bacterial persistence. AMPs may play an important role in OM pathogenesis.Recurrent and chronic otitis media (OM) are often refractory to antibiotics due to bacterial persistence in biofilm within the middle ear.  Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis, with NTHi being the most frequently detected species from the middle ear of children with recurrent and chronic OM [Otitis media (OM) is a common paediatric disease, and in high income countries OM is the primary reason that young children visit their General Practitioner, are prescribed antibiotics and undergo surgery . Complicronic OM . Bacterironic OM , 8. To rjunbo mouse model of OM, deletion of the gene encoding an AMP from the bactericidal permeability increasing (BPI) family (BPIFA1) resulted in exacerbation of OM severity due to increased epithelial remodelling [et al suggest that AMPs play a homeostatic role in maintaining healthy middle ear epithelium. Formulations of AMPs that could be applied intranasally or through the tympanic membrane  offer a potential antimicrobial therapy for children with recurrent and chronic OM, which may also reduce antibiotic use and curb the global rise in antibiotic resistance [Antimicrobial peptides and proteins (AMPs), otherwise known as host defence peptides, are rapid mediators of the innate immune system with broad-spectrum antimicrobial, antibiofilm and chemotactic activity that can influence the microbiota of mucosal surfaces –11. Therodelling . Based osistance , 18, 19.This study measured the concentrations of six AMPs in middle ear effusion (MEE) samples from children with a history of recurrent acute OM (rAOM). We hypothesised that children with no bacterial otopathogens detected in their MEE would have higher AMP titres than children with bacterial otopathogens in the MEE, due to the demonstrated antimicrobial activity of AMPs. We also measured a panel of fourteen cytokines in MEE from the same children to assess inflammatory responses and because there is important interplay between AMPs and pro-inflammatory cytokines particularly in epithelial cell and monocyte responses , 20, 21.This study used MEE samples collected from children enrolled in the GROMIT study . All chiMEE samples were collected from anaesthetised children as previously described . In brie2 (sPLA2). HBD1, HBD2, LL-37 and lactoferrin levels were measured using in-house multiplex fluorescent bead-based assays on the Bio-Plex® 200 system  [2 levels were measured using commercial ELISA kits  and according to the manufacturers’ instructions.An aliquot of MEE was thawed and used to quantify the following AMPs: Human beta defensin 1 (HBD1), Human beta defensin 2 (HBD2), LL-37, lactoferrin, bactericidal/permeability-increasing protein (BPI) and secreted phospholipase Aia, USA) . BPI andFourteen cytokines were quantified in another aliquot of thawed MEE: IL-1α, IL-1β, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFNγ, TNFα and TFGβ1, 2 and 3. IL-1β, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IFNγ, and TNFα levels (pg/mL) were measured using an in-house multiplex cytokine bead assay on the Bio-Plex® 200 system  using previously published methods . TFGβ1, 2 = 1.88 pg/mg of protein; IL-1β, IL-5, IL-12p70, IFNγ and TNFα = 0.09 pg/mg of protein; IL-6 and IL-10 = 0.57 pg/mg of protein; IL-8 = 3.17 pg/mg protein; IL-13 and IL-17A = 0.18 pg/mg of protein; TFGβ1 = 3.51pg/mg protein; TFGβ2 = 1.35pg/mg protein TFGβ3 = 5 pg/mg protein and IL-1a = 0.32 pg/mg protein.Total protein was previously determined in each MEE sample using the Micro BCA Protein Assay Kit and according to the manufacturers’ instructions  . Levels P-value asymptotic significant 2-sided) for categorical variables . Data were checked for normal distribution. AMP and cytokine levels were compared between PCR-ve/culture-ve; PCR+ve/culture-ve; PCR+ve/culture+ve children using Kruskal-Wallis tests with post-hoc pairwise comparisons for non-parametric data.Statistical analyses were performed using the IBM SPSS® software platform version 24. Population demographics were compared between the 3 groups of children: 1) PCR-ve/culture-ve, 2) PCR+ve/culture-ve and 3) PCR+ve/culture+ve, using Kruskal-Wallis tests for continuous variables  and Pearson Chi-square analysis . Ninety-seven percent of children had received all 3 doses of the 7-valent pneumococcal conjugate vaccine according to the Australian immunisation schedule. Nucleic acid from at least one of 11 common upper respiratory tract viruses was detected in 54 of the 67 children. The frequency of respiratory virus detection was similar between all 3 groups: PCR-ve/culture-ve = 73.1%, PCR+ve/culture-ve = 80.8% and PCR+ve/culture+ve 93.3% . Median titres of lactoferrin were 9-fold higher in PCR+ve/culture+ve compared to PCR-ve/culture-ve MEE (P < 0.0001), and 6-fold higher in PCR+ve/culture-ve compared to PCR-ve/culture-ve MEE (P = 0.022). Median concentrations of LL-37, BPI and sPLA2 were 15-, 12- and 56-fold higher respectively in PCR+ve/culture+ve MEE compared to PCR-ve/culture-ve MEE . LL-37 and sPLA2 levels were 5- and 56-fold higher (respectively) in PCR+ve/culture-ve MEE compared to MEE from children with no bacterial otopathogen detected (PCR-ve/culture-ve MEE), P = 0.018; P = 0.015. Median BPI levels were 4-fold higher in PCR+ve/culture-ve MEE compared to PCR-ve/culture-ve MEE (P = 0.015) and 3-fold higher in PCR+ve/culture+ve MEE compared to PCR+ve/culture-ve MEE (P = 0.006). Median concentrations of HBD2 were approximately 2-fold higher in PCR+ve/culture+ve MEE compared to PCR-ve/culture-ve MEE but this was not significant.All of the 6 AMPs tested could be measured and most children had levels of each AMP above the limit of detection for each assay. Except for HBD2, median concentrations were significantly higher in MEE from children that had detectable bacterial otopathogen by PCR and were highest in children with otopathogen that could be detected in their MEE by both PCR and culture . IL-1β, IL-6, IL-8 and IL-10 levels were at least 5-fold higher in PCR+ve/culture-ve MEE compared to PCR-ve/culture-ve MEE (P ≤ 0.044). IL-12p70 and TNFα were only measurable in 7/67 and 10/67 children respectively but these children all had detectable bacterial otopathogen in their MEE by both PCR and culture. Similarly, IL-1α was only measureable in 13/35 children but these children all had bacterial otopathogen detected by both PCR and culture or PCR alone. TGFβ3 was not detected in MEE while concentrations of TGFβ1 and TGFβ2 were similar across groups . Concentrations of IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNFα were always significantly higher in MEE from children when bacterial otopathogen was detected by culture and PCR compared to when bacterial otopathogen was not detected  bacterial otopathogens in their MEE. Contrary to our hypothesis, levels of AMPs and cytokines were lowest in MEE from children with no bacterial otopathogen detected; while higher AMP and cytokine titres were observed in MEE from children with bacterial otopathogen detected by PCR, and the highest titres were found in MEE from children with bacterial otopathogen detected by both PCR and culture. This indicates endogenous AMP levels are not adequate to clear bacterial otopathogen or that bacterial persistence mechanisms are at play (e.g biofilms). This has implications for helping to understand rAOM pathogenesis and suggests that elevated concentrations of AMPs and pro-inflammatory cytokines in MEE may be a marker of inflammation associated with bacterial persistence and/or presence of neutrophil extracellular traps (NETs), which are decorated in AMPs .OM pathogenesis is complex and multifactorial. Nasopharyngeal and middle ear biofilms are increasingly recognised as playing a central role in OM pathogenesis by providing a bacterial reservoir, enabling bacterial persistence and recurring or chronic infection , 33. PCRPseudomonas aeruginosa and NTHi were also present in sputum [in vitro assays using chinchilla middle ear epithelial cells have revealed that NTHi challenge results in upregulation of mRNA for the chinchilla equivalent of LL-37, cCRAMP, which has bactericidal activity against the three major otopathogens [in vitro and in vivo models are required [Our finding, that a greater inflammatory cytokine response was associated with bacterial otopathogen detection, particularly NTHi, is supported by several studies. Firstly, in adults with chronic obstructive pulmonary disease (COPD), higher levels of LL-37, IL-8, TNFα and greater inflammatory infiltrate were found in sputum with culturable NTHi compared to culture-negative sputum [n sputum . Thirdlyathogens . Finallyathogens . Togetherequired , 39, 40.S. pneumoniae OM resulted in more potent middle ear granulation tissue formation [In vitro studies have demonstrated that NTHi induces the secretion of IL-1α by human middle ear epithelial cells and that this acts synergistically with NTHi to upregulate HBD2 mRNA transcription [et al., suggest TGF-β is more important in epithelial remodelling after resolution of inflammation [Previous studies have implicated the TGF-β pathway and IL-1α actions in OM pathogenesis. In rat models of OM, microarray experiments have revealed that bacterial OM rather than Eustachian tube obstruction drives TGF-β signalling. Furthermore, NTHi OM rather than ormation . In a laormation . In vitrcription . Surprisammation .The higher concentrations of AMPs and inflammatory cytokines associated with bacterial otopathogen detection in our study may be indicative of more severe or persistent disease and the presence of non-resolving NETs . AlthougWhether elimination of bacterial otopathogensfrom the middle ear of children could be achieved through application of AMP doses that exceed physiological levels through ventilation tubes as an adjunct to traditional antibiotic treatment remains elusive. Preclinical models have provided critical proof-of-concept data for the therapeutic application of AMPs. Synergy between synthetic AMPs and antibiotics have been more effective than antibiotics alone for reducing the severity of cutaneous abscesses in a murine infectious abscess model . In chinIn conclusion, we have provided the first details on the presence and biological range of AMPs that are present in MEE from children with a history of rAOM. We have also shown that AMPs, pro-inflammatory cytokines and the regulatory cytokine IL-10 are associated with presence of bacterial otopathogens in the middle ear. To reduce the global burden of recurrent and chronic OM, new therapies that focus on eradicating bacterial otopathogens from the middle ear to reduce inflammation are urgently needed. Whether this can be achieved through the therapeutic use of AMPs requires further research, but our data provide important insight into the physiological concentrations of AMPs which may need to be exceeded in a therapeutic setting.We acknowledge that our study has some minor limitations predominantly due to sample size and sample volume constraints. Due to the rarity of this sample type and volume needed for immune and microbiological assays, there were small numbers in this study. This may have been a contributing factor to the similar levels of TGFβ observed between groups when numbers were further restricted. Additionally, due to volume limitations, we did not quantify immune cells, specifically neutrophils, in MEE from children in this study. However, we have previously identified neutrophil infiltrate and NETs in MEE from other children in this cohort , thus, h


Centaurea scabiosa and Inula salicina placed at different heights above ground in two types of surrounding vegetation: short and tall. Even at such a small-scale, we detected significant shift in total visitation rate of inflorescences in response to their vertical position. In short vegetation, inflorescences close to the ground were visited more frequently, while in tall vegetation, inflorescences placed higher received more visits. Moreover, we found major differences in the composition of the pollinator community on flowers at different heights. In a second experiment, we measured flower visitation rate in inflorescences of Salvia verticillata of variable height. Total flower visitation rate increased markedly with inflorescence height in this case. Data on seed set of individual plants provide evidence for a corresponding positive pollinator-mediated selection on increased inflorescence height. Overall, our results demonstrate strong vertical stratification of plant–pollinator interactions at the scale of mere decimetres. This may have important ecological as well as evolutionary implications.Visitation of plants by different pollinators depends on individual plant traits, spatial context, and other factors. A neglected aspect of small-scale variation of plant–pollinator interactions is the role of vertical position of flowers. We conducted a series of experiments to study vertical stratification of plant–pollinator interactions in a dry grassland. We observed flower visitors on cut inflorescences of  Interactions between plants and their pollinators play an important role in the evolution  and mainMany plants show high levels of phenotypic plasticity. Inflorescence height is thus highly variable at the intraspecific level and may be important in driving visitation of individual plants. However, the importance of inflorescence height is little understood in grasslands, where the vertical distance between different flowers is rarely more than a few decimetres. In communities of multiple plants species, a few observational studies found that different bee species tend to visit flowers at different heights . In addiDactylorhiza lapponica plants in tall vegetation, while there was no significant selection on plant height in short vegetation. Similarly, in field experiments with Primula farinosa, P. farinosa plants increased their fruit and seed production apparently because of an increase in their nutritional status rather than increased pollination. The role of vegetation height for pollination thus remains unclear. These studies also looked at the topic entirely from the plant’s point of view and did not measure the effects of vegetation height on visitation frequency or pollinator foraging behaviour. Apart from vegetation height, local density of the same or other plant species . The study site is a species rich calcareous grassland on a southwest-facing slope managed by occasional pasture by cows and sheep. The area is state-owned and publicly accessible. No permits were needed for this study.Centaurea scabiosa and Inula salicina, at different heights above ground. To avoid confounding factors, e.g. taller plants having a different size of inflorescences than shorter plants, we used inflorescences cut from plants in the local population. We selected inflorescences of a similar size and general appearance and placed them in 15 ml tubes with water. We attached each tube to a bamboo stick of different length and attached the stick to the ground. This way, we manipulated the height of the inflorescence between 5 and 105 cm above ground. We placed the inflorescences along two short transects, each containing seven inflorescences placed 50 cm apart. One transect was surrounded by short and the other by tall and dense vegetation; the transects were ca. 10 m apart. The area of short vegetation was grazed by cows in the spring, while the area of tall vegetation was not managed. Short vegetation was characterized by most plants <10 cm tall; the average height of inflorescences of all plants growing within 50 cm from the transect in all directions was 7.2 cm (SD = 6.00). Tall vegetation was composed of a dense layer of plants reaching ca. 50 cm; plants growing within 50 cm from the transect had flowers on average 50.1 cm above ground (SD = 14.73). We individually placed seven inflorescences of either C. scabiosa or I. salicina in each transect at 5, 15, 25, 45, 65, 85, and 105 cm above ground in a randomized order.In the first experiment, we observed visitation of inflorescences of two plant species, I. salicina, 10 in short and 10 in tall vegetation, and 16 transects in C. scabiosa, eight in short and eight in tall vegetation. In total, this amounts to 18 h of observations.We observed visitation of the inflorescences by insects between 10:30 and 16:00 h, for 30 min in each transect, and identified all visitors at the species level or classified them into taxonomical groups with the highest precision we could achieve without capturing the insects. Both transects were observed simultaneously, one person observed each transect. After the 30 min period, we replaced the inflorescences and randomized the order of their vertical position along each transect and took another set of observations. In total, we measured inflorescence visitation in 20 transects in S. verticillata at the same site. In this case, we did not cut the inflorescences so that we could test whether flower visitation rate varied between inflorescences within the natural limits of their height above ground and to test whether variation in flower visitation rate translated into differences in seed set; i.e. whether female fitness was affected by the vertical position of the inflorescence. We used the following approach to minimize confounding effects, such as taller plants having more resources, different display size, etc. We selected 17 plants of S. verticillata with multiple ramets of approximately the same size and with the first several flowers open or with buds ready to start flowering on 18 July 2017. We took advantage of the fact that the inflorescences grow on relatively long and flexible stems. We bent one of them close to the ground, where it was attached to a stick so that the bottom of the inflorescence was positioned just above ground. We made sure that the orientation of the inflorescence remained unchanged. The second stem was attached to another stick so that it reached a maximum height and the third inflorescence was positioned at an intermediate height. This way, the only difference between the inflorescences was their vertical position.In the second experiment, we focused on the effect of inflorescence height for flower visitation rate and its consequences for seed set in S. verticillata plants at one of three dates , depending on when they reached the peak of flowering. The three manipulated ramets per plant were observed simultaneously during one 30 min period and filmed using three digital cameras, which gave a total of 25 h of recordings. Afterwards, we measured the height of each inflorescence as a distance of the highest open flower from the ground and counted the number of open flowers. We then watched the recordings and counted and identified all flower visitors. For each visitor, we also counted the number of flowers visited during each inflorescence visit.We performed observations of flower visitation in individual S. verticillata to ripen and then harvested them on 14 or 24 August 2017 depending on seed development in individual plants. We counted the number of developed seeds and the maximum potential seed set by multiplying the number of flowers by four, which is the number of seeds the plant can produce per flower. We counted the flowers and seeds in individual whorls within each inflorescence separately to gain data on potential differences in percentage seed set along the inflorescence from the lowest to the highest whorl.We waited for the seeds of Bombus spp., some of which are difficult to identify alive. Fortunately, we have extensive collections from the study site, so we know that there are three species, which we could not distinguish from Bombus terrestris, specifically B. lucorum, B. cryptarum, and B. magnus. However, over 85% of individuals of this species group in our collections from this site belong to B. terrestris. Similarly, B. lapidarius could be confused with B. confusus and B. ruderarius, but they have rarely been found on the site. A similar level of uncertainty exists in our identification of B. sylvarum. The number of potential bumblebee misidentifications during the field observations was thus low and unlikely to confound our results.As already mentioned, we identified flower-visiting insects without capturing them. Naturally, we could not identify all individuals to the species level, so we classified some of them into higher taxa or categories, such as ‘small solitary bees.’ The most abundant flower visitors were bumblebees, S. verticillata; i.e. generalized additive mixed models (GAMM) were used in this case. Poisson distribution with overdispersion (quasi poisson distribution) was used for the number of visits, while overdispersed binomial  distribution was used for data on proportions. These analyses were performed using mgcv 1.8-17 package  to account for the non-linear nature of these relationships. The identity of individual plants, each having three ramets manipulated and observed, was included as a random factor in analyses of data from the experiment with  package  in R 3.2 package .To gain insights into the effects of inflorescence height on plant fitness, we tested how seed set of individual ramets depended on the number of flowers and the inflorescence height using a generalized linear model. Similarly as in analyses of selection gradients , we stanC. scabiosa and I. salicina  and I. salicina . Comparison of the results presented in C. scabiosa and 286 visits (13 taxa) on I. salicina.In the first experiment, we observed a strong, mostly non-linear, dependence of the total visitation rate on inflorescence height in both salicina . Analysisalicina  showed tgetation . Also, tC. scabiosa, we observed a significant effect of inflorescence height on the number of inflorescence visits by B. lapidarius, which preferred mostly inflorescences close to the ground . On the contrary, small solitary bees did not shift their visitation  visited mostly inflorescences >40 cm above ground and the height of the surrounding vegetation had no effect on the relationship between the number of visits and inflorescence height . On the other hand, small solitary bees favoured inflorescences close to the ground in short vegetation and shifted higher above ground in tall vegetation  , followed by B. sylvarum  . Apis mellifera, which was excluded from the previous analyses because it was too rare, visited on average over 60% of flowers during one inflorescence visit and the three bumblebee species over 40%. On the contrary, other visitors, which we classified as small solitary bees, Syrphidae, and other Diptera, visited less than 20% of the flowers per inflorescence visit  ; Table 3rrestris ; Table 3ce visit .F = 6.21, P = 0.0165; F = 6.09, P = 0.0175; F = 1.55, P = 0.2201), while seed set significantly increased with the number of visits , whose height was experimentally adjusted. Vertical position was thus the only apparent difference between the inflorescences. This is important because taller plants usually have higher percentage seed set even when they are hand-pollinated because they have more resources than shorter plants . TestingA. mellifera) visited a majority of flowers within an inflorescence by moving along the inflorescence and probing one flower after another. The three bumblebee species exploited slightly lower percentage of flowers, while small solitary bees and Diptera usually probed only a few flowers per inflorescence (S. verticillata, we would expect higher level of geitonogamous pollination in inflorescences higher above ground because of shifts in the flower visitor community (see Flower visitation by different insect species can have different effects on plant fitness not only because different species differ in the number of pollen grains deposited per visit , but alsrescence . Differerescence . High lerescence . In our nity see , perhapsS. verticillata. However, in other plant species, florivory and seed predation can have a large negative effect on plant fitness (Inflorescences which are more attractive for pollinators may also attract higher numbers of florivores and seed predators . Seed se fitness . For exaS. verticillata, we also observed increased seed set with inflorescence height, which supports the hypothesis of selection for increased inflorescence height. Overall, we detected pronounced vertical stratification of plant–pollinator interactions at a scale of mere decimetres in a temperate grassland.In conclusion, we experimentally demonstrated that both total flower visitation and the composition of the community of insect visitors changed with the vertical position of inflorescences in three common plant species growing in a dry grassland. Moreover, we found that the dependence of visitation rate on inflorescence height was mediated by the height of the surrounding vegetation. In one species, 10.7717/peerj.4998/supp-1Supplemental Information 1The number of inflorescence visits on individual plants in experimental transects surrounded by short or tall vegetation is shown for each flower visitor species or group.Click here for additional data file.10.7717/peerj.4998/supp-2Supplemental Information 2Total number of visitors arriving in each inflorescence during one recording period of approximately 30 minutes is provided for three ramets per plant differing in inflorescence height. Total number of flower visits during the recording period is also provided because most visitors probed multiple flowers per inflorescence visit.Click here for additional data file.10.7717/peerj.4998/supp-3Supplemental Information 3Identity of a flower visitor and the number of flowers probed is provided for each inflorescence visit on individual plants. Three ramets per plant were observed and can be distinguished by different inflorescence height.Click here for additional data file.10.7717/peerj.4998/supp-4Supplemental Information 4Salvia verticillata plant. Three ramets differing in inflorescence height were used in each plant. Inflorescences were composed of the main stem and usually 1-2 side branches; which were counted separately.The number of flowers and the number of developed seeds was counted in individual whorls in inflorescences of each Click here for additional data file.


Background: Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by degeneration of spinal motor neurons leading to muscular weakness. The antisense oligonucleotide nusinersen was approved for the treatment of patients with 5q-associated SMA. Treatment must be repeatedly administered intrathecally by lumbar puncture. So far, data regarding cerebrospinal fluid (CSF) parameters are sparse and examinations of CSF cytology during nusinersen treatment are completely missing.Methods: 87 CSF samples from 19 adult SMA patients who underwent repeated lumbar punctures for intrathecal injections of nusinersen were investigated. CSF specimens were quantitatively assessed regarding leukocyte subpopulations by routine cytology after Pappenheim staining. A control group with 38 CSF samples from 10 patients with repeated lumbar punctures due to other diseases was used.Results: Treatment with nusinersen did not result in persistent inflammatory cellular changes or a relevant shift of leukocyte subpopulations in the CSF. During nusinersen therapy unique macrophages with numerous sharply defined purple and granular inclusions were detected in all patients. These macrophages were not found in CSF of patients with other diseases who underwent repeated lumbar punctures.Discussion: Routine CSF cytology performed by experienced personnel represents an important and feasible tool for safety monitoring during treatment with intrathecally administered therapeutics. Analysis of leukocyte subpopulations did not raise safety concerns during nusinersen therapy. The potential significance of the unique phagocytic cells for disease course and treatment response needs to be further elucidated in the future. SMN1) gene on chromosome 5q13  is an autosomal recessive neurodegenerative disorder characterized by degeneration of lower motor neurons in the spinal cord with subsequent progressive muscular atrophy and weakness . The disome 5q13 . The antenotypes . During enotypes , 8. Aparenotypes  and the enotypes , data ren = 8), meningitis (n = 1) or chronic inflammatory CNS disorder (n = 1) served as control group. Median age was 35 years, ranging from 28 to 61 years. This investigation was approved by the Ethics Committee of Hannover Medical School (No. 3142-2016).Routine CSF cytology specimens of adult patients with genetically proven 5q-SMA who were treated with nusinersen at the Department of Neurology at Hannover Medical School between November 2017 and January 2019 were analyzed. In total, we collected 87 CSF samples from 19 adult patients with SMA  who underwent repeated lumbar punctures (ranging from three to seven) for intrathecal injections of nusinersen on days 0, 14, 28, 63, and at month 6, 10, and 14. Median age was 35 years, ranging from 19 to 64 years. Seven female and 12 male patients were included into the analysis. 38 CSF cytology samples from mostly age-matched patients who underwent repeated lumbar punctures at least twice within 6 months between 2016 and 2019 with diagnosis of idiopathic intracranial hypertension (IIH) , prussian blue staining for iron was performed regularly. Dried cells were immersed in a solution of 2% potassium hexacyanoferrate and 1% hydrochloric acid  followed by a counterstaining with nuclear fast red aluminum sulfate solution  (CSF was drained prior to administration of nusinersen and was subjected to standard diagnostic procedures within 1 h after lumbar puncture in the neurochemistry laboratory at the Department of Neurology as described previously . BrieflyGermany) .Specimens were independently examined by two raters experienced in CSF cytology who were blinded to condition and time point. In samples from SMA patients, the following leukocyte populations were assessed quantitatively: lymphocytes, plasma cells, monocytes, macrophages, erythrophages, erythrosiderophages, siderophages, neutrophils, eosinophils, and basophils. Cartilage and bone marrow cells were present in some specimens but were not analyzed. In cases where raters either disagreed on the presence or absence of any cell type or the percentage of lymphocytes and/or monocytes differed by more than 10% between the two raters' assessments, the specimen was reviewed and discussed by both raters together. After consensus was reached and data were corrected accordingly, mean values of both raters' assessments were calculated for each specimen and cell population. Specimens from repeatedly punctured patients with other diseases were assessed as a control. Prussian blue stainings were analyzed regarding iron containing siderophages. Specimens were examined using an x40 objective .All statistical analyses were performed using GraphPad Prism 8.0 . Data are given as arithmetic means ± standard error of the mean (SEM).At the time of first lumbar puncture before treatment with nusinersen, all patients showed a normal CSF leukocyte cell count (<4 cells/μl). Evaluation of all 87 CSF samples revealed mild pleocytosis (ranging from 5 to 21 cells/μl) in 4 specimens which did not persist at the subsequent lumbar punctures . The proBeginning with the second lumbar puncture, macrophages with sharply defined purple and blue granular inclusions were detected whereas these cells were not present in any specimen of the first lumbar puncture before treatment with nusinersen . These mThese cells, which we termed “nusinophages” for convenience, were characterized by numerous sharply demarcated inclusions varying in size and in color between purple and blue . PrussiaThe antisense oligonucleotide nusinersen (Spinraza®) must be repeatedly administered intrathecally for the treatment of patients with 5q-associated SMA. Monitoring of potential alterations of CSF parameters and especially changes of the cellular composition of the CSF is crucial.Here, we systematically analyzed leukocyte subpopulations in adult SMA patients treated with nusinersen. Since no evidence for sustained inflammatory cellular reactions or a relevant shift of the cellular composition in the CSF was found, cytological analysis did not reveal safety issues of nusinersen treatment in adult patients with SMA. These findings complement the extensive clinical data demonstrating safety and feasibility of nusinersen therapy , 13–15.Interestingly, during the treatment with nusinersen unique macrophages with numerous sharply delineated blue and purple inclusions were detected in all patients. These cells were not found in patients who had undergone repeated lumbar punctures due to other diseases and we are not aware of the existence of similar CSF macrophages in other conditions. However, different intervals between the lumbar punctures of SMA patients and control group patients represent a limitation of this comparison. Since stainings for oligonucleotides in an infant treated with nusinersen indicated the presence of nusinersen in neuronal and non-neuronal cells in brain and peripheral tissue  we specuIn summary, cells with unique inclusions were detected in the CSF of patients after repeated administration of nusinersen. Further investigations are needed to clarify whether these inclusions might contain nusinersen and if these cells may influence the response to therapy. We suggest that analysis of CSF parameters should supplement clinical measures of safety and therapy response during treatment with intrathecally delivered drugs.All datasets generated for this study are included in the manuscript and/or the This study was carried out in accordance with the recommendations of the institutional ethics committee of Hannover Medical School with written informed consent from all subjects (No. 3142-2016). All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the institutional ethics committee of Hannover Medical School.SG, MH, SA, LB, and TS evaluated the specimens. SG, MH, SA, KJ, PS, K-WS, LM, AO, OS-K, MB, MS, SP, and TS analyzed the data. SG, MH, SA, KJ, MS, SP, and TS drafted and wrote the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.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.


Parkinson’s disease (PD) is the second most common neurodegenerative disorder in elderly people. Currently, the diagnosis of PD is based on neurological examination, neuroimaging, and the response to dopaminergic medication. The diagnosis can be challenging, especially at early disease stages, when the symptoms of patients with atypical parkinsonism (APD) may strongly overlap. Therefore, reliable biomarkers that are able to identify patients with PD are much needed. Here, we aimed to identify and validate new biomarkers for PD in cerebrospinal fluid (CSF). We performed a profiling experiment using mass spectrometry (MS) of CSF from ten PD patients and ten matched non-neurological controls. We selected one protein, galectin-1 , which was differentially expressed in PD vs. controls, and quantified its concentrations in CSF by enzyme-linked immunosorbent assay (ELISA) in three new cohorts of 37 PD patients, 21 APD patients, and 44 controls. CSF levels of Gal-1 were lower in PD in both the discovery and validation experiments and discriminated PD from controls with moderate–high accuracy levels (ELISA: area under the curve = 0.7). Similar levels of Gal-1 were found in PD and APD. Gal-1 levels were correlated to age in all groups and correlated in the PD patients to CSF levels of total tau, phosphorylated tau, neurofilament light chain (NFL), and the mini-mental state examination (MMSE) score. We conclude that MS profiling of proteins may be a useful tool to identify novel biomarkers of neurological diseases and that CSF Gal-1 levels may discriminate PD from non-neurological controls.The online version of this article (10.1007/s12035-018-1426-9) contains supplementary material, which is available to authorized users. Parkinson’s disease (PD) is one of the α-synucleinopathies and the most prevalent neurodegenerative movement disorder. Accumulation of α-synuclein (α-syn) in dopaminergic neurons in PD leads to neuronal death causing motor and non-motor dysfunction. PD motor symptoms include bradykinesia, muscular rigidity, impaired balance, and resting tremor. Non-motor symptoms include sleep disorders, olfactory dysfunction, autonomic dysfunction, and cognitive impairment , 2.PD diagnosis is based on neurological evaluations, neuroimaging, and the response to dopaminergic medication following the current international clinical criteria , 4. HoweCerebrospinal fluid (CSF) is the body fluid that is closest to the brain, and its composition may reflect the  processes in the brain. Several proteins that were already known to be involved in PD have been investigated in CSF as potential biomarkers for diagnosis, disease progression, or cognitive decline, such as α-syn, neurofilament light chain (NFL), DJ-1, tau, and amyloid β42 (reviewed in ). A consWe aimed to identify new CSF biomarkers that have the potential to discriminate PD from controls. For this purpose, we selected PD patients from a unique cohort of patients with uncertain diagnosis of parkinsonism at presentation, which is very representative of the daily situations when clinicians are confronted with patients with the suspicion of a movement disorder at the first visit to a specialist. We performed a profiling experiment by mass spectrometry in CSF of ten PD patients and ten non-neurological controls. We selected the protein galectin-1 , which was differentially expressed in PD vs. controls, for further validation studies, using an enzyme-linked immunosorbent assay (ELISA).We have selected CSF samples from ten PD and ten non-neurological control patients for the discovery experiment matched for sex and age. For the validation experiment, we have included CSF from 37 PD patients, 21 APD patients , and 44 non-neurological controls. Samples were matched for sex in all groups and for age in PD and controls, since age was higher in the APD group. We used separate cohorts of patients for the discovery and validation phases of the study, with the exception of only one PD CSF sample, which was used for both discovery and validation experiments. CSF samples for the discovery experiment were also selected based on low number of leukocytes (0–5 cells per μL) and erythrocytes (≤ 200 cells per μL). Demographic characteristics for both cohorts are shown in Table PD and APD patients for the discovery and validation cohorts were selected from a longitudinal study performed at the Radboud University Medical Center, previously described in detail  . These cThe non-neurological control group consisted of patients who were referred to our center with a suspicion of neurological disease, but after extensive neurological examination had no neurological disorder and were diagnosed with other disorders, such as non-neuronal sarcoidosis, diabetes, radiculopathy, or headache. Their CSF did not show any abnormality for the following parameters: cell count, glucose, total protein, lactate, hemoglobin, bilirubin, and oligoclonal IgG bands.CSF samples of PD, APD, and non-neurological controls were collected in polypropylene tubes, centrifuged, aliquoted, and stored in polypropylene tubes at − 80 °C until experiments. All participants provided written informed consent, and the study was approved by the local institutional review board Arnhem-Nijmegen.Total protein concentration in CSF was determined using the 2D Quant kit , according to the manufacture’s protocol, and 400 μg of total protein for each sample was used as input for the discovery experiment. CSF samples were applied to an affinity removal column for depletion of 14 most abundant proteins  to enrich low-abundant proteins.Samples were diluted in 8 M urea to denature proteins prior to reduction in 10 mM dithiotreitol for 20 min at room temperature and alkylation with 50 mM chloroacetamide for 20 min at room temperature in the dark. Proteolytical digestion was performed by a first incubation with LysC protease for 3 h at 37 °C after which the sample was diluted 1:3 with 50 mM ammonium bicarbonate prior to overnight incubation with trypsin at 37 °C. Peptides were concentrated and desalted using C18 Omix tips (Agilent Technologies), eluted in 20 μL 80% acetonitrile, and dried using a SpeedVac centrifuge at 45 °C. Peptides were suspended in ammonium hydroxide buffer (pH 10) and subsequently fractionated using C18-reversed phase liquid chromatography . Peptides were eluted from the column at 100 μL/min using a 15-min linear gradient of 5 to 45% acetonitrile adjusted to pH 10 with ammonium hydroxide. The 20 collected fractions were adjusted to pH 2.7 using formic acid prior to LC-MS/MS analysis.m/z, 2-Hz spectrum acquisition rate) followed by six data-dependent MS/MS experiments acquired at intensity scaled spectral acquisition rates . Only precursor ions in the range of 400–1400 m/z with charge state of z = 2+ or higher were considered for collision-induced dissociation experiments with dynamic exclusion set to 2 min.Samples were analyzed by nanoflow liquid chromatography  connected online to an ultra high-resolution quadrupole time-of-flight tandem mass spectrometer  via an axial desolvation vacuum-assisted electrospray ionization source . Peptides were loaded onto the trapping column  at 10 μL/min with 0.1% formic acid using two loop volumes of solvent (20 μL). Peptides were eluted from the analytical column  using a 20-min linear gradient of 5 to 35% acetonitrile in 0.1% formic acid at 600 nL/min. The mass spectrometer was operated in positive ion mode for data-dependent MS/MS acquisition. Each AutoMSn duty cycle consisted of one full MS spectrum  and should be quantified in at least 50% of the samples of any group.Raw MS data files were subsequently analyzed by MaxQuant software version 1.5  using thGal-1 was quantified in CSF by using a commercial ELISA , according to the company’s protocol, with the exception of prolonged incubation times, as suggested by the company. Biotinylated anti-human galectin-1 antibody was incubated for 90 min, avidin–biotin–peroxidase complex (ABC) for 50 min, and color-developing agent for 30 min. CSF was diluted three times, and all samples were analyzed in duplicate.We performed a partial validation of the ELISA according to previous recommendations , 21. TheThe recovery was evaluated by spiking two different concentrations (6.7 ng/mL and 4.5 ng/mL) of Gal-1 recombinant protein in three times diluted CSF samples or in sample buffer. The percentage of spiked recombinant protein that was recovered from CSF was calculated. Values between 80% and 120% were considered satisfactory.Precision was determined based on (1) intra-assay variation by measuring five diluted CSF samples in four replicates within one plate, and (2) inter-assay variation by measuring three diluted CSF samples in duplicate at identical positions in five different plates on five different days. The mean coefficient of variation (CV) was calculated; a CV below 20% was considered satisfactory.p = 0.004). Total protein concentration in CSF was measured by turbidimetric benzethonium chloride method using a Cobas 8000 instrument  for automated measurement.CSF Gal-1 concentrations in the validation cohort were normalized by CSF total protein concentration to correct for a small significant difference of total protein concentration between PD (mean 530 ± 182 mg/L), APD (mean 591 ± 273 mg/L), and controls  or Student’s t test (for parametric data) was used when data of only two groups were available. Analysis of covariance was performed with age and intake of dopaminergic medication as confounding factors. Correlations and partial correlations, with age and total protein concentration as covariates, were investigated by Spearman’s test. Receiver operating characteristic (ROC) curve was constructed to determine the diagnostic accuracy by the area under the curve (AUC) and the Youden index for the optimal cutoff values of sensitivity and specificity.Data analysis was performed using IBM SPSS Statistics 22  and GraphPad Prism 5 . Differences between groups were determined by ANOVA followed by Bonferroni post hoc test for normally distributed data and Kruskal–Wallis test followed by Dunn’s post hoc test for not normally distributed data. Mann–Whitney p < 0.05) (Table p = 0.02)  were considered for further analysis, resulting in 482 proteins to be analyzed. Among these, 32 proteins were present at significantly different levels in PD and controls (5) Table . More de02) Fig. a.Fig. 1CThe performance of the ELISA assay was as follows. The detection limit was determined at 460 ng/L. The recovery of recombinant Gal-1 protein spiked in CSF was considered satisfactory with CV ranging from 85 to 118%. Both the intra-assay and inter-assay variation were considered satisfactory with a mean CV% of 4% (± 0.01) and 14% (± 0.05), respectively.p = 0.02). Total protein concentration was lower in PD and higher in APD patients than in controls (p < 0.001); therefore, Gal-1 levels were corrected for the total protein level. Gal-1 levels were lower in PD  compared to non-neurological controls . The difference in Gal-1 levels between the PD and controls remained significant after correction for age and intake of dopaminergic medication (p = 0.013). CSF Gal-1 levels in PD/APD patients were similar when they were either on dopaminergic medication or not at the time of lumbar puncture (p = 0.15). CSF Gal-1 levels in the PD group were positively correlated to CSF concentrations of total tau , phosphorylated tau , and NFL , but α-syn levels were not significantly correlated to Gal-1 . CSF Gal-1 levels correlated to MMSE scores determined at 3 years of follow-up . No significant correlations between Gal-1 and other clinical parameters were observed. Partial analysis, taking age and total protein concentration as covariates, confirmed the correlations of Gal-1 in the PD group with total tau, phosphorylated tau, but not with NFL or MMSE. Correlation of Gal-1 levels to disease duration in the APD group was also retained.Age was significantly different between groups due to older patients in the APD group . OverexOne limitation to our study may be the relatively small number of APD samples available for this study, which might have underestimated the power of Gal-1 to discriminate PD from APD. However, the design of our prospective study did not allow for an inclusion of a larger number of APD patients. Further confirmation in larger, independent, cohorts remains necessary.Another limitation is that our CSF samples were stored for a long period since its collection, between 2003 and 2006, and long-term storage may affect protein stability. Protein levels are generally low in CSF, and differences in sample processing could affect the results of mass spectrometry and ELISA analysis. However, we followed international guidelines for CSF collection and storage that were described later in a consensus paper . PreviouAn important strong aspect of our study is that, although the number of parkinsonism patients we included in this study is moderate, they were selected from a unique longitudinal study. Unlike many other biomarker studies, which use highly selected cohorts of patients with clinically undisputable diagnosis, this study included patients with uncertain diagnosis at baseline, closely representing the daily situation for clinicians when they are confronted with a patient suspected of a movement disorder . In thesIn summary, we successfully profiled proteins present in CSF of PD and non-neurological controls by mass spectrometry. Among the proteins that were differentially expressed in PD vs. controls, Gal-1 was selected as a potential biomarker for PD. Our validation experiment for Gal-1 confirmed our findings from the discovery study, indicating that mass spectrometry profiling of proteins in CSF may be a useful tool to indentify novel biomarkers of neurological diseases, but this validation also demonstrated that CSF Gal-1 levels were similar in PD and APD. Furthermore, the correlations of Gal-1 with both NFL and tau suggested that Gal-1 may be involved in axonal function; however, further studies should clarify this association.ESM 1(DOCX 18 kb)


Colony-stimulating factor-1 (CSF-1) is a homodimeric glycoprotein. The main role of CSF-1 is as a hematopoietic growth factor that modulates proliferation, differentiation, and survival of macrophages. Moreover, CSF-1 has also been reported to be aberrantly expressed in several human cancers. However, the precise role of CSF-1 in upper tract urothelial carcinomas (UTUC) has not been studied. In this research, we examined the clinical significance of CSF-1 expression in UTUC.  One hundred twelve cancer tissue samples of UTUC from patients were included in this study, and the other cohort of 35 UTUC were paired cancer-adjacent normal samples. CSF-1 expression was evaluated by immunohistochemistry, and the association of CSF-1 expression with different clinicopathological variables was analyzed. P = 0.005). The CSF-1 expression was primarily localized in the nucleus and was significantly correlated with tumor size (P = 0.04) and patients who had a high stage (P < 0.001), distant metastasis (P = 0.006), recurrence (P = 0.003), and cancer death (P = 0.005). High CSF-1 expression was correlated with poor disease-free survival (P = 0.008) and cancer-specific survival (P = 0.001). Our results also used univariate and multivariable analyses, which found that high CSF-1 expression was an independent predictor of poor disease-free survival  and cancer-specific survival .  CSF-1 expression was higher in UTUC than in the normal urothelium ( Our findings indicate that the expression of CSF-1 is a potential prognostic marker for predicting patient survival and recurrence in UTUC. Upper t(HIF-1α) , leptin (HIF-1α) , and sig(HIF-1α)  associatColony-stimulating factor-1 (CSF-1), also called “macrophage colony-stimulating factor” (M-CSF), is an important hematopoietic growth factor. CSF-1 binds to its receptor—the colony-stimulating factor-1 receptor (CSF-1R/c-fms)—and regulates the survival, differentiation, and proliferation of the monocyte-macrophage lineage , 17. AddBecause there is no published research investigating the role of CSF-1 in UTUC, we aim to examine the association between the clinicopathological behavior of UTUC and CSF-1 expression in cancer tissues.One hundred twelve formalin-fixed UTUC tissues and thirty-five paired noncancerous urothelial samples were obtained from the Department of Urology, Kaohsiung Medical University Hospital, from 1997 to 2006 as previously described , 15. AllFour-micrometer-thick sections from paraffin-embedded blocks were cut onto precoated slides, followed by deparaffinization, rehydration, and antigen retrieval as previously described , 15. EndScoring for CSF-1-positive staining was decided based on the percentage of positively stained cells in 4 quantitative categories as previously described , 15: scoP values less than 0.05 were regarded as statistically significant.All statistical analyses were executed using the SPSS statistical package for PC  as previously described . As a re2. UM-UC-14, a human transitional cell carcinoma of the renal pelvis, was purchased from the European Collection of Authenticated Cell Cultures (ECACC). This cell line was cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS, 2 mM glutamine, 1% nonessential amino acids (NEAA), and antibiotic-antimycotic and incubated at 37°C, 5% CO2.BFTC909, a human renal pelvis transitional cell line , was pur2. Immunofluorescence was performed using the Image-iT™ Fixation/Permeabilization Kit (Invitrogen™). We removed the culture medium from the cells and then performed cell fixation, permeabilization, and a blocking procedure per the manufacturer's protocol. After blocking, we aspirated the blocking solution and incubated the cells with an anti-CSF-1 monoclonal antibody (M-CSF Antibody (D-4), sc-365779, Santa Cruz) at a 1 : 50 dilution in blocking solution at 4°C overnight. The cells were then incubated with fluorescein isothiocyanate- (FITC-) conjugated secondary antibody diluted in phosphate-buffered saline (PBS) for 1 h at room temperature (protected from light). Next, the cells were incubated with DAPI (Thermo Scientific™) diluted in PBS for 10 min at room temperature in the dark. Finally, cells were mounted by ProLong™ Gold Antifade Mountant (Thermo Scientific™) and observed using a fluorescence microscope.BFTC909 and UM-UC-14 cell lines were seeded in a 35 mm Glass Bottom Dish (ibidi) and incubated at 37°C, 5% COP = 0.005)  . Positiv= 0.005) . We also= 0.005) .n = 112) was examined by immunohistochemistry and categorized into four scores (quartiles). On the basis of the scoring, tumor tissues were further sorted into low  and high  CSF-1 expression groups . Next, we examined the CSF-1 expression for indication of correlation with different clinicopathologic characteristics including tumor stage, grade, gender, age, tumor location, tumor side, lymphovascular invasion, distant metastasis, recurrence, cancer death, and serum creatinine level. The correlations between these clinicopathologic variables and CSF-1 expression are listed in P < 0.001), distant metastasis (P = 0.006), recurrence (P = 0.003), and cancer death (P = 0.005).The expression of CSF-1 in UTUC tissues (n groups . We founP = 0.046) and CSF-1 expression  in univariate analysis (P = 0.007) . In the multivariate analysis, we found that cancer-specific survival was also related to CSF-1 expression . Next, we explored whether the CSF-1 expression in human UTUC tissue samples was correlated to disease-free survival and cancer-specific survival of patients using Kaplan-Meier survival analysis. Kaplan-Meier survival curves showed that higher CSF-1 expression correlated with a significantly lower disease-free survival (P = 0.008) and cancer-specific survival (P = 0.001)  . Univari= 0.007) . High tu= 0.001) .We offered the first evidence that high CSF-1 expression is a potential prognostic marker for predicting patient survival and recurrence of UTUC. First, the expression of CSF-1 was higher in UTUC tissues than in cancer-adjacent normal tissues. Second, positive staining of CSF-1 was mainly expressed in the nucleus. Third, a high level of CSF-1 positively correlated with tumor stage, tumor size, distant metastasis, and recurrence. Finally, CSF-1 expression was associated with poor disease-free and cancer-specific survival, and univariate and multivariate proportional hazard analyses indicated that it was also an independent prognostic biomarker for patients with UTUC.c-fms protooncogene [CSF-1 is a cytokine generated by different types of cells, and it regulates the biological functions of monocytes and macrophages, including cell proliferation, differentiation, and survival , 17, 31.oncogene . The CSFoncogene , 34. Stuoncogene . Interesoncogene . STAT3 ioncogene –38. Impooncogene . Based oIn this study, the immunohistochemistry analysis revealed that the staining of CSF-1 was primarily expressed in the nucleus, although previous studies indicated the staining position of CSF-1 was also in the cytoplasm of various cancer cells such as renal cell carcinoma, soft tissue sarcomas, and gastric cancer , 40, 41.High CSF-1 expression was found to be an independent predictor of poor survival rates in patients with UTUC. We hope our results will help determine the prognosis for UTUC patients and may also help indicate a plan for aggressive treatment.


In tuberculous meningitis (TBM), a higher pretreatment CSF bacterial load was associated with increased disease severity and host inflammation. The bacterial load can be used to predict new neurological events but not death. Owing to the divergent pathogenesis of TBM-associated neurological complications and deaths, strategies to reduce them may need reassessment. Mycobacterium tuberculosis load in the brain of individuals with tuberculous meningitis (TBM) may reflect the host’s ability to control the pathogen, determine disease severity, and determine treatment outcomes.The M. tuberculosis load in cerebrospinal fluid (CSF) specimens from 692 adults with TBM. We sought to understand the relationship between CSF bacterial load and inflammation, and their respective impact on disease severity and treatment outcomes.We used the GeneXpert assay to measure the pretreatment M. tuberculosis load was associated with increased disease severity , CSF neutrophil count (r = 0.364 and P < .0001), and cytokine concentrations (r = 0.438 and P < .0001). A high M. tuberculosis load predicted new neurological events after starting treatment  but not death. Patients who died had an attenuated inflammatory response at the start of treatment, with reduced cytokine concentrations as compared to survivors. In contrast, patients with high pretreatment CSF bacterial loads, cytokine concentrations, and neutrophil counts were more likely to subsequently experience neurological events.A 10-fold higher M. tuberculosis load may be a useful predictor of neurological complications occurring during TBM treatment. Given the evidence for the divergent pathogenesis of TBM-associated neurological complications and deaths, therapeutic strategies to reduce them may need reassessment.The pretreatment GeneXpert-determined  Mycobacterium tuberculosis to the brain, resulting in meningoencephalitis with necrotizing, granulomatous inflammation predominantly affecting the basal meninges. Inflammation can lead to life-threatening complications of hydrocephalus, infarcts, and tuberculomas ) was higher than in HIV-uninfected participants , although M. tuberculosis loads in these 2 groups were not significantly different (P = .087). Pretreatment GeneXpert CSF bacterial loads varied from very low to medium; a high load was not recorded. In the 404 HIV-uninfected participants, 276 (68.3%) had a negative test result, 63 (15.6%) had very low bacterial loads, 58 (14.4%) had very low bacterial loads, and 7 (1.7%) medium bacterial loads. In the 288 HIV-infected participants, 123 (42.7%) had a negative test result, 72 (25.0%) had very low bacterial loads, 74 (25.7%) had low bacterial loads, and 19 (6.6%) had medium bacterial loads . Specifically, higher bacterial loads were observed in individuals with more severe coma, and an increased M. tuberculosis load was associated with a lower GCS . The bacterial load (per 10-fold increase) affected disease severity in all participants. For all participants, the odds ratio (OR) was 0.71  for grade 1 versus grade 2 and 1.59  for grade 1 versus grade 3. For HIV-uninfected participants, the OR was 1.56  for grade 1 versus grade 2 and 1.84  for grade 1 versus grade 3. For HIV-infected participants, the OR was 1.0  for grade 1 versus grade 2 and 1.5  for grade 1 versus grade 3. The duration of illness before treatment was not associated with BMRC grade (P = .977) or M. tuberculosis load (P = .651).In addition, an increased M. tuberculosis load before treatment was related to inflammatory responses, we first explored the relationship between M. tuberculosis load and blood and CSF cell counts. Bacterial loads stratified by Ct values showed negative correlations with neutrophil numbers in blood and CSF specimens from both HIV-infected participants and uninfected participants  and with respect to HIV infection status .PCA was used to evaluate 10 baseline CSF cytokine concentrations, to identify correlated measurements that accounted for the variance of the data set. The first component, PC1, covered 71.4% variance of the cytokine profile, PC2 covered 6.4%, PC3 covered 4.9%, PC4 covered 4.4%, and other PCs covered less . In othend TNF-α . InteresLTA4H genotype was associated with CSF IL-1β, IL-2, and IL-6 concentrations, with low concentrations for genotype CC, intermediate concentrations for genotype CT, and high concentrations for genotype TT [LTA4H genotype and bacterial load and found that the M. tuberculosis load in participants with genotype TT was slightly lower than that in those with genotype CC (P = .051) or CT (P = .015), but the linear trend test showed that the LTA4H genotype was not associated with the M. tuberculosis load overall  or standard (n = 345) antituberculosis treatment. As previously reported, the intensive regimen was not associated with any improvement in any measure of treatment response or outcomes, including survival . During M. tuberculosis loads in the GeneXpert-positive data set  and compared the bacterial load between these 2 groups. Bacterial loads were not significantly different between early and late neurological events . The M. tuberculosis load was significantly associated with new neurological events in HIV-uninfected participants  and with a trend in HIV-infected participants . Results stratified by HIV infection lost statistical significance after adjustment for multiple testing. GCS was also strongly associated with new neurological events . These findings highlight that pretreatment CSF M. tuberculosis load can be used as a predictor, together with GCS, of new neurological events occurring during TBM treatment.The results  indicateP = .731 for the HIV-uninfected group, and P = .099 for the HIV-infected group; Death occurred in 192 participants (27.7%), with a median time to death of 39 days  after the start of treatment B. The baM. tuberculosis load was associated with more-severe disease at baseline and strongly predicted new neurological events after the start of treatment, but it did not influence 9-month mortality . Surprisingly, we found that the CSF M. tuberculosis load. Drugs with the potential to target neutrophils  may be An additional limitation of our study was that we could not follow the decline of bacterial loads after the start of treatment. Even before the start of antituberculosis treatment, CSF bacillary loads are very low. In our participants, pretreatment bacillary loads in CSF specimens were at least 100-fold lower than loads reported in sputum specimens from patients with pulmonary tuberculosis . FurtherIn summary, our data shed new light on the pathogenesis of TBM and suggest divergent mechanisms that lead to death and neurological events occurring after the start of treatment. Death from TBM is associated with an attenuated inflammatory response at the start of treatment, with reduced CSF leukocytes and cytokine concentrations, compared with those in survivors. In contrast, patients with high pretreatment CSF bacterial loads, cytokines, and neutrophils are more likely to experience new neurological events or paradoxical inflammatory complications. These findings may have important implications for the selection of future host-directed therapies.Supplementary MaterialClick here for additional data file.