Altered Cerebrospinal Fluid Neurofilament Light Chain but Not Neurogranin Levels Are Associated with Response to Ocrelizumab Treatment in Relapsing-Remitting Multiple Sclerosis: A Preliminary Study.

INTRODUCTION: Ocrelizumab is a CD20-targeting monoclonal antibody used for treatment of multiple sclerosis (MS). Serum and cerebrospinal fluid (CSF) neurofilament light (NFL) chain levels are reduced in MS patients under ocrelizumab treatment indicating a preventive action against neuro-axonal degeneration. Our aim, in this preliminary study, was to explore the impact of ocrelizumab treatment on synaptic integrity through assessment of neurogranin levels. METHODS: Thirteen relapsing-remitting multiple sclerosis (RRMS) patients resistant to first-line immunomodulating agents were enrolled and followed up for 24 months under ocrelizumab treatment. Disease activity was monitored by periodic EDSS, MSSS, and cranial-spinal MRI assessments. No evidence of disease activity (NEDA)-3 was determined, and CSF levels of NFL (marker of neuro-axonal integrity) and neurogranin (marker of synaptic integrity) were measured by ELISA at baseline and 12-month ocrelizumab treatment. RESULTS: Seven RRMS patients, who preserved NEDA-3 status during 24-month follow-up, showed ≥30% NFL level decrease, whereas 6 patients with stable/increased NFL levels displayed relapse, MRI lesion, or disability progression. Although most RRMS patients exhibited increased CSF levels of neurogranin under ocrelizumab treatment, patients with and without neurogranin level increase did not differ in terms of clinical features and NEDA-3 status. Baseline neurogranin levels negatively correlated with baseline EDSS scores. CONCLUSION: Our results confirm that NFL effectively monitors treatment response of RRMS patients under ocrelizumab treatment. Neurogranin does not appear to exhibit a similar benefit in screening of RRMS disease activity. Nevertheless, lower neurogranin levels are associated with increased disability in RRMS indicating a potential disease activity biomarker function.

Immunomodulatory lipid mediator profiling of cerebrospinal fluid following surgery in older adults.

Arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) derived lipids play key roles in initiating and resolving inflammation. Neuro-inflammation is thought to play a causal role in perioperative neurocognitive disorders, yet the role of these lipids in the human central nervous system in such disorders is unclear. Here we used liquid chromatography-mass spectrometry to quantify AA, DHA, and EPA derived lipid levels in non-centrifuged cerebrospinal fluid (CSF), centrifuged CSF pellets, and centrifuged CSF supernatants of older adults obtained before, 24 h and 6 weeks after surgery. GAGE analysis was used to determine AA, DHA and EPA metabolite pathway changes over time. Lipid mediators derived from AA, DHA and EPA were detected in all sample types. Postoperative lipid mediator changes were not significant in non-centrifuged CSF (p > 0.05 for all three pathways). The AA metabolite pathway showed significant changes in centrifuged CSF pellets and supernatants from before to 24 h after surgery (p = 0.0000247, p = 0.0155 respectively), from before to 6 weeks after surgery (p = 0.0000497, p = 0.0155, respectively), and from 24 h to 6 weeks after surgery (p = 0.0000499, p = 0.00363, respectively). These findings indicate that AA, DHA, and EPA derived lipids are detectable in human CSF, and the AA metabolite pathway shows postoperative changes in centrifuged CSF pellets and supernatants.

Relation of intrathecal oligoclonal band production to inflammatory mediator and immunotherapy response in 208 children with OMS.

In 208 children with opsoclonus-myoclonus syndrome (OMS), CSF IgG oligoclonal bands (OCB) and 22 immunomarkers in CSF and 21 in serum/blood were measured. In 36 untreated OMS, 58% were OCB(+), whereas 55% of treated OMS were OCB(-). OCB positivity or negativity did not alter concentrations or frequencies of immunomarkers. The phenotypes of OCB(+) and OCB(-) patients were not distinctive. CSF B cells were expanded in untreated OMS regardless of OCB positivity. These data reveal a much higher frequency of OCB positivity in untreated OMS than previously realized and a disconnect between intrathecal OCB and inflammatory mediator production.

Kappa free light chains in cerebrospinal fluid to identify patients with oligoclonal bands.

BACKGROUND AND PURPOSE: The gold standard for detection of intrathecal immunoglobulin synthesis is the measurement of oligoclonal bands (OCB). In the diagnosis of multiple sclerosis, the kappa free light chains (KFLC) index has a similar sensitivity and specificity as OCB. This study investigated whether determination of the KFLC index could be used to predict the presence of OCB. METHODS: The KFLC index was determined prospectively from 295 paired serum and cerebrospinal fluid samples. KFLC were determined by nephelometry using the N Latex FLC kappa kit (Siemens Healthcare Diagnostics Products GmbH) on the BN Prospec analyzer (Siemens Healthcare Diagnostics Products GmbH) (cohort I). A cut-off value was determined using receiver operating characteristic analysis in relation to OCB positivity. These results were validated prospectively in 96 samples (cohort II) as well as retrospectively in samples of 46 patients known to be OCB positive (cohort III). We also compared the agreement of two commercially available nephelometric KFLC assays. RESULTS: In cohort I, a KFLC index of 3.61 yielded 100% sensitivity and 88% specificity. Prospective validation of this cut-off value in cohort II showed 92% sensitivity and 96% specificity. In cohort III, a sensitivity of 93% was achieved. Comparison of Siemens and Binding Site (Birmingham, UK) assays revealed good agreement (r2  = 0.86). CONCLUSIONS: The KFLC index with a cut-off value of 3.61 had high diagnostic accuracy to predict immunoglobulin G synthesis via OCB analysis. Determination of the KFLC index provided a quantitative parameter that could be used as an initial diagnostic step in inflammatory central nervous system disorders before measuring OCB.

New insights into the pharmacokinetics and pharmacodynamics of natalizumab treatment for patients with multiple sclerosis, obtained from clinical and in vitro studies.

BACKGROUND: The monoclonal antibody natalizumab (NAT) inhibits the migration of lymphocytes throughout the blood-brain barrier by blocking very late antigen (VLA)-4 interactions, thereby reducing inflammatory central nervous system (CNS) activity in patients with multiple sclerosis (MS). We evaluated the effects of different NAT treatment regimens. METHODS: We developed and optimised a NAT assay to measure free NAT, cell-bound NAT and VLA-4 expression levels in blood and cerebrospinal fluid (CSF) of patients using standard and prolonged treatment intervals and after the cessation of therapy. RESULTS: In paired CSF and blood samples of NAT-treated MS patients, NAT concentrations in CSF were approximately 100-fold lower than those in serum. Cell-bound NAT and mean VLA-4 expression levels in CSF were comparable with those in blood. After the cessation of therapy, the kinetics of free NAT, cell-bound NAT and VLA-4 expression levels differed. Prolonged intervals greater than 4 weeks between infusions caused a gradual reduction of free and cell-bound NAT concentrations. Sera from patients with and without NAT-neutralising antibodies could be identified in a blinded assessment. The NAT-neutralising antibodies removed NAT from the cell surface in vivo and in vitro. Intercellular NAT exchange was detected in vitro. CONCLUSIONS: Incorporating assays to measure free and cell-bound NAT into clinical practice can help to determine the optimal individual NAT dosing regimen for patients with MS.

The effects of intrathecal rituximab on biomarkers in multiple sclerosis.

OBJECTIVES: Clinical trials of IV-rituximab have proved successful. It is unclear whether intrathecal (IT)-rituximab is more efficacious at lower doses. We examine its effects on B-cell biomarkers. METHODS: MS patients received IT-rituximab at 3 time-points. CSF and serum samples were obtained at up to 5 time-points (days 0, 7, 14, 56 and 112). Serum and CSF BAFF and CXCL13, and CSF kappa and lambda free light chains (FLC) were measured. Flow cytometry was performed, examining effects on lymphocytes, CD3-19+ and CD3-20+ cells. RESULTS: CSF BAFF fell following rituximab (p=0.0091 absolute values, p=0.0284 change from baseline) whilst serum BAFF increased across time-points 1-4 (p=0.0005 absolute values, p=0.0017 change from baseline). There were significant reductions in CD20+ and CD19+ cells in blood from baseline (p<0.0001) but not in CSF. CSF kappa FLC levels significantly increased (p=0.0480). CONCLUSIONS: BAFF levels fall in CSF but increase in serum following IT-rituximab. Rituximab appears to act peripherally with dramatic decreases in peripheral CD20+ and CD19+ cells. It is likely that CSF B-cell counts were too low to enable differences to be seen. The rapid reduction in B-cells suggests rituximab has immediate effects. The profound depletion of B-cells, despite low doses of rituximab, underlines rituximab's efficacy.

Sex differences in soluble markers vary before and after the initiation of antiretroviral therapy in chronically HIV-infected individuals.

OBJECTIVE: To evaluate differences in soluble inflammatory markers between chronically HIV-infected men and women, with or without cognitive impairment, and in response to treatment. DESIGN: Soluble biomarkers were measured in cryopreserved plasma and cerebrospinal fluid (CSF) of 60 treatment-naïve individuals (25 men and 35 women) with chronic HIV infection and 18 HIV-uninfected controls (9 men and 9 women) from Thailand. Following enrollment, participants began combination antiretroviral therapy and were evaluated for expression of these markers after 48 weeks. METHODS: Plasma and CSF levels of 19 soluble biomarkers (IFN-γ, TNFα, TNF-RII, IL-1α, IL-1ß, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-15, MCP-1, t-Tau, IP-10, neopterin, IFNα, I-FABP, and sCD14) were measured using either a multiparameter or standard ELISA assay. RESULTS: Prior to combination antiretroviral therapy, women with impaired cognition had elevated levels of neopterin and TNF-RII compared with women with normal cognition in both the plasma and CSF; however, levels did not differ between cognitively impaired or normal men. In a secondary outcome-hypothesis generating analysis, sex differences were also pronounced in plasma levels of MCP-1, IL-10, I-FABP, and sCD14 in response to treatment. Neopterin, IP-10, TNFα, TNF-RII, IFNα, MCP-1, IL-8, I-FABP, and sCD14 plasma levels remained elevated following 48 weeks of therapy in both sexes compared with uninfected controls. CONCLUSION: We provide evidence of sustained immune activation after 48 weeks of treatment and identify possible sex differences in biomarkers previously linked to cognitive impairment, chronic inflammation, and gut integrity that may contribute to immunological differences between sexes in relationship to disease progression and response to therapy.

Determination of seven selected neuro- and immunomodulatory steroids in human cerebrospinal fluid and plasma using LC-MS/MS.

Dehydroepiandrosterone (DHEA) and its 7-oxo- and 7-hydroxy-metabolites occurring in the brain are considered neurosteroids. Metabolism of the latter is catalysed by 11ß-hydroxysteroid dehydrogenase (11ß-HSD) which also interconverts cortisol and cortisone. The concurrent metabolic reaction to DHEA 7-hydroxylation is the formation of 16α-hydroxy-DHEA. The LC-MS/MS method using triple stage quadrupole-mass spectrometer was developed for simultaneous quantification of free DHEA, 7α-hydroxy-DHEA, 7ß-hydroxy-DHEA, 7-oxo-DHEA, 16α-hydroxy-DHEA, cortisol and cortisone in human plasma and cerebrospinal fluid (CSF). The method employs 500 µL of human plasma and 3000 µL of CSF extracted with diethyl ether and derivatized with 2-hydrazinopyridine. It has been validated in terms of sensitivity, precision and recovery. In plasma, the following values were obtained: limit of detection: 2-50p g/mL; limit of quantification: 5-140 pg/mL; within-day precision 0.58-14.58%; between-day precision: 1.24-13.89% and recovery: 85-113.2%). For CSF, the values of limit of detection: 2-28 pg/mL; limit of quantification: 6-94 pg/mL; within-day precision; 0.63-5.48%; between-day precision: 0.88-14.59% and recovery: 85.1-109.4% were acquired. Medians and concentration ranges of detected steroids in plasma and CSF are given in subjects with excluded normal pressure hydrocephalus (n=37; 65-80 years). The method enables simultaneous quantification of steroids important for the estimation of 11ß-HSD activity in human plasma and CSF. It will be helpful in better understanding various degenerative diseases development and progression.

[YEL-AND meningoencephalitis in a 4-year-old boy consecutive to a yellow-fever vaccine]. / Méningo-encéphalite après vaccination anti-amarile : syndrome de YEL-AND.

Yellow fever is a vector-borne disease transmitted by an endemic mosquito in sub-Saharan Africa and tropical South America. It causes fever and possibly liver and renal failure with hemorrhagic signs, which may be fatal. The yellow-fever vaccine is an attenuated vaccine that is recommended for all travelers over the age of 9 months in high-risk areas. Adverse effects have been reported: minor symptoms (such as viral syndrome), hypersensitivity reactions, and major symptoms such as viscerotropic disease (YEL-AVD) and neurotropic disease (YEL-AND). The yellow-fever vaccine-associated autoimmune disease with central nervous system involvement (such as acute disseminated encephalomyelitis) associates fever and headaches, neurologic dysfunction, seizures, cerebrospinal fluid (CSF) pleocytosis, and elevated protein, with neuroimaging consistent with multifocal areas of demyelization. The presence of antibodies or virus in CSF, within 1-30 days following vaccination, and the exclusion of other causes is necessary for diagnosis. We describe herein the case of a 4-year-old child who presented with severe encephalitis consecutive to a yellow-fever vaccine, with favorable progression. Diagnosis is based on the chronology of clinical and paraclinical signs and the presence of yellow-fever-specific antibodies in CSF. The treatment consists of symptomatic treatment and immunoglobulin injection.

Comparative study of the cytokine/chemokine response in children with differing disease severity in enterovirus 71-induced hand, foot, and mouth disease.

BACKGROUND: Enterovirus 71 (EV71) infection can lead to a rapidly progressing, life-threatening, and severe neurological disease in young children, including the development of human hand, foot, and mouth disease (HFMD). This study aims to further characterize the specific immunological features in EV71-mediated HFMD patients presenting with differing degrees of disease severity. METHODOLOGY: Comprehensive cytokine and chemokine expression were broadly evaluated by cytokine antibody array in EV71-infected patients hospitalized for HFMD compared to Coxsackievirus A16-infected patients and age-matched healthy controls. More detailed analysis using Luminex-based cytokine bead array was performed in EV71-infected patients stratified into diverse clinic outcomes. Additionally, immune cell frequencies in peripheral blood and EV71-specific antibodies in plasma were also examined. PRINCIPAL FINDINGS: Expression of several cytokines and chemokines were significantly increased in plasma from EV71-infected patients compared to healthy controls, which further indicated that: (1) GM-CSF, MIP-1ß, IL-2, IL-33, and IL-23 secretion was elevated in patients who rapidly developed disease and presented with uncomplicated neurological damage; (2) G-CSF and MCP-1 were distinguishably secreted in EV71 infected very severe patients presenting with acute respiratory failure; (3) IP-10, MCP-1, IL-6, IL-8, and G-CSF levels were much higher in cerebrospinal fluid than in plasma from patients with neurological damage; (4) FACS analysis revealed that the frequency of CD19(+)HLADR(+) mature B cells dynamically changed over time during the course of hospitalization and was accompanied by dramatically increased EV71-specific antibodies. Our data provide a panoramic view of specific immune mediator and cellular immune responses of HFMD and may provide useful immunological profiles for monitoring the progress of EV71-induced fatal neurological symptoms with acute respiratory failure.

Deciphering the physiology underlying the rapid clinical effects of perispinal etanercept in Alzheimer's disease.

Excess tumor necrosis factor (TNF) plays a pivotal role in the pathogenesis of Alzheimer's disease(AD). Clinical improvement following perispinal administration of etanercept in patients with Alzheimer's disease and other forms of dementia and brain dysfunction is characteristically evident within minutes. The rapidity and constellation of the clinical effects across multiple domains (cognition, mood, memory, motor function, and attention) suggest they are mediated by non-synaptic signaling mechanisms previously unrecognized for etanercept. These mechanisms likely extend beyond the known roles of TNF as a gliotransmitter that modulates synaptic strength, synaptic scaling, and AMPA receptor trafficking. Preliminary basic science and clinical investigation suggests that perispinal administration of etanercept may lead to its rapid penetration into the cerebrospinal fluid (CSF) within the cerebral ventricles. Diffusion of large molecules into the periventricular brain parenchyma is known to occur, but this process may not be sufficient to explain the rapidity of the clinical effects. There exist populations of cells, including CSF-contacting neurons and modified ependymal cells called tanycytes, that have receptive surfaces in direct contact with the CSF. It is hypothesized that the rapid clinical effects of perispinal etanercept involve non-synaptic signal transduction across the ependymal barrier and into neuronal networks via these CSF-contacting cells. This hypothesis challenges the dogma that penetration of a therapeutic into the cerebral parenchyma through the endothelium of the cerebral vasculature (the so-called blood- brain barrier) is necessary to produce rapid clinical effects in AD. CSF-contacting cells may constitute a therapeutic target for a diverse group of brain, psychiatric and spinal disorders.

[The immunologic factors in cerebrospinal liquor under bacterial, fungous or parasitic neuro-infections: a literature review].

The prognosis of course and outcome of inflectional inflammatory diseases of central nervous system, indicators of inflammatory reaction and immune response in cerebrospinal liquor in patients has been investigated.

[The utility of QuantiFERON TB Gold for diagnosing tuberculous meningitis in children]. / Utilitatea testului quantiFERON TB gold in diagnosticul meningitei tuberculoase la copil.

MATERIAL AND METHODS: We performed QuantiFERON-TB Gold in Tube (QFT-G) in blood and CSF at 40 children diagnosed with TB meningitis and at 39 children with non TB meningitis, admitted between October 2006 and December 2009. RESULTS: The CSF analyses were suggestive for TB at 27 patients and only 14 had positive culture. The sensitivity of QFT-G in CSF was 72.72% and 69.44% in blood; specificity 96.96% in CSF and 89.18% in blood; the positive predictive value was 96% in CSF and 86.2% in blood; negative predictive value was 78.04% in CSF and 75% in blood. The sensitivity of TST was 61.76% and specificity 82.05%. The sensitivity of the culture from CSF was only 35%. The sensitivity and specificity of QFT-G was higher than TST and culture and better in CSF than in blood. CONCLUSIONS: The determination of alpha-interferon in serum and CSF is useful diagnostic marker of tuberculosis who could improve the management of TB meningitis.

Correlation of CSF proinflammatory cytokines with MRI in tuberculous meningitis.

RATIONALE AND OBJECTIVES: To demonstrate the correlation of proinflammatory cytokines (PCs), intercellular adhesion molecule (sICAM-1), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) in CSF of tuberculous meningitis (TBM) patients with magnetic resonance imaging (MRI) including diffusion tensor imaging (DTI) and also to look for the changes in imaging parameters after antitubercular treatment (ATT) in these patients. MATERIALS AND METHODS: Forty patients with TBM (median age, 27.7 years) and 30 age-/sex-matched controls were included in this study. PCs were quantified from the CSF of TBM patients at the time of hospital admission (baseline). MRI including DTI was performed at the time of baseline study and 6 months after ATT. RESULTS: Significant positive correlation of PCs with fractional anisotropy (FA) values and post-contrast signal intensity (PCSI) collected from cerebral cortical regions was observed in TBM patients. A significant positive correlation of FA values with PCSI was also observed at both time points in patient groups. At baseline study significantly high FA values were observed in patients compared to controls. Significantly decreased FA values and PCSI were observed in the patients after 6 months of ATT compared to the baseline study. CONCLUSIONS: Results of this study suggest that the DTI-derived anisotropy have the potential to delineate meningeal inflammation and it may be used in assessment of therapeutic response in TBM patients as an additional method to conventional imaging.

Rituximab levels in cerebrospinal fluid of patients with neurological autoimmune disorders.

BACKGROUND: Rituximab, a monoclonal antibody against the B-cell-specific surface protein CD20, is being evaluated for treatment of multiple sclerosis and neuromyelitis optica. Both diseases are restricted to the brain and cerebrospinal fluid (CSF). Whereas the ability of rituximab to deplete B cells in peripheral blood and tissue is well known, little information is available about the ability of rituximab to penetrate the barriers separating brain and CSF from the serum compartment. OBJECTIVE: To measure rituximab levels in serum and CSF of rituximab-treated patients and correlate them with CSF and response markers. METHODS: Fourteen paired serum/CSF samples of patients with autoimmune nervous system disorder were analyzed for up to 43 weeks after rituximab application. RESULTS: Rituximab remains detectable within the CSF after i.v. application for up to 24 weeks. Furthermore, levels of rituximab in CSF correlate significantly with the integrity of the blood CSF barrier.

Interleukin-10 and tumor necrosis factor-alpha: model of immunomodulation in multiple sclerosis.

OBJECTIVES: The mechanisms involved in the pathogenesis of relapsing-remitting multiple sclerosis are still unclear. The aim of the present study was to evaluate both cerebrospinal fluid (CSF) CD4+ CD7+ T cells and peripheral blood (PB) interleukin-10 (IL-10) as well as tumor necrosis-alpha (TNF-alpha) levels in patients with definite multiple sclerosis of the relapsing-remitting type. METHODS: To assess the above-mentioned cytokine levels we performed our test by the means of ELI-spot assay; the T-helper cell subset was assayed using flow cytometry. RESULTS: PB IL-10 levels of multiple sclerosis (MS) patients in remission were significantly (p<0.001) higher than in MS patients in the active phase. There was significant and increased evidence of TNF-alpha levels only in the MS patients in the active phase. CD4+ CD7+ T cells, characterized by a preferential Th1-like cytokine profile, were detectable only in seven patients in the active phase without evidence of a statistical significance with respect to cytokine levels. CONCLUSION: The data indicate that the production of different cytokines characterized the expression of relapsing-remitting MS. The data also suggest that is it possible to control MS using the regulatory cytokine balance.