In the case presented, a patient has an unexplained episode of hypertension during aneurysm clipping. Following the procedure, the patient was discovered to have bilateral thalamic infarctions unrelated to the vascular location of the aneurysm. After a review of the case, it becomes apparent that intracranial hypotension caused by lumbar over drainage of cerebrospinal fluid (CSF) is the likely cause of both the episode of intraoperative hypertension and the thalamic infarcts. It is often presumed that having an open dura protects against intracranial hypotension and subsequent herniation. We present this case to suggest that opening the dura might not be protective in all cases and anesthesiologists must pay particular attention to the rate of CSF drainage. Lumbar CSF drainage is a technique frequently employed during neurological surgery and it is important for anesthesiologists to understand the signs, symptoms, and potential consequences of intracranial hypotension from rapid drainage. Lumbar cerebrospinal fluid drainage (CSF) drainage is a commonly used approach during cerebrovascular and skull base neurosurgical procedures to aid in brain relaxation and surgical exposure. With a closed dura, one well-known complication of lumbar drainage is the abrupt increase in an aneurysm's transmural pressure gradient caused by acute CSF drainage which can lead to catastrophic rebleeding . A much A 64-year-old, 87 kg female presented for clipping of a 4 mm wide neck unruptured saccular anterior communicating artery aneurysm which was discovered incidentally during the evaluation of headaches and memory difficulties. Her past medical history was significant for remote breast carcinoma, hypertension, obstructive sleep apnea, and gastroesophageal reflux disease. She was a lifelong nonsmoker and notable preoperative medications included aspirin, furosemide, and propanolol. Preoperative imaging had no evidence of prior infarcts. After induction of anesthesia, a right internal jugular central venous catheter and radial arterial catheter were placed and maintenance of anesthesia was performed with a combination of 0.5 MAC sevoflurane, propofol 50 mcg/kg/min, and remifentanil 0.125 mcg/kg/min infusions. Mannitol 1 gram per kilogram for a total of 80 grams was administered. The neurosurgeon placed an 80 cm closed-tip, barium impregnated lumbar drainage catheter with a 0.7 mm inner diameter  at the L3-4 level prior to the start of surgery.The surgery commenced and was progressing unremarkably. Prior to opening of the dura, 20 mL of CSF was drained over 15 minutes per neurosurgeon request. After dural opening and during dissection of the aneurysm, the surgeon requested the lumbar drain to be opened allowing further drainage of CSF. After approximately 15 minutes, the blood pressure sharply increased over the course of 1 to 2 minutes from a baseline systolic blood pressure of 130 mmHg to over 205 mmHg with an associated decrease in heart rate from 60 bpm to 50 which resolved over the course of minutes. This acute hypertension was treated with a number of interventions including 250 mg propofol and 1 mcg/kg remifentanil boluses aimed at treating light anesthesia as well as a bolus of 7.5 mg labetalol. At the time of the hypertensive event, the propofol, remifentanil, and Sevoflurane dosing had been stable and unchanged for over an hour. There was also no change in the level of surgical stimulation at this time as the dura had been incised and the neurosurgeons were using the operative microscope to expose the aneurysm. After the aneurysm was successfully clipped, the lumbar drain was closed and the surgery was completed without further episodes of hypertension. A total of 60 mL of CSF was drained via the lumbar drain during the case in addition to the losses from the surgical field.At conclusion of the operative procedure, the patient remained comatose and unresponsive despite an hour in the operating room awaiting emergence from anesthesia. A postoperative head CT was obtained prior to transport to the intensive care unit which demonstrated mild cerebral edema and borderline inferior transtentorial herniation, but no significant hemorrhage or focal abnormalities. She was then transported to the Neurocritical Care Unit intubated and ventilated. Initial arterial blood gas analysis on arrival to the ICU did not reveal a cause to her delayed emergence . She did not receive any benzodiazepines during the case, but did receive 50 mcg of fentanyl on induction and 1 gram of levetiracetam in addition to the propofol, remifentanil, and Sevoflurane maintenance. Initial neurological exam demonstrated midline and equal 4 mm pupils which were reactive to light bilaterally. She withdrew to painful stimuli in all four extremities. On postoperative day (POD) 1, her mental status continued to be depressed with a Glasgow Coma Scale of 7T (E2:V1T:M4). That day, an MRI was obtained which revealed bilateral thalamic infarctions on the diffusion weighted imaging which can be seen in  Anaesthesia, John Barker notes that with the use of an 18-gauge malleable needle, “Drainage of cerebrospinal fluid is controlled by a gate clamp in order to avoid the intense arterial pressor response and cardiac arrhythmia which can occur with free flow” [The case presented describes a patient who had sudden and severe unexplained hypertension during aneurysm clipping without obvious changes in anesthetic depth or surgical stimulation, but did have an open and draining lumbar CSF catheter. Although the causality between CSF drainage and hypertension is not clear, there are two important features which argue for it as the etiology of the observations made. Firstly, this case demonstrates the phenomenon of systemic hypertension related to CSF drainage. Although not commonly seen with the relatively low flow rates of modern lumbar drainage catheters, it was well recognized with previous types of CSF drainage devices. In his 1975 letter to the editor ofee flow” . With raee flow” , 7. In oThis case illustrates an important point: dural opening may not protect against intracranial hypotension and subsequent Cushing's response as well as commonly thought. CSF over drainage should be included in the differential diagnosis of any unexplained hypertension in the presence of an open lumbar drainage catheter after other causes of hypertension such as aneurysm rupture and light anesthesia are excluded. It is also imperative to alert our neurosurgical colleagues if we notice a possible Cushing's response whether being from intracranial hypotension or other causes such as brain compression.Although this episode of hypertension cannot be definitively linked to brain sagging and transient transtentorial herniation, the evidence strongly suggests that it is the likely etiology and anesthesiologists should be aware of this phenomenon. With the utilization of endovascular treatment for cerebral aneurysms, future anesthesiologists will continue to have decreased exposure to open surgery and the nuances associated with it. It will be increasingly important to better describe in the literature these nuances where previous generations such as that of John Barker received from clinical practice.


There is currently a renaissance of interest in the many functions of cerebrospinal fluid (CSF). Altered flow of CSF, for example, has been shown to impair the clearance of pathogenic inflammatory proteins involved in neurodegenerative diseases, such as amyloid-β. In addition, the role of CSF in the newly discovered lymphatic system of the brain has become a prominently researched area in clinical neuroscience, as CSF serves as a conduit between the central nervous system and immune system.This article will review the importance of CSF in regulating normal brain development and function, from the prenatal period throughout the lifespan, and highlight recent research that CSF abnormalities in autism spectrum disorder (ASD) are present in infancy, are detectable by conventional structural MRI, and could serve as an early indicator of altered neurodevelopment.The identification of early CSF abnormalities in children with ASD, along with emerging knowledge of the underlying pathogenic mechanisms, has the potential to serve as early stratification biomarkers that separate children with ASD into biological subtypes that share a common pathophysiology. Such subtypes could help parse the phenotypic heterogeneity of ASD and map on to targeted, biologically based treatments. Until recently, it was thought that the main purpose of cerebrospinal fluid (CSF) was merely to provide protective cushioning of the brain, but new discoveries in the last 5 years have revealed that CSF plays a critical role in brain development and function, both prenatally and throughout the lifespan. It is now recognized that the two primary functions of normal CSF circulation are as follows: 1) delivery of growth factors and other signaling molecules necessary for healthy neural growth  delivery and (2) The CSF system originates in the first few weeks of gestation when the neural tube closes and is filled with CSF . As the 3 each day [3 of CSF at any given time [Throughout early development and the lifespan, the production of CSF needs to be continuously balanced by the corresponding absorption of CSF. The brain continuously produces CSF at a rate of 500 cmeach day  and thenven time . In factven time . Freshlyven time . From thven time  and throven time . Thus, wven time . This “cven time . See FigIn vivo structural magnetic resonance imaging (MRI) can measure the volume of different CSF compartments, which can serve as indirect markers of altered CSF production and absorption.Deviation from typical levels of CSF production, represented through an enlargement or reduction in lateral ventricle (LV) volumes, has been a research focus in multiple neurodevelopmental disorders . Howevern = 10) had increased “extra-axial CSF,” which is an excessive amount of CSF in the subarachnoid space surrounding the cortical surface of the brain , and thus warranted replication in a larger, independent sample. In Shen et al. 2017 [N = 343 infants, 47 of whom developed ASD). In this second study, infants who later developed ASD had 18% more EA-CSF at 6 months than the control groups (HR-negative and LR groups). EA-CSF volume remained persistently elevated through 24 months of age, relative to controls , who were scanned at 2–4 years of age and had 15% more EA-CSF at this age than children with typical development (N = 77) [We recently published a third study of a large cohort of preschool-aged children with ASD ((N = 77) . This th(N = 77) . In summ(N = 77) , 20, 22.While these studies were the first to report an association between increased EA-CSF and ASD, several previous reports in the general pediatrics literature have reported an association between increased EA-CSF and motor delays –26. SincIn both studies of infants who developed ASD, LV volume was not significantly enlarged compared to controls, despite increased volume of extra-axial CSF , 20. Furpositive association between extra-axial CSF volume and total cerebral volume [negative association between CSF and brain volume would be expected instead. Thus, increased EA-CSF in young children with ASD is likely related to different mechanisms than what is observed in degeneration of brain tissue.Is increased EA-CSF volume simply the result of brain tissue loss? In neurodegenerative conditions like dementia, increased extra-axial CSF arises because CSF fills in the subarachnoid space that was previously occupied by atrophic brain tissue , 35. Howl volume , 20, 22,While EA-CSF volume has been found to be positively associated with overall brain volume , 20, theOne of the most consistent findings from previous neuroimaging studies in ASD has been that brain size is significantly enlarged in early childhood, so it is important to evaluate CSF abnormalities during infancy in the context of early brain enlargement. The first direct MRI evidence of brain enlargement prior to age 2 years was reported in the same 2013 cohort of 55 infants (10 of whom developed ASD), who were longitudinally imaged between 6 and 24 months . The HR-A larger study evaluated the individual trajectories of 15 HR-ASD infants, who had data at all three serial MRI scans at 6, 12, and 24 months of age , compareTaken together, these infant studies show that infants who later develop ASD have elevated levels of EA-CSF at 6 months , 20, incHow might increased extra-axial CSF in infancy and early brain overgrowth be related? CSF circulation serves as a means of transporting important growth factors and signaling molecules throughout the brain that are required for the normal development of the neocortex, such as insulin-like growth factors IGF1 and IGF2)  and IGF2, 4, 9. IIs it possible that increased accumulation of CSF over the surface of the brain leads to neuroinflammation? The primary function of continuous CSF outflow is the removal of inflammatory byproducts of brain metabolism, such as amyloid-β (Aβ) and tau protein , 9. In nThere are three clearance systems that are responsible for CSF outflow and clearance of Aβ see Fig. . BecauseThe link between CSF flow, neuroinflammation, and brain development has been informed by recent discoveries in neurodegenerative disorders. Failure of Aβ clearance is increasingly recognized in the pathogenesis of Alzheimer disease . The patOn the other end of the lifespan, there is a marked increase in CSF production in the first year of normal brain development , which mN=159) were associated with greater EA-CSF volume [In ASD, there are three lines of converging evidence that support a potential link between increased extra-axial CSF, impaired CSF circulation, and buildup of neuroinflammation . First, there is mounting evidence of increased levels of Aβ in individuals with ASD, which has been found in neurons from postmortem brain tissue, blood, and peripheral CSF –65. SecoF volume . During F volume .Third, the recent discovery of the meningeal lymphatic system , and itsCurrently, diagnosis of autism spectrum disorder (ASD) relies on the presence of behavioral impairments that do not emerge until the latter part of the first and during the second year of life , and thuprediction biomarkers, but also stratification biomarkers to parse the phenotypic heterogeneity in ASD, which is a well-recognized impediment to developing targeted treatments. For example, a fully cross-validated machine learning algorithm relying on the amount of EA-CSF volume at 6 months predicted later ASD diagnosis at 24 months with 66% sensitivity and 68% specificity [single stand-alone marker for all cases on the autism spectrum. However, the replication and reliability of the findings between three independent cohorts (consisting of both high- and low-risk children with ASD) [Thus, there is a need for both early and biologically derived markers for ASD in infancy to help identify which children need what type of treatment. It is important to identify not only cificity . This prcificity  (in ordecificity . This idcificity . The finith ASD) , 20, 22 In order to validate EA-CSF as a potential stratification biomarker, several future studies must be conducted to (a) determine its specificity , (b) elucidate the underlying biology , and (c) test potential mechanisms using experimental approaches such as those established by studies described above of the glymphatic and meningeal lymphatics systems , 10, 68.The phenotypic heterogeneity in ASD has hampered efforts toward targeted treatments, which has led to major initiatives by the child psychiatry field and the U.S. National Institute of Mental Health to identify biological subtypes of ASD . Thus, t


The majority of children undergoing Hematopoietic Stem cell Transplantation (HSCT) require conditioning therapy to make space and prevent rejection of the donor stem cells. The exception is certain children with Severe Combined immune deficiency, who have limited or no ability to reject the donor graft. Transplant conditioning is associated with significant morbidity and mortality from both direct toxic effects of chemotherapy as well as opportunistic infections associated with profound immunosuppression. The ultimate goal of transplant practice is to achieve sufficient engraftment of donor cells to correct the underlying disease with minimal short- and long-term toxicity to the recipient. Traditional combinations, such as busulfan and cyclophosphamide, achieve a high rate of full donor engraftment, but are associated with significant acute transplant-related-mortality and late effects such as infertility. Less “intensive” approaches, such as combinations of treosulfan or melphalan with fludarabine, are less toxic, but may be associated with rejection or low level chimerism requiring the need for re-transplantation. The major benefit of these novel approaches, however, which we hope will be realized in the decades to come, may be the preservation of fertility. Future approaches look to replace chemotherapy with non-toxic antibody conditioning. The lessons learnt in refining conditioning for HSCT are likely to be equally applicable to gene therapy protocols for the same diseases. Conditioning is required for the majority of children with non-malignant diseases.Some children with severe combined immune deficiency require little or no conditioning—but this usually only corrects T cell function.Mixed chimerism may indicate that re-transplantation may be required.○ Less toxic.○ Associated with more mixed chimerism and may require further procedures.○ May allow long term preservation of fertility.Less intensive conditioning is:conditioning therapy plays a vital role in allowing engraftment of new Hematopoietic progenitor cells (HPC) in the patient. These new HPC can correct some, or occasionally all, of the manifestations of a non-malignant disease.With the exception of some children with severe combined immunodeficiencies , all patients who undergo allogeneic Hematopoietic Stem cell Transplantation (HSCT) require therapy prior to receipt of the graft. This myeloablative combinations (MAC) achieved prolonged aplasia, and were associated with full donor chimerism (DC). However, such therapy is associated with a significant burden of early and late toxicities, making MAC less suitable for older patients, or those with significant co-morbidities. This led to the concept of reduced intensity (RIC) regimens, which are defined as regimens containing reduced doses of myeloablative drugs (or radiotherapy), which are therefore less likely to achieve marrow ablation and more likely to produce mixed chimerism (MC). For the vast majority of HSCTs, namely older adults with malignant disease, the balance between MAC and RIC is a clear trade off: more transplant–related mortality (TRM) is seen with MAC, and more relapse with RIC; these 2 often counter-balancing each other. Multiple attempts to define RIC in terms of specific drug doses were made during 2006–2009, however, there is a spectrum of conditioning and it is preferable to define truly non-myeloablative or minimally intensive conditioning protocols (MIC) where the initial neutrophil recovery is frequently recipient, MAC protocols which mostly achieve sustained full donor chimerism, and then RIC protocols which comprise all those in-between , with a partial or complete inability to reject a graft, can achieve MC or full DC with no conditioning or RIC. The first successful allogeneic transplantation reported was performed without conditioning (or graft-versus-host disease prophylaxis) in a patient with X-linked Severe Combined Immunodeficiency (SCID) in 1968 . AlthougFor decades, a combination of myeloablative doses of busulfan (Bu) and cyclophosphamide was used for many children with a wide variety of non-malignant diseases. Its efficacy, in reliably achieving full DC, is not questioned. However, the profile of toxicity, in particular with endothelial injury, most often manifested as veno-occlusive disease (VOD) means we have continued to look for alternatives.Reducing the Bu dose and increasing the immunosuppression, e.g., by replacing cyclophosphamide with fludarabine (mirroring the RIC approach), has produced good results , particureduced toxicity” was first used to describe a Treo-based regimen (Substituting treosulfan (Treo) for Bu is undoubtedly less toxic, with a low rate of VOD. Although the term “ regimen , the com regimen . Treo ma regimen , 9. This regimen –12, for  regimen . In addi regimen  and a va regimen .The combination of Flu with Melphalan was the first widely used RIC protocol in PID . HoweverIn addition to the conditioning therapy given to allow engraftment of donor cells, it is also important to prevent allogeneic T-lymphocytes from the graft reacting against the donor (or host), which causes graft-versus-host disease (GvHD).ex-vivo Sheep red cell rosetting and soybean lectin . In general, the more myeloablative combinations are associated with gonadal failure and infertility; whereas the RIC combinations may be associated with preservation of these important long term toxicities. Currently, long term follow up data for children treated with Treo are lacking, but early data looks promising .The development of malignancy is another major long term consequence of transplant, But the majority of these appear to be secondary malignancies in patients transplanted for malignant disease, with the exception of Fanconi anemia.As mentioned above, for the sick infant with newly diagnosed SCID, an unconditioned graft may be required, to achieve some T-lymphocyte function and control of infection, and then allow an older child in better shape to tolerate a conditioned graft and achieve a better functioning (donor) immune system. With the adoption of newborn screening for SCID, younger, well infants are being diagnosed earlier, at an age where the use of conventional chemotherapy is less studied and with diagnoses (such as Artemis) where the adverse consequences of chemotherapy are well-known . To achiPreclinical studies identified CD45  and CD11A synergistic combination of two unconjugated rat anti-human CD45 antibodies has been tested in two clinical studies. A Phase 1 dose escalation study used these mAbs in patients with hematologic malignancies that received standard conditioning  and as pClinical testing of radioisotope-labeled anti-CD45 mAbs are in the advanced phase in adults with myeloid disorders, and second generation agents continue to be developed  CD45 antAntibodies that target the cell surface receptor CD117 can also effectively clear host HSC niches. CD117 is a receptor tyrosine kinase expressed on the surface of HSC and early hematopoietic progenitors. Interaction of CD117 with its ligand, stem cell factor (SCF), provides signals necessary for HSC survival, proliferation, and differentiation. Treatment of mice with anti-CD117 mAbs results in transient depletion of HSC with return of normal blood formation within 2 weeks of antibody administration . This wiA humanized anti-CD117 mAb, AMG 191 has been developed and is currently undergoing testing as sole conditioning for patients with SCID. Large animal studies support the safety and efficacy of this agent in depleting HSC . Early dPre-clinical studies have focused on increasing the niche clearing potency of the naked anti-CD117 antibodies. The combination of a second mAb directed against the macrophage checkpoint inhibitor, CD47, with an anti-CD117 mAb demonstrated marked increase in myeloablation and donor cell engraftment compared to the CD117 mAb alone . Other sAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The host response to intruders in the central nervous system (CNS) may be beneficial but could also be harmful and responsible for neurologic symptoms and sequelae in CNS infections. This immune response induces the activation of the kynurenine pathway (KP) with the production of neuroactive metabolites. Herein, we explored cytokine and KP responses in cerebrospinal fluid (CSF) and serum in patients with encephalitis, aseptic, and bacterial meningitis.n = 10), aseptic meningitis , acute bacterial meningitis , and disease control patients with similar symptoms but without pleocytosis in CSF (n = 42). Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) was used to measure KP metabolites in CSF and serum.Cytokines were measured in CSF and serum by multiplex assay in adult patients with encephalitis of infectious, autoimmune or unknown etiology , showed markedly elevated levels in CSF compared with serum, indicating production within the CNS. In contrast to most mediators, the highest level of IP-10 was found in the ASM group, suggesting a potential role for IP-10 in aseptic/viral meningitis. Neopterin and IP-10 were associated with marked changes in KP metabolites in CSF with increasing kynurenine/tryptophan ratio reflecting indoleamine 2,3-dioxygenase activity. Neopterin, a marker of IFN-γ activity, was associated with an unfavorable balance between neuroprotective and neurotoxic tryptophan metabolites.We show that parenchymal and meningeal inflammations in CNS share a characteristic cytokine profile with a general immune response in the CSF with limited influence from the systemic circulation. IFN-γ activity, assessed by neopterin and IP-10 levels, may play a role in the activation of the KP pathway in these patients, potentially mediating neurotoxic effects.The online version of this article (10.1186/s12974-018-1366-3) contains supplementary material, which is available to authorized users. The host inflammatory response to intruders to the central nerve system (CNS) plays an important role for neuronal injury in encephalitis and meningitis. The cytokine profiles of aseptic meningitis (ASM) and acute bacterial meningitis (ABM) have been investigated in several studies, in general showing increased levels of inflammatory mediators –8. HowevH1 cells, activation of regulatory T cells (Tregs) and inhibition of natural killer (NK) cells , 1514, 1K) cells –19. In tK) cells , linkingAltered cytokine levels and associated imbalance of neurotoxic and neuroprotective metabolites in the KP have been suggested to contribute to the pathogenesis of several chronic conditions in the CNS, such as schizophrenia , 22, bipThe aim of the present study was to elucidate the inflammatory network and KP metabolites in ASM and ABM, characterized by meningeal inflammation, and in encephalitis, characterized by brain parenchymal inflammation. Parallel samples of serum and CSF were examined in patient groups and control patients, i.e., patients with similar symptoms but without CSF pleocytosis or other signs of CNS infection.6 /L was found in 68 patients. Of these, 23 did not fulfill the case definition of encephalitis of viral, autoimmune or unknown cause, aseptic meningitis (ASM), or bacterial meningitis (ABM) . In the control group, patients with delirium, chronic or acute psychiatric disease, Parkinson’s disease, Huntington’s disease, CNS malignancy, dementia, epilepsy or seizures, cerebral vascular disease, transient global amnesia, and septicemia were not included. For detailed case definitions, see Additional file This cross-sectional study was performed at the Oslo University Hospital (OUS), Ullevaal, a regional hospital for 2.7 million people. Patients were eligible for inclusion if they had 1) onset of symptoms of CNS infection within less than 7 days and (2) clinical indication for a diagnostic lumbar puncture (LP). Patients were included between January 2014 and December 2015. CSF leukocyte counts ≥ 5 × 10 onset ofAll patients, or their next of kin when the patient was not able to consent, gave written informed consent to participate in the study. The study was approved by The Regional Committees for Medical and Health Research Ethics  and the ethical council of the hospital.For all included patients, CSF leukocyte counts (CSF WBC), CSF protein, and CSF glucose were measured. Bacterial culture of CSF and analyses for identification of causative agent were performed in all individuals by a panel of specific PCR for common neurotropic virus and bacteria.g for 10 min. Serum samples were collected in endotoxin-free tubes without any additives, allowed to clot at room temperature, and centrifuged at 3000×g for 10 min. Supernatants from CSF and serum were centrifuged within 10 h after collection and immediately frozen in triplicates of approximately 700 μL each at − 80 °C. All analyses were performed on previously unthawed samples. For six patients, no serum was available.CSF samples were collected in endotoxin-free polypropylene tubes and stored at 4 °C until centrifugation at 2000×A multiplex cytokine assay  was used to measure the concentrations of 27 different cytokines: tumor necrosis factor (TNF), IFN-γ, interleukin (IL)-1β, IL-1 receptor antagonist (IL-1Ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17A, monocyte chemoattractant protein (MCP)-1/CCL2, IFN-inducible protein-10 (IP-10)/CXCL10, eotaxin/CCL11, macrophage inflammatory protein-1α and -1β , regulated on activation, normal T cell expressed and secreted (RANTES/CCL5), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), basic fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF). The assay was performed using the instructions of the manufacturer. CSF samples were tested undiluted. Only cytokines with less than 20% missing values were included in further analyses. Undetectable levels were assigned the lowest detectable level (LDL) measured in the cohort for the respective marker. In the CSF samples, IFN-γ, IL-5, PDGF, Basic FGF, and RANTES, and for serum, IL-2, IL-5, IL-7, IL-15, G-CSF, GM-CSF, and FGF were excluded from further analyses based on the criteria stated above.Tryptophan and kynurenine metabolites were measured only in the patients with encephalitis, ASM with confirmed viral etiology , ABM, and controls  and analyzed by Pearson’s chi-square test. Correlations were analyzed using Spearman’s rank correlation coefficient. All data analyses were performed in SPSS version 24  and graphs generated by GraphPad Prism 7 .Continuous data are presented as median . Due to lack of normal distribution, analysis of variance (ANOVA) with the Kruskal-Wallis test for multiple groups was used. If the Kruskal-Wallis test revealed significant differences, the Mann-Whitney Streptococcus pneumoniae (n = 2), Staphylococcus aureus (n = 2), Neisseria meningitidis (n = 1), and Haemophilus influenzae (n = 1) were detected in CSF by growth and/or PCR, and for all these patients, the causative bacteria was also detected in blood culture. Patients with encephalitis presented with less headache and more focal neurology compared to the other groups, and impairment of consciousness was observed in significantly fewer patients with ASM and in the control group. Importantly, the majority of the control group had fever, headache, and neck stiffness, similar to most of the patients with CNS infection.Ten patients had encephalitis of viral, autoimmune, or unknown etiology according to the case definition , cytokines with potent effect on T cell function , and growth factors  Fig. . AlthougThe encephalitis group and the control group generally showed lower cytokine levels in CSF than in serum, with exceptions for IL-6 and IL-8 in the encephalitis group and MCP-1 and IP-10 for both groups Figs. . In factp = 0.03), IL-1Ra , IL-6 , and MCP-1  suggesting intrathecal production of most of the examined mediators , a significant correlation between serum and CSF levels were found only for TNF , those in the ASM group with verified viral cause , ABM (n = 6), and controls (n = 22). The median levels of most KP metabolites were higher in patients with encephalitis, VM, and ABM compared with the controls, with the highest median levels observed in the ABM group , indicating an increased conversion of TRP , QA , and PIC , for all other KP metabolites, there was no significant correlation between CSF and serum levels. Furthermore, all CSF/serum ratios for KP metabolites, except for TRP, KYNA, and PIC ratio, were positively correlated with CSF WBC count, while only the ratio of PIC was correlated with CSF albumin/serum ratio  ratio in patients with CNS infection. The lack of correlation of IP-10 to KYNA/(3-HK + QA) could be related to low statistical power. However, whereas IP-10 is induced by IFN- γ in several cell types including monocytes, stromal cells, and endothelial cells [In the present study, we observed very low levels of TRP for several patients with CNS infection, especially in patients with ASM. We hypothesize that this results from increased IDO activity, as these patients had a significantly higher level of KYN compared to patients with detectable TRP levels. The strong correlation of KYN/TRP ratio with neopterin and IP-10 indicates that this IDO activity is driven by IFN-γ. IDO has been found to exhibit an immunosuppressive effect by upregulation of Tal cells , neopteral cells , 61.Finally, when looking at the CSF/serum ratios of KP metabolites, we found very narrow ranges in the control group, suggesting strict control of KP in healthy subjects.Studies of the inflammatory profile in human CNS infections including both meningeal and parenchymal infections are relatively scarce –65, and In conclusion, we found a marked increase in a wide range of inflammatory mediators in CSF in aseptic and bacterial meningitis with a more modest increase in encephalitis. The markedly higher levels in CNS than in serum for most of the mediators indicate compartmentalization of immune responses in the CSF. Our data may also suggest that increased IFN-γ activity, as assessed by neopterin and IP-10, may contribute to neurotoxicity through enhanced TRP catabolism. In particular, dysregulation of the KP with signs of an increased formation of neurotoxic QA in encephalitis should be explored further in these conditions.Additional file 1:Table S1. Case definitions of encephalitis, aseptic and viral meningitis and bacterial meningitis. (PDF 179 kb)Additional file 2:Figure S1. Flowchart of inclusion of patients and overview of various analyses performed in the study population. (PDF 342 kb)Additional file 3:Table S2. Cytokine levels in CSF and serum. (PDF 215 kb)Additional file 4:Table S3. Correlations of cytokines in CSF with serum levels, CSF WBC, albumin ratio and KYN/TRP ratio. (PDF 197 kb)Additional file 5:Table S4. KP metabolites in CSF and serum. (PDF 198 kb)Additional file 6:Figure S2. Serum levels of KP metabolites. (PDF 81 kb)Additional file 7:Table S5. CSF/serum ratios of KP metabolites and the correlation with CSF WBC and albumin ratio. (PDF 194 kb)


Does ambroxol cross the blood-brain barrier, and what are the biochemical changes associated with ambroxol therapy in patients with Parkinson disease with and without glucocerebrosidase gene mutations?In this open-label clinical trial of 17 patients with Parkinson disease, ambroxol crossed the blood-brain barrier and bound to the β-glucocerebrosidase enzyme, and it increased β-glucocerebrosidase enzyme protein levels and cerebrospinal fluid α-synuclein levels in patients both with and without glucocerebrosidase gene mutations.Ambroxol therapy has potential for study as a neuroprotective compound for the treatment of patients with Parkinson disease both with and without glucocerebrosidase gene mutations. GBA1 (OMIM 606463), are the most important risk factor for Parkinson disease (PD). In vitro and in vivo studies have reported that ambroxol increases β-glucocerebrosidase (GCase) enzyme activity and reduces α-synuclein levels. These observations support a potential role for ambroxol therapy in modifying a relevant pathogenetic pathway in PD.Mutations of the glucocerebrosidase gene, GBA1 mutations.To assess safety, tolerability, cerebrospinal fluid (CSF) penetration, and target engagement of ambroxol therapy with GCase in patients with PD with and without An escalating dose of oral ambroxol to 1.26 g per day.This single-center open-label noncontrolled clinical trial was conducted between January 11, 2017, and April 25, 2018, at the Leonard Wolfson Experimental Neuroscience Centre, a dedicated clinical research facility and part of the University College London Queen Square Institute of Neurology in London, United Kingdom. Participants were recruited from established databases at the Royal Free London Hospital and National Hospital for Neurology and Neurosurgery in London. Twenty-four patients with moderate PD were evaluated for eligibility, and 23 entered the study. Of those, 18 patients completed the study; 1 patient was excluded (failed lumbar puncture), and 4 patients withdrew (predominantly lumbar puncture–related complications). All data analyses were performed from November 1 to December 14, 2018.Primary outcomes at 186 days were the detection of ambroxol in the CSF and a change in CSF GCase activity.GBA1 mutations and 9 without GBA1 mutations) were included in the primary analysis. Between days 0 and 186, a 156-ng/mL increase in the level of ambroxol in CSF  was observed. The CSF GCase activity decreased by 19% . The ambroxol therapy was well tolerated, with no serious adverse events. An increase of 50 pg/mL (13%) in the CSF α-synuclein concentration  and an increase of 88 ng/mol (35%) in the CSF GCase protein levels  were observed. Mean (SD) scores on part 3 of the Movement Disorders Society Unified Parkinson Disease Rating Scale decreased  by 6.8 (7.1) points . These changes were observed in patients with and without GBA1 mutations.Of the 18 participants  who completed the study, 17 (8 with The study results suggest that ambroxol therapy was safe and well tolerated; CSF penetration and target engagement of ambroxol were achieved, and CSF α-synuclein levels were increased. Placebo-controlled clinical trials are needed to examine whether ambroxol therapy is associated with changes in the natural progression of PD.NCT02941822; EudraCT identifier: 2015-002571-24ClinicalTrials.gov identifier:  This single-center open-label nonrandomized, noncontrolled trial assesses the safety, tolerability, cerebrospinal fluid penetration, and biochemical changes associated with ambroxol therapy for the treatment of patients with Parkinson disease with and without mutations in the glucocerebrosidase gene. GBA1 (OMIM 606463), cause the autosomal recessive lysosomal storage disorder, Gaucher disease.1 These mutations are the most important genetic risk factor for Parkinson disease (PD),2 exhibiting penetrance of 10% to 30%.4 They are present in 5% to 15% of Caucasian patients with PD, 25% of Ashkenazi Jewish patients with PD, and 1% of individuals without PD.5Mutations in the glucocerebrosidase gene, GBA1 cell and animal models, an increased α-synuclein accumulation and a reciprocal relationship between α-synuclein levels and β-glucocerebrosidase (GCase) enzyme activity have been reported.11 Although GCase activity is reduced in the brains of patients with PD with and without GBA1 mutations, it is lower in those with the mutations.12 Reduced GCase activity in the brain is associated with increased levels of α-synuclein.13 In addition, GCase activity is decreased in the cerebrospinal fluid (CSF) of patients with PD with and without GBA1 mutations compared with controls.14 Upregulation of brain cytosolic/lysosomal GCase activity may reduce α-synuclein levels, mediating a neuroprotective effect in patients with PD both with and without GBA1 mutations.17In 18Ambroxol therapy has been safely used as a cough linctus since the 1970s  in patients with PD of moderate severity. The clinical trial was conducted at the Leonard Wolfson Experimental Neuroscience Centre, a dedicated clinical research facility at the University College London Queen Square Institute of Neurology from January 11, 2017, to April 25, 2018. A clinical trial steering committee provided clinical oversight. Statistical support was provided by the Peninsula Clinical Trials Unit of the University of Plymouth in the United Kingdom. The study was approved by the Institutional Joint Research Office of University College London, the UK Medicines and Healthcare products Regulatory Agency, and the London-Bloomsbury Research Ethics Committee. Written informed consent was obtained from all participants before study-related procedures were initiated. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki28 were judged able to self-administer the clinical trial drug, and were at stage 3 or less on the Hoehn and Yahr Scale, which classifies the progression of PD symptoms in 5 stages . A portion of participants were preselected on the basis of their known GBA1 mutation–carrier status. Key exclusion criteria included the use of an interventional medicinal product within the last 30 days or exposure to 3 or more interventional medicinal products within the last 12 months. All participants underwent confirmatory sequencing of exons 1 to 11 of the GBA1 gene. A full list of exclusion and inclusion criteria can be found in eMethods 2 in Patients were recruited from the established research databases of the National Hospital for Neurology and Neurosurgery and the Royal Free London Hospital. Eligible participants were aged 40 to 80 years, had idiopathic PD as defined by Queen Square Brain Bank criteria,At the evaluation for clinical trial entry, each patient underwent a physical and neurological examination, an electrocardiogram, and blood sampling for clinical laboratory tests. Women of childbearing age also received a pregnancy test. After confirmation of patient eligibility, clinical visits were held at baseline, day 11, day 93, day 186, and day 279, with telephone contact at predetermined intervals , 120 mg (days 8-14), 180 mg (days 15-21), and 300 mg (days 22-28). This exposure period was followed by 158 days of administration of ambroxol at 1.26 g per day (420 mg 3 times per day). Patients were issued ambroxol therapy in 2 batches, 1 at baseline and 1 at 93 days. Ambroxol tablets were donated by PRO.MED.CS Praha a. s. .All patients were taking dopaminergic therapy, and levodopa dosage equivalents have been included in am and 9 am by a single assessor (S.M.). Measurement instruments included (1) the Movement Disorder Society Unified Parkinson Disease Rating Scale , which was administered on days 0, 93, 186, and 279; (2) the Montreal Cognitive Assessment , which was administered on days 0 and 186; (3) the Non-Motor Symptoms Scale , which was administered on days 0 and 186; and (4) the Non-Motor Symptoms Questionnaire , which was administered on days 0 and 186.Assessments were performed between 8 Empty ambroxol blister packs were collected at 93 and 186 days to assess participant adherence to treatment. All adverse events (AEs), biochemical results , the change in blood leucocyte GCase activity, the change in CSF GCase protein levels, the CSF total glucosylceramide levels, the CSF and serum α-synuclein levels, and the CSF and serum tau levels. We also recorded the results of the MoCA, NMSS, and NMSQuest assessments that were conducted at baseline and 186 days.am from participants after they had fasted overnight. Samples were frozen at –80 °C within 60 minutes of collection in the case of CSF and within 90 minutes of collection in the case of leucocyte pellets. Samples were only defrosted immediately before the performance of assays. Cerebrospinal fluid GCase assays were all performed within 7 and 14 days of sample collection. Leucocyte GCase assays were performed between 7 and 28 days of sample collection.Cerebrospinal fluid was collected before 10 Details of the biochemical assays are provided in eMethods 3 and 4 in t test (significance threshold of P < .05) of the change in CSF GCase activity. The lower CI and 1-sided t test results (significance threshold of P < .05) that were presented for the change in CSF ambroxol levels reflected the fact that the change was lower bounded by 0 because of anticipated 0 concentrations at baseline. To register as a change, ambroxol concentrations were required to be greater than the assay limit of detection of 0.5 ng/mL at 186 days. A descriptive analysis that correlated the change (Pearson coefficient) in ambroxol levels and the change in CSF GCase activity was also performed. The changes in secondary outcomes between baseline and 186 days were presented with 95% CIs. All data analyses were performed from November 1 to December 14, 2018.The statistical analyses followed a predefined statistical analysis plan written by the clinical trial statisticians (A.H.V.S. and J. Hosking). As the result of unforeseen circumstances, the statistician specified in the original protocol was not available to perform the final analysis. Therefore, a revised statistical analysis plan  completed the study. One patient was excluded after unsuccessful LP attempts that were performed by 2 experienced operators. Two patients withdrew because they experienced headaches after LP and before the beginning of ambroxol treatment. One participant withdrew at day 1 after treatment began, citing the high ambroxol tablet count (21 tablets per day), and 1 withdrew at 93 days after treatment began, citing family illness. Of the 18 patients who completed the study, 1 was excluded from the CSF analyses  because of a red blood cell contamination of more than 500 cells per cmGBA1 mutations had the following variants (an asterisk indicates a phenotype that is known to cause severe Gaucher disease): p.E326K wild type (3 patients), p.N370S wild type (1 patient), p.R463C* wild type (2 patients), p.T369M/p.W393X* (1 patient), and RecNcil * wild type (1 patient).Participants with GBA1 mutations [GBA1+] and 4 without mutations [–GBA1]) underwent a third LP at 279 days. These 7 participants included the patient who was excluded from the analyses because of a blood-contaminated baseline CSF sample. As a result, 18 participants (8 GBA1+ and 10 –GBA1) were included in blood and clinical analyses at 186 and 279 days. Seventeen participants (8 GBA1+ and 9 –GBA1) were included in the CSF analyses at 186 days, and 6 participants (3 GBA1+ and 3 –GBA1) were included in the CSF analyses at 279 days.Seven participants  adherence to treatment of 89% (13%). Blood and CSF ambroxol levels at baseline confirmed that no participants had taken the drug before the treatment start date.GBA1+ and –GBA1 subgroups are available in eTables 3 and 4 in t test, P < .001). At 186 days, the mean (SE) CSF ambroxol levels were 11% (6%) of the mean blood levels. As anticipated by our pretrial CSF studies, which indicated inhibition of GCase in CSF rather than enhancement in tissues, the mean (SE) CSF GCase activity decreased by 0.059 (0.026) nmol/mL per hour , vomiting (2 patients), a burning sensation after swallowing the interventional medicinal product (1 patient), and loose stool (1 patient). Definitely related AEs were acid reflux (1 patient), nausea (1 patient), and a transitory skin condition on the chest, back, and arms (1 patient). A full list of AEs is available in eTable 5 and a list of recorded AEs is available in eTable 6 in P = .01; P = .002; P = .36) or glucosylceramide levels . The correlation between ambroxol concentration and the change in CSF GCase activity was not significant . The CSF analysis of the 6 participants from whom CSF was collected at baseline, 186 days, and 279 days is presented in eTable 4 in Between baseline and 186 days, mean (SE) increases of 50 (17) pg/mL (13%) in total CSF α-synuclein concentration  (P = .46), although a mean (SE) decrease of 0.20 (0.08) pg/mL was observed in serum tau levels .Between baseline and 186 days, mean (SE) blood leucocyte GCase activity increased by 1.0 (1.4) nmol/mg/h  D. Wide vP = .01) with a mean (SD) rebound of 7.2 (9.8) points between 186 and 279 days. This change appeared to be associated primarily with the MDS-UPDRS part 3 motor score , which showed a mean (SD) decrease of 6.8 (7.1) points between baseline and 186 days  and a mean (SD) increase of 7.6 (7.0) points between 186 and 279 days. Seven of 18 patients increased their dopaminergic therapy during the course of the study, and the same deflection of MDS-UPDRS changes was observed in those on stable therapy and in those who increased their medication therapy  total MDS-UPDRS score decreased  by 8.7 (11.8) points , but the mean (SD) change in the NMSQuest score was only 0.2 (2.6) points  between baseline and 186 days.As anticipated, the change in MoCA scores were skewed and bound by 0 because of the preponderance of participants who maintained their scores from baseline. An increase  in the mean (SD) MoCA scores of 1.7 (1.3) points was recorded between baseline and 186 days. In addition, the mean (SD) NMSS score increased  by 11.5 (18.5) points .Before the clinical trial, we performed in vitro assays to estimate the association of ambroxol, an inhibitory chaperone, with changes in GCase activity in acellular CSF. We added 500nM 29 The experiment comprised 5 technical repeats for CSF derived from 5 participants for each condition. Compared with the control samples, a mean (SD) decrease of 42% (12%) in CSF GCase activity  was observed after the addition of ambroxol. The denatured and conduritol B epoxide–inhibited samples registered no residual activity.In addition, we performed a positive control by thermodynamically denaturing and chemically inhibiting GCase with conduritol B epoxide.17 Our study indicates that ambroxol therapy is well tolerated in patients with PD. This finding is important, as the administered dose was approximately 10 times higher and was administered for a longer duration than specified in its license.To our knowledge, this study represents the first clinical trial of personalized therapy for a stratified  subtype of PD and the first use of ambroxol therapy in patients with PD. The study met its primary outcomes, which were to confirm that ambroxol was able to penetrate CSF and that it had a modulatory effect on CSF GCase. The finding that ambroxol penetrates CSF is consistent with a recent clinical trial of ambroxol  in 5 patients with a mean age of 18 years who had neuronopathic Gaucher disease.8Ambroxol has an inhibitory effect on GCase activity within acellular human CSF, which is consistent with its known activity as an inhibitory chaperone with a neutral pH. Within largely acellular CSF, the GCase protein is free, which is in contrast to its normal intracellular lysosomal location. Ambroxol is a pH-dependent inhibitory small molecular chaperone that binds to the active site of the GCase protein and reduces activity. Binding enables transportation to the lysosome and elution of free active enzymes under acidic conditions. Therefore, in acellular CSF, ambroxol will bind to and inhibit free GCase. However, in tissues, including those in the brain, ambroxol will increase intracellular GCase activity, as reported in studies of rodent and primate models15 that ambroxol has also had an association with α-synuclein metabolism. These results suggest an increase in GCase activity within the brain itself, although this activity cannot, of course, be measured in vivo. No clear consensus exists on the association of PD with changes in total CSF α-synuclein, but reduced levels of total CSF α-synuclein have been described, while oligomeric and phosphorylated α-synuclein have increased.30 Ambroxol upregulates the expression of GCase, probably via the transcription factor EB pathway, and increases vesicular export.23 The increase in CSF α-synuclein could be interpreted as an increase of extracellular export of the protein from the brain parenchyma.The sustained upregulation of expression of CSF GCase protein levels indicates target engagement of ambroxol with the GCase pathway. The increase in total CSF α-synuclein concentration implies, based on previous in vitro and in vivo data,Interpretation of the changes in MDS-UPDRS and MoCA results is difficult in the context of a nonplacebo-controlled study. However, the changes support the clinical impression that no substantial deleterious effect of ambroxol was observed among participants taking ambroxol, including any adverse effect on the motor features of their PD.GBA1 study group, but this homogeneity may have limited the interpretation of the data. The mean age of our participants was 60.2 years, which is relatively young for patients with PD. However, this mean age was consistent with our inclusion criteria of a Hoehn and Yahr stage of 3 or less and was comparable with many drug intervention studies of patients with PD. Participants were recruited from databases of patients with PD with and without GBA1 mutations, but we do not believe this recruitment method resulted in a bias relevant to the biochemical biomarkers used as end points in this study.Our study has several limitations. The sample was relatively small, although the complexity of the study and its nature as a proof-of-concept clinical trial were factors in its design. A placebo arm was not used, so the clinical outcomes should be interpreted with caution. We could have elected to have a more genetically homogenous GBA1 mutation and potentially in those without a GBA1 mutation. Larger placebo-controlled studies are warranted.In conclusion, we confirm that ambroxol has potential as a drug to target the glucocerebrosidase pathway in PD and increase GCase activity in the brain. These findings concur with cell and animal modeling, which indicate that ambroxol modulates α-synuclein levels. We believe ambroxol therapy has promise for further investigation as a drug to improve outcomes, particularly in patients who have PD with a


Bacterial meningitis is a life-threatening disease that evokes an intense neutrophil-dominated host response to microbes invading the subarachnoid space. Recent evidence indicates the existence of combinatorial V(D)J immune receptors in neutrophils that are based on the T cell receptor (TCR). Here, we investigated expression of the novel neutrophil TCRαβ-based V(D)J receptors in cerebrospinal fluid (CSF) from human patients with acute-phase bacterial meningitis using immunocytochemical, genetic immunoprofiling, cell biological, and mass spectrometric techniques. We find that the human neutrophil combinatorial V(D)J receptors are rapidly induced in CSF neutrophils during the first hours of bacterial meningitis. Immune receptor repertoire diversity is consistently increased in CSF neutrophils relative to circulating neutrophils and phagocytosis of baits directed to the variable immunoreceptor is enhanced in CSF neutrophils during acute-phase meningitis. Our results reveal that a flexible immune response involving neutrophil V(D)J receptors which enhance phagocytosis is immediately initiated at the site of acute bacterial infection. Bacterial meningitis is associated with considerable mortality and morbidity worldwide and survivors are at increased risk of experiencing serious long term disabling sequelae –5. The pneutrophil TCRαβ. The TCRαβ immunoreceptor in neutrophils is expressed across the entire human life span, undergoes a dramatic decline during aging . Unlike other sites of purulent infection, the latter provides conditions in which degraded tissue is absent and thus facilitates precise analysis of the immune function of neutrophils at the site of inflammation.all of the above criteria. In addition, one patient meeting the above criteria was included for neutrophil activation experiments, and one patient with varicella zoster meningitis was recruited as a positive control representing an acute non-bacterial meningitis. All patients were free of immunosuppressive medication. One patient (patient 5) was diagnosed with monoclonal gammopathy shortly after admission to the hospital. A total of 10 healthy individuals was randomly selected for additional neutrophil experiments.Adult patients were recruited to the study in the random order they were admitted to the emergency rooms or the intensive care units of the Hospital of the University of Heidelberg, Campus Mannheim. Inclusion criteria were: (i) suspected bacterial meningitis, (ii) initial CSF leukocyte count ≥300/μl, (iii) CSF neutrophil fraction >75%, (iv) age >17 years, and (v) positive CSF cultures for bacterial pathogens. Under these conditions, a total of 10 patients  CSF and peripheral blood samples were collected at a second time point 5 and 7 days after symptom onset, respectively, to monitor the response to antimicrobial therapy. All CSF and peripheral blood samples were processed within 3 h of collection. The study was approved by the Ethics Committee of the Medical Faculty Mannheim, University of Heidelberg (Permit Number: 2007-254N-MA). Written informed consent had been obtained from all individuals included in this study.The samples analyzed included acute-phase CSF obtained by lumbar puncture and peripheral blood collected by venipuncture at the same time point that were submitted to the laboratory for routine testing. From residual aliquots of CSF and peripheral blood CD15Bacterial pathogens in the CSF samples were identified by light microscopy of Wright-Giemsa or Gram stained cytospin smears, microbiological culturing techniques, and MALDI-TOF biotyping, respectively. Viral pathogens were detected using routine PCR testing.+ neutrophils and CD3+ lymphocytes were purified by density gradient centrifugation  and magnetic cell sorting  from freshly obtained CSF and peripheral blood that was drawn at the same timepoint. Purity of CD15-MACS isolated neutrophils was routinely >99.5% as assessed by flow cytometry . Light microscopic inspection of Giemsa stained CD15+ cytospin preparations from all patients by two independent trained hematologists  confirmed these results. Viability of CD15-MACS purified neutrophils was >98% for up to 12 h even in the presence of bacterial pathogens as assessed by flow cytometric 7-AAD staining (data not shown). Flow cytometric analyses were performed on a FACSCalibur flow cytometer (BD Biosciences) using the following antibodies: anti-human CD3-APC, anti-human CD15-PE, anti-human CD2-FITC, and anti-human CD19-PercP (BD Biosciences).CD15For immunostaining, cytospins were fixed for 10 min in icecold acetone. After 20 min of drying the cells were blocked with 5% goat serum in 1% BSA in PBS, incubated with mouse anti-human monoclonal anti-TCRβF1 , rabbit anti-human CD11a, rabbit anti-human CD11c, rabbit anti-human CD18, rabbit anti-human CD83  or mouse anti-human CD11b  at 4°C overnight, washed in PBS for 15 min, and incubated with Alexa 488-labeled goat anti-mouse IgG , Cy3-labeled donkey anti-rabbit IgG , and DRAQ5 , respectively. Mouse IgG1 (BD Biosciences) or rabbit IgG-isotype control antibodies (Abcam) were used as negative control. Positive staining was visualized by confocal microscopy as previously described . For 3D SDS-PAGE and transfer to nitrocellulose membranes was conducted as previously reported , 11, 17.Interleukin-8 levels were measured in serum and CSF samples collected from patients with acute bacterial meningitis and healthy age-matched controls using a Human IL-8 Single Analyte ELISArray Kit (SABiosciences) according to the manufacturers' protocol.+ neutrophils and CD3+ lymphocytes the “CDR3 length spectratyping” method published by Pannetier et al.  were PCR amplified and subsequently fluorescence labeled in a run-off reaction for 4 cycles using the D4-labeled primer D4-TTGGGTGTGGGAGATCTCTGC specific for the TCRβ constant region. The expressed Vβ length variants (“spectratypes”) were analyzed on a CEQ™ 8000 Genetic Analysis System (Beckman Coulter)  , 11, 17.Total RNA of the PB and CSF cells was extracted with Trizol reagent and subjected to RT-PCR using a One-step RT-PCR kit (Qiagen). cDNA was then subjected to Amplicon rescued multiplex PCR (ARM-PCR) using human T cell beta receptor primers according to the manufacturer's instructions (iRepertoire Inc.). The PCR products were run on 2% agarose gel and the DNA was extracted from the gel using a Gel Extraction kit (Qiagen). After library preparation high-throughput sequencing of the CDR3 regions was performed on a HiSeq2000 DNA sequencer (Illumina) by iRepertoire Inc. The raw data were analyzed by iRepertoire using IRmap programs with a modified Smith-Waterman algorithm. For data analyses, paired-end sequence reads were joined together and the merged sequences were mapped to germline V, D and J reference sequences according to the method we previously reported . A newlyS. pneumoniae (ATCC 47619), N. meningitidis ] were propagated overnight, resuspended in RPMI 1640 (PAA Laboratories) and subsequently heat-inactivated for 1 h at 65°C. Freshly isolated CD15+ neutrophils from three healthy individuals  were then infected with the bacteria at a ratio of 1:10. After 9 h incubation at RT cells were harvested in TRI Reagent BD (Sigma-Aldrich).Bacteria [6 freshly collected CD15+ PB neutrophils or CSF neutrophils were incubated in the presence of standardized polystyrene bead baits  for 1.5–3 h at room temperature in RPMI medium (cell/bead ratio 1:1). Bead baits were coated with endotoxin-free anti-TCRαβ antibodies (5 μg per 107 bead baits) or equal quantities of endotoxin-free non-specific IgG antibodies (isotype control) and albumin (irrelevant protein), respectively. Subsequently, cytospins were prepared and neutrophil bead phagocytosis was quantitated by bright field microscopy. For blockade of phagocytosis cytochalasin D was used at a concentration of 5 μg/ml. Neutrophils that had ingested or bound ≥1 bead were considered phagocytosis positive. The total number of beads associated with the cell (bound and ingested) was determined for each individual cell. For each cytospin preparation the beads/cell ratio and the percentage of phagocytosing cells were assessed from at least 12 randomly selected fields of vision.Aliquots of 3 x 106 purified PB neutrophils from healthy individuals (n = 5) were pre-incubated with anti-CD3ε  and anti-CD28  antibodies or non-specific IgG antibodies (isotype controls) for 10 min, and subsequently infected with FITC-labeled non-opsonized E. coli bacteria  for 10 min. Respiratory burst activity was assessed in the presence of PMA (1 μg/ml), CD3/CD28 costimulation, non-specific IgG or the absence of a stimulus. All incubations were performed in serum-free medium at 37° C.For quantitative determination of neutrophil phagocytic and respiratory burst activities modified protocols from the flow cytometry-based Phagoburst kit (Glycotope Biotechnology) were used. To assess phagocytic activity, aliquots of 10Leukocyte counts in the 10 CSF samples recruited to the study ranged from 300 to 15,418 cells/μl . The tim+ CSF-leukocytes were authentic neutrophils, we tested for surface expression of additional neutrophil markers. Because PB neutrophils have been shown to adopt characteristics of dendritic cells (DC) under specific long-term culturing conditions . Importantly, Western blot demonstrated that TCRαβ protein levels were markedly increased in the CSF neutrophils compared to PB neutrophils irrespective of the causative bacterial pathogen indicating that the TCRαβ is induced in the CSF during acute bacterial meningitis  for 3 h  and the phagocytosed bead/cell ratios  in both individuals when TCRαβ-directed phagocytosis was induced  and subsequently infected with non-opsonized fluorescence-labeled E. coli baits. In addition, we investigated whether CD3/CD28 mediated costimulation of the TCR augments the respiratory burst activity of neutrophils. CD3/CD28 costimulation resulted in a consistent increase (1.1–2.8 fold) in the percentage of phagocytosing neutrophils in all individuals  TCRαβ-directed phagocytosis was significantly increased in CSF neutrophils relative to PB neutrophils. Collectively, these findings demonstrate that phagocytosis is markedly enhanced in normal and CSF neutrophils when baits are directed to the TCRαβ.Finally, we investigated whether the observed increase in TCRαβ-directed phagocytosis in normal neutrophils was also detectable in neutrophils during acute meningitis. For this, circulating neutrophils and CSF neutrophils from an individual with suspected acute bacterial meningitis (patient 8) were collected upon admission and immediately challenged with TCRαβ-directed baits. As expected by the general activation of neutrophils in the subarachnoid space, phagocytic activity was generally increased in CSF neutrophils compared to PB neutrophils . As obseWe report that the acute phase of bacterial meningitis is characterized by immediate initiation of a flexible host response in the subarachnoid space that relies on the recently identified neutrophil TCRαβ immune receptor. This is the first demonstration that variable neutrophil based host defense mechanisms are implicated in the initial phase of bacterial infection.+ neutrophils is routinely present in the circulation , we speculate that the findings presented here reflect a general as yet unrecognized host defense mechanism operative in acute bacterial infection.Conceptually, the observation of an immediate variable TCR response in neutrophils during acute bacterial meningitis identifies a rapidly responding flexible element in the host defense armamentarium of a classic myeloid phagocyte. This lends further evidence to the emerging view that RAG recombinase  diversifThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.TF, DY, VC, and JM performed the experiments. KP, DD, AP, PF, AB, MN, and WK contributed conception and design of the study. BR, CS, and OW collected clinical data. CW and JH performed next-generation sequencing and bioinformatics. TF, WK, and KP wrote the manuscript and prepared figures and tables. 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.


PVI and ECS measured by CSF drainage were 99.8 ± 89.5 and 0.4 ± 0.2 cmH2O/mL. Among the demographic factors, body weight was correlated with OP (r = 0.27), CP (r = 0.35), and PVI (r = 0.20). Height was weakly correlated with CP (r = 0.199) During the study period, 22 participants (21.34%) developed PDPH. None of the CSF pressure factors were significantly different between the PDPH and non-PDPH group and did not contributed to the development of PDPH. CSF pressure factors might not be related to the development of PDPH.Post-dural puncture headache (PDPH) is an unfavorable situation seen in considerable number of patients even though atraumatic and small needle reduces its incidence. CSF pressures measured at the time of puncture change after CSF drainage. In the present study, we investigated relationships between CSF pressure-related factors and occurrence of PDPH. We prospectively enrolled 103 participants who underwent CSF studies for meningitis. Using a standardized protocol, CSF opening pressure (OP) and closing pressure (CP) were measured, and cerebrospinal elastance (ECS) and pressure-volume index (PVI) were investigated. Within 14 days after dural puncture, we confirmed PDPH. According to PDPH development, the CSF pressure factors and clinical variables were compared between PDPH and non-PDPH group. Of the 103 participants, 100 (97.0%) had decreased CP, 16 (15.5%) had values below 6 cmH Dural puncture is essential for the diagnosis of CNS infections and disorders related to CSF dynamics . Post-duSeveral hypotheses have been presented explaining the pathogenesis of PDPH, such as traction of pain-sensitive structures such as the meninges or cranial nerve occurring when upright and vasodilation to maintain intracranial volume according to the Monro-Kellie doctrine , 5. AlthAlong with opening pressure (OP), pressure measured immediately after CSF drainage is referred to as closing pressure (CP). It is plausible to hypothesize that CP, more than OP, reflects the CSF pressure environment at the time of PDPH generation. Measurements of CP might be helpful in situations of idiopathic intracranial hypertension (IIH) and hydrocephalus, because the primary purpose of management is reducing pressure through CSF drainage , 10. In The change in pressure between OP and CP (OP-CP) roughly reflects volume of the CSF reservoir . CraniosIn the present study, we investigated whether CSF pressure factors at the time of CSF examination are associated with subsequent development of PDPH.This study was conducted prospectively from October 2014 through June 2018. We enrolled participants who were scheduled to undergo CSF studies to detect meningitis.Before CSF study, all participants were evaluated by brain CT, basic laboratory tests, clinical history, and neurologic examinations. We excluded participants with a history of neurologic disease , connective tissue disorders, previous dural puncture, or abnormal structural lesions on neuroimaging studies. Participants who underwent traumatic tap and failed dural puncture were also excluded. After confirmatory CSF analysis, we enrolled participants with aseptic meningitis or normal CSF findings.To evaluate the relationship between pressure factors and PDPH, we performed dural puncture while controlling for the following factors related to PDPH. (1) Needle shape: Quincke needle, (2) needle size: 20–23 gauge, (3) bevel orientation: parallel to the long axis of the spine, (4) needle insertion location and angle: L4-5 interspace with 10-degree angle toward the umbilicus, (5) CSF drainage volume: 15 ml, (6) dural puncture position: side-lying position, (7) resting duration and hydration after dural puncture: 4 h and 80 ml/h with normal saline. Skilled neurologists performed all dural puncture procedures.Once the CSF flow was established, a stopcock valve and manometer were attached to the spinal needle. To avoid false rises and drops of CSF pressure, we asked participants to relax and straighten their legs slowly. CSF OP was checked at the peak level of the manometer that showed slight up-down changes with respiration. Then, we opened the valve and collected CSF into a test tube. After CSF drainage was complete, we opened the manometer valve and measured CP in the same way.According to ICHD-3, the diagnostic criteria of PDPH include a headache that occurs within 5 days after dural puncture as well as criteria for identifying headaches attributed to low CSF pressure. Some previous studies reported that PDPH could develop between 5 and 14 days after dural puncture . TherefoThis study was approved by the local ethics committee of Ujeongbu St. Mary's Hospital, Ujeongbu, Republic of Korea . Written informed consent was obtained from all participants. For participants who were 18 years old or younger, we obtained written informed consent from their parents.When the lumbar puncture procedure ended, the operator checked and described the pressure , procedure  and demographic factors. The craniospinal elastance (ECS) was calculated using OP, CP, and removed CSF volume .Pressure-volume index (PVI) is the CSF volume required to raise CSF pressure by a factor of 10.U-test was used for numerical variables. Categorical variables were analyzed by the chi-square test with Fisher's correction when appropriate. According to diagnosis, statistical analysis performed between PDPH and non-PDPH in each aseptic meningitis and normal CSF group. Spearman correlation analysis was used to evaluate the relationships between factors that may be related to PDPH. Binary logistic regression analysis was performed to assess significant related factors for PDPH. All tests were two-tailed with a significance level of p < 0.05.The data described in the mean ± standard deviation. All statistical analyses were performed to test relationships between PDPH and controls using R (version 3.5.1). The Mann-Whitney For this study, we enrolled 133 participants who visited our center with diagnoses of meningitis. We excluded 30 participants who did not match inclusion criteria, had incomplete data, or were lost to follow-up. Finally, 103 participants  were included in the analysis . CSF stu2O, and CP was 10.2 ± 3.5 cmH2O. After draining CSF, 100 participants (97.1%) exhibited decreased CSF pressure with decreases of pressure (OP-CP) of 6.1 ± 3.1 cmH2O. CSF CP was lower than 6 mmH2O in 16 participants (15.5%) (CSF OP was 16.3 ± 4.7 cmH (15.5%) .r = 0.270, p = 0.006), and CP was also significantly correlated with weight  and height . ΔOP-CP showed a negative relationship with puncture trial number  and PVI was significantly correlated with weight  (CSF OP showed a significant relationship with weight (= 0.046) .n = 22) and a non-PDPH group (n = 81). CSF pressure factors include OP, CP, OP-CP, ECS, and PVI. None of these factors differed between groups  After CSF drainage, CSF CP significantly decreased compared to OP. (2) Most CSF pressure factors were correlated with body weight. (3) CSF pressure factors are not related to PDPH development.2O of pressure requires removing 0.91 ml of CSF . We may make several observations about these results. First, CSF pressure evaluations are not performed at the time of PDPH diagnosis, so restorative changes may occur in CSF pressure over time. Second, when PDPH occurs in a patient without a concomitant significant decrease of CP, we should consider the possibility of additional pressure drop due to persistent CSF leakage at the initial dural puncture point. In SIH patients, various imaging studies may be used to detect CSF leaks and focal CSF collection . Written informed consent was obtained from all participants. For participants who were 18 years old or younger, we obtained written informed consent from their parents.SK, HS, SL, DR, TK, and JP conceived and designed the present study. HS, SL, and DR performed dural puncture procedures and acquired data. SK, TK, and JP analyzed and interpreted the data. SK and JP drafted the manuscript and made final decisions regarding the version to be published. JP agreed to be accountable for all aspects of the work and to ensure that questions related to the accuracy or integrity of any part of the work would be appropriately investigated and resolved.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: Parkinson's disease (PD) is characterized by proteinopathies and these proteinopathies seem to interact synergistically and lead to protein aggregations and changes in protein cerebrospinal fluid (CSF) levels. In this study, we aimed to explore the longitudinal changes of CSF a lpha-synuclein (α-syn), total tau (t-tau), phosphorylated tau (p-tau), and beta-amyloid (Aβ1−42) and their relationships with each other and with baseline clinical entities like REM sleep behavior disorder (RBD), cognitive impairment, motor symptoms, and olfaction dysfunction.Method: One hundred and twelve non-demented PD patients and 110 controls were recruited from Parkinson's Progression Markers Initiative (PPMI).We used a linear mixed model within groups to assess longitudinal protein changes over 6 and 12 months and a random regression coefficient within the linear mixed model to investigate the correlation between proteins and their baseline clinical characteristics.Results: P-tau was lower in PDs only at baseline, but during a year, p-tau increased more rapidly in PDs than controls. Aβ1−42 was not significantly different between groups at any separate timepoint; however, when assessed longitudinally, Aβ1−42 showed significant changes in both groups. Conversely, t-tau and α-syn differed significantly between groups, but their longitudinal changes were not significant in either of the groups. Moreover, all proteins' baseline levels, except p-tau, could determine estimated longitudinal tau changes. Baseline RBDSQ scores but not UPDRS III, MoCA, or UPSIT scores were predictive of longitudinal increase in α-syn levels.Conclusion: Longitudinal changes in levels of CSF proteins are related to each other and could help researchers further understand PD pathology. In addition, RBD seems to be a potential prognostic factor for PD progression. However, in order to reach a consensus, longer follow-up times are required. As thelife expectancy of population increases, neurodegenerative diseases like Parkinson's disease (PD) become the spotlight in medicine . PD is cMoreover, it is known that PD symptoms progress heterogeneously  which cain vivo and accelerate cognitive dysfunction  have been previously shown to be decreased or normal in PD patients  in PD and other synucleinopathies compared to healthy controls –20. In apatients –21. Aparbaseline , 23.1−42) levels in PD patients have reached heterogeneous results e.g., the study conducted by Hall et al. has shown significantly increasing levels of all proteins except Aβ1−42 during a two-year follow-up, in which increase of p-tau was associated with motor symptoms aggravation and cognitive decline  database freely available at rotocols . The PPMCSF was collected by using standardized lumbar puncture procedures. CSF was collected into siliconized polypropylene tubes and the first 1–2 mL of CSF of sent to the site's local laboratory for routine testing for cell count, total protein level, and glucose level. An additional 15–20 mL of CSF was transferred into 15-mL conical propylene tubes at room temperature, mixed gently, centrifuged at 2,000 g for 10 min at room temperature, and transferred into 1.5-mL pre-cooled siliconized polypropylene aliquot tubes followed by immediate freezing on dry ice. The frozen aliquots of CSF were shipped overnight to the PPMI Biorepository Core Laboratories on dry ice and then thawed, aliquoted into 0.5-mL siliconized polypropylene tubes, refrozen once, and stored at −80°C. The coded frozen aliquots of CSF were transferred from the PPMI Biorepository Core laboratories to the University of Pennsylvania and to Covance for analyses. CSF Aβ1-42, t-tau and p-tau were measured using the xMAP-Luminex platform with INNOBIA AlzBio3 immunoassay kit-based reagents . CSF α-syn and CSF hemoglobin levels were analyzed using appropriate commercially available sandwich type ELISA kits  .http://www.R-project.org/. A p-value of < 0.05 was considered statistically significant.We used Kolmogorov-Smirnov test as well as a Q-Q plot to check for the normal distribution of data. To present data, we used mean, standard deviation, median, and range. To find the difference between the two groups during the study period, we used the Mann-Whitney test. To assess the changes within each group  considering the correlation of measurements , we used a linear mixed model within each group. In this analysis, multiple comparison corrections were performed by Bonferroni method. Other linear mixed models with the interaction of time and groups were used to test the difference of trends in parameters during the study course between the two groups. Correlation of different parameters and their changes during the study course was assessed with Pearson correlation. To adjust for the probable confounding effect of age, sex, and disease duration (in PD group) we used partial correlation coefficient. Finally, to evaluate the effect of baseline values of biomarkers on their own changes and the changes of other biomarkers, we used a two-step procedure: in the first step, we obtained the estimated mean change of each parameter on each subject with the use of random regression coefficients of a linear mixed model. In the next step, we used correlation coefficient to evaluate the relation of these estimations with baseline values of all parameters. Also, the correlation of different biomarkers' estimated changes was calculated. All statistical analyses were performed by R , Demographics and clinical scores of study participants and their comparison are given in Table On the whole, age and disease duration were correlated with levels of some CSF biomarkers at different time points in PD and controls.1−42 were correlated with age in the control group and levels of t-tau showed correlation with age in PD group at all time points. Percentage of changes in levels of t-tau and Aβ1−42 were correlated with age in both groups.Values of Aβ1−42 during 6 months was correlated with disease duration. Percentage of changes during one year, only for t-tau and Aβ1−42, was correlated with disease duration.Baseline values of p-tau and t-tau in PD groups were correlated with disease duration. Also, percentage of changes of p-tau and Aβ1−42 showed no significant difference between the two groups at any time point. . AβOn the whole, the pattern of changes (increase or decrease) of biomarkers was not different between controls and PD group, except for total tau for which there was a different pattern of change between the two groups. Also, the trend of changes based on interaction analysis of time and group, within the linear mixed model, was not significantly different between the two groups. Table .None of the CSF biomarkers showed any significant correlation with each other in control group when adjusted for age and sex although several significant correlations were observed in control group when not adjusted. Correlations between levels of the same biomarker at different time points were significant in the PD group whether adjustment for age, gender, and disease duration was done or not. However, there were some exceptions for t-tau.1−42 (P = 0.002). . Moreover, estimated changes of p-tau and Aβ1−42 were correlated positively (P < 0.001). (Table Estimated changes of p-tau and t-tau were positively correlated to each other . Table .p = 0.0013) (data not shown).Using linear mixed models for changes of p-tau, t-tau, α-syn, and Aβ CSF levels, with interaction of baseline UPDRS III, RBDSQ, UPSIT, and MoCA scores, no significant association between these clinical scores with estimated changes of each CSF marker was observed except for the significant correlation between estimated changes in alpha-synuclein level and RBDSQ scores  in prediction of longitudinal changes of CSF markers in PD patients. In brief, all CSF markers were lower in PDs compared to controls except AβThis study has shown a reduced level of α-syn and t-tau at all time-points and p-tau only at baseline in patients' CSF compared to healthy controls'. This observation is in line with the most recent studies , 20, 21;P-tau levels were significantly lower in the PD group compared to controls and rose significantly during the 1-year follow-up time, only in the PD group. Therefore, the p-tau level differences between PD and control groups vanished at the 6-month and one-year time points. On the other hand, t-tau levels were different between the two groups at all time points despite their non-significant longitudinal changes. In the study by Boungiorno et al., t-tau but not p-tau longitudinal increase was significant. Hall et al., however, found an increase in both. Thus, due to the high rate of p-tau changes during the disease course, at least in some time points, there would be an overlap with p-tau levels in controls, which challanges its diagnostic value. Thus, we hypothesize that CSF t-tau is a better candidate as a diagnostic tool than p-tau, which needs to be further investigated.Our study does not provide the means to discover the underlying mechanism of these protein level differences; however, we speculate that at the very early stages of PD, i.e., at our baseline, there is an accumulation of toxic p-tau in form of neurofibrillary tangles (NFTs) in neurons, which causes absorption of more functional tau molecules from extracellular space as a compensatory mechanism in order to reverse back the function of neuronal trafficking. The similar mechanisms are the case for α-syn. In contrast, it has been shown that due to the altered processing of α-syn and occasionally increased transcription of SNCA gene, neurons secrete exosomal α-syn and propagate PD pathology , 33. MayIt is worthy of note that in AD, CSF tau levels are elevated from the earliest phases of the disease , 37 exce1−42 levels were not significantly different between the two groups at any time point although significant longitudinal increase of Aβ1−42 was observed in both groups. However, the study done by Buongiorno et al. has shown the opposite, i.e., a decrease in Aβ levels during 18 months, which was associated with cognitive decline in non-demented PD patients (1−42) in contrast to the mentioned study, which evaluated the level of Aβ42, and this might explain the discrepancies in results. The progressive reduction of Aβ1−42 levels in other studies (both in AD and PD) can be mainly attributed to its extracellular accumulation but we can not justify the observed increase in Aβ1−42 in our study.On the other hand, Aβpatients . In addiOn the whole, it seems that the difference in clinical and pathophysiological characteristics contributes to variability in disease progression and different results of such studies.42, non-significantly changed level of CSF proteins in patients with short disease duration (<5 years from diagnosis) and controls were observed  . This isobserved . SimilarThis study showed the correlation between different CSF markers adjusted for age and gender at different time points in PD patients. No significant correlation found in control group when adjusted for age and gender. In addition, lower baseline levels of p-tau, t-tau, and α-syn were correlated with lower rate of estimated decrease of t-tau within a linear mixed model.The disappearance of any association between CSF biomarkers in controls after adjusting for age and gender made us infer that any observed correlation between protein concentrations in healthy people was confounded by age and gender. However, in PD group correlations remained significant after this adjustment; meaning that there must be a pathological and disease-specific link between these proteinopathies. Therefore, we suspect that any correlation observed between levels of different biomarkers in control cases of previous PPMI studies was the result of not adjusting them for age and gender .t-tau decrease might be due to lower baseline α-syn CSF levels being associated with more toxic α-syn accumulation in Lewy bodies. Actually, accumulation of α-syn in Lewy body has a seeding effect on tau oligomerization and accumulation  Aβ level, which is necessary for tau phosphorylation and accumulation  sleep Behavior Disorder (RBD) can be a clue to early diagnosis of neurodegenerative diseases , specifiIn this study, we detected significant increase in CSF levels of alpha-synuclein in PD-RBD patients. Although a previous cross-sectional study in PD patients have revealed no association between CSF alpha-synuclein levels and RBD , prior iImportantly, changes in other CSF protein levels were not associated with UPDRS III, MoCA, and UPSIT scores. This might refer to the fact that motor symptoms, cognitive impairment, and olfaction dysfunction are not appropriate predictors of progression of neuronal degeneration in PD patients. In this study we have not assessed progression of different PD symptoms in association with the baseline clinical scores and it is worthy of note that although these CSF biomarkers pattern of changes in patients with various clinical entities at baseline might elucidate the speed of neurodegeneration to some extent, it does not necessarily mean that clinical manifestations progress is in line with CSF marker changes. However, it might help identify the speed of neurodegeneration at a molecular level and design more targeted therapies.Although there are some positive points to this study like recruitment of drug-naïve patients, it suffers from some limitations like short follow-up time. To reach a more comprehensive conclusion, further longitudinal assessments regarding the predictive role of CSF proteins and their different species in patients at different stages of the disease and with longer follow-ups are suggested.In addition, the patients in this study were quite heterogeneous in terms of disease duration which renders this study to some limitations; for instance, the patterns of CSF protein changes of different subgroups at different disease stages may be different.In addition, in the manuscript we have assumed that lower CSF concentration of proteins is due to their accumulation in extracellular and intracellular spaces. This assumption might not be the case and several undiscovered mechanisms may play a role in determining the CSF concentrations of these proteins. There are some other limitations such as various degrees of CSF concentrations due to altered states of hydration and the position of proteins in a caudo-rostral column from the lumbar region to the cerebral origin. Moreover, clinical application of the difficult and invasive procedure of lumbar puncture to determine CSF markers as a way to follow-up the patients suffering from neurodegenerative diseases seems unlikely and such studies may just help enlighten the pathological mechanisms of proteinopathies.In sum, it seems that α-syn, t-tau, p-tau, and Aβ play a role in PD pathology and have bidirectional interactions with each other, which mostly converge on tau pathology and lead to changes of CSF tau levels. Most noticeably, colocalization of α-syn and t-tau molecules in intracellular inclusions cause a reduction in CSF α-syn and t-tau levels, in contrast to AD without synucleinopathies which is associated with higher levels of CSF tau.Importantly, among the clinical measures we applied, only RBD was predictive of α-syn increase during time. This might refer to the fact that evaluating patients for RBD at baseline is far more important than motor, olfaction, or cognitive assessment for prognostic purposes.We suggest that different subtypes of PD patients progress heterogeneously. Thus, conducting similar studies in patient subtypes with various clinical symptoms and genetic, epigenetic, and environmental predispositions may be helpful.In addition, most of the studies have shown the role of lower Aβ levels at baseline in predicting cognitive decline in the future . HoweverAll procedures performed here, including human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.MD, MA, AA-G, SP, AK contributed to the conception and design of the study; MY, AA-G, MD, SP contributed to data collection and analysis; and MD, SP, AK, AA-G contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CH and handling Editor declared their shared affiliation.