Increased REDD1 facilitates neuronal damage after subarachnoid hemorrhage.

Regulated in development and DNA damage responses 1 (REDD1) is a highly conserved stress-response protein and can be induced by hypoxia/ischemia and DNA damage. However, it is not known whether REDD1 involves in neuronal damage caused by subarachnoid hemorrhage (SAH) that is known as one of the most important causes of disability and death worldwide. Here, we first found that SAH markedly induced the increase of REDD1 (35.467 ng/ml) in cerebrospinal fluid (CSF) of patients at acute stage (within 24 h from bleeding) compared to that of control (0.644 ng/ml). And, REDD1 level was positively correlated with severity of brain injuries (Hunt-Hess grade of SAH), but it showed an obvious decline at recovery stage 6.201 ng/ml (before discharge from hospital) because of good recovery. Moreover, it was found that the expression of REDD1 was significantly induced by hemolysate in a dose-dependent way in neurons. Knockdown of REDD1 by lentivirus encoded REDD1-shRNA could inhibit the neuronal apoptosis and LDH leakage caused by hemolysate. Importantly, the level of REDD1 in peripheral blood of SAH patients was significantly higher (4.364 ng/ml) than that of healthy persons (1.317 ng/ml) and also was positively correlated with that in CSF. Taken together, our findings provide the novel and direct evidence that REDD1 could play a critical role of process of neuronal damage caused by SAH, suggesting a new molecular target to protect brain function from SAH injury.

Cerebrospinal fluid IL-10 as an early stage discriminative marker between multiple sclerosis and neuro-Behçet disease.

Multiple Sclerosis (MS) and Neuro-Behçet's Disease (NBD) are two recurrent disorders affecting the central nervous system (CNS) by causing inflammation and irreversible damage. Inaugural clinical symptoms for both diseases might be very similar and definitive diagnosis could be delayed. The present study aimed to find out possible differences at early stages in the transcription factors/cytokines expression profiles in blood and cerebrospinal fluid (CSF) of MS and NBD patients which could be useful discriminative markers. Cytokines and transcription factors related to Th1, Th2, Th17 and T regulatory populations were studied by quantitative RT-PCR simultaneously in PBMCs and CSF, from 40 patients presenting a first episode of clinical features related to CNS inflammation and 22 controls with non inflammatory neurological diseases enrolled mainly for severe headache. The follow up of 12 months did allow a definitive diagnosis of remitting relapsing MS (RRMS) in 21 patients and of NBD in the other 19 among those with CNS inflammation compared to controls. In initial blood samples, T-bet was significantly increased in NBD patients only while IFN-γ was elevated in patients who evolved into RRMS or NBD. IL-17a, GATA-3 and IL-4 were significantly lower in RRMS patients than in the NBD group. In initial CSF samples, ROR-γt, IL-17a and IFN-γ were significantly elevated in patients compared to controls. The most striking finding was the significant increase of CSF IL-10 that we did observe in NBD patients only. Thus, we propose CSF IL-10 as a predictive marker to help clinicians discriminating between these two neurological disorders.

Elevated LRRK2 autophosphorylation in brain-derived and peripheral exosomes in LRRK2 mutation carriers.

Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene can cause late-onset Parkinson disease (PD). LRRK2 mutations increase LRRK2 kinase activities that may increase levels of LRRK2 autophosphorylation at serine 1292 (pS1292) and neurotoxicity in model systems. pS1292-LRRK2 protein can be packaged into exosomes and measured in biobanked urine. Herein we provide evidence that pS1292-LRRK2 protein is robustly expressed in cerebral spinal fluid (CSF) exosomes. In a novel cohort of Norwegian subjects with and without the G2019S-LRRK2 mutation, with and without PD, we quantified levels of pS1292-LRRK2, total LRRK2, and other exosome proteins in urine from 132 subjects and in CSF from 82 subjects. CSF and urine were collected from the same morning clinic visit in 55 of the participants. We found that total LRRK2 protein concentration was similar in exosomes purified from either CSF or urine but the levels did not correlate. pS1292-LRRK2 levels were higher in urinary exosomes from male and female subjects with a LRRK2 mutation. Male LRRK2 mutation carriers without PD had intermediate pS1292-LRRK2 levels compared to male carriers with PD and controls. However, female LRRK2 mutation carriers without PD had the same pS1292-LRRK2 levels compared to female carriers with PD. pS1292-LRRK2 levels in CSF exosomes were near saturated in most subjects, ten-fold higher on average than pS1292-LRRK2 levels in urinary exosomes, irrespective of LRRK2 mutation status or PD diagnosis. These results provide insights into the effects of LRRK2 mutations in both the periphery and brain in a well-characterized clinical population and show that LRRK2 protein in brain exosomes may be much more active than in the periphery in most subjects.

Utility of CSF Cytokine/Chemokines as Markers of Active Intrathecal Inflammation: Comparison of Demyelinating, Anti-NMDAR and Enteroviral Encephalitis.

BACKGROUND: Despite the discovery of CSF and serum diagnostic autoantibodies in autoimmune encephalitis, there are still very limited CSF biomarkers for diagnostic and monitoring purposes in children with inflammatory or autoimmune brain disease. The cause of encephalitis is unknown in up to a third of encephalitis cohorts, and it is important to differentiate infective from autoimmune encephalitis given the therapeutic implications. AIM: To study CSF cytokines and chemokines as diagnostic biomarkers of active neuroinflammation, and assess their role in differentiating demyelinating, autoimmune, and viral encephalitis. METHODS: We measured and compared 32 cytokine/chemokines using multiplex immunoassay and APRIL and BAFF using ELISA in CSF collected prior to commencing treatment from paediatric patients with confirmed acute disseminated encephalomyelitis (ADEM, n = 16), anti-NMDAR encephalitis (anti-NMDAR E, n = 11), and enteroviral encephalitis (EVE, n = 16). We generated normative data using CSF from 20 non-inflammatory neurological controls. The sensitivity of CSF cytokine/chemokines to diagnose encephalitis cases was calculated using 95th centile of control values as cut off. We correlated CSF cytokine/chemokines with disease severity and follow up outcome based on modified Rankin scale. One-way hierarchical correlational cluster analysis of molecules was performed in different encephalitis and outcome groups. RESULTS: In descending order, CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 had the best sensitivity (>79.1%) when all encephalitis patients were included. The combination of IL-6 and IFN-α was most predictive of inflammation on multiple logistic regression with area under the ROC curve 0.99 (CI 0.97-1.00). There were no differences in CSF cytokine concentrations between EVE and anti-NMDAR E, whereas ADEM showed more pronounced elevation of Th17 related (IL-17, IL-21) and Th2 (IL-4, CCL17) related cytokine/chemokines. Unlike EVE, heat map analysis showed similar clustering of cytokine/chemokine molecules in immune mediated encephalitis (ADEM and anti-NMDAR E). Th1 and B cell (CXCL13 and CXCL10) molecules clustered together in patients with severe encephalopathy at admission and worse disability at follow up in all encephalitis. There was no correlation between CSF neopterin and IFN-γ or IFN-α. CONCLUSION: A combination panel of cytokine/chemokines consisting of CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 measured using multiplex immunoassay may be used to diagnose and monitor intrathecal inflammation in the brain. Given their association with worse outcome, certain key chemokines (CXCL13, CXCL10) could represent potential therapeutic targets in encephalitis.

Increased levels of MIP-1α in CSF and serum of ALS.

OBJECTIVES: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease with complicated pathogenesis. No effective diagnostic test and cure exists for the disease at present. We detected the levels of MIP-1α in cerebrospinal fluid (CSF) and serum and then further evaluated whether MIP-1α levels correlate with the severity and progression of ALS. METHODS: We used ELISAs to detect MIP-1α levels from 58 patients with ALS and 45 age- and gender-matched controls. The patients with ALS were also clinically evaluated with the revised ALS functional rating scale (ALSFRS-r). Moreover, we followed up with 40 cases of ALS by way of call or clinic visit 4 years after enrollment in this study. Finally, we assessed the correlations between MIP-1α levels and various clinical parameters. RESULTS: We found that the levels of MIP-1α in patients with ALS significantly increased compared to controls and they were positively correlated with duration. MIP-1α showed negative correlations with disease progression rate and the decrease in ALSFRS-r. Furthermore, the cumulative survival of patients with ALS with high levels of MIP-1α exceeded patients with low MIP-1α levels. CONCLUSIONS: MIP-1α levels increased in both CSF and serum of patients with ALS, and it may be a potential neuroprotective biomarker in ALS.

Biomarkers of central nervous system inflammation in infantile and juvenile gangliosidoses.

BACKGROUND: The gangliosidoses (Tay-Sachs disease, Sandhoff disease, and GM1-gangliosidosis) are progressive neurodegenerative diseases caused by lysosomal enzyme activity deficiencies and consequent accumulation of gangliosides in the central nervous system (CNS). The infantile forms are distinguished from the juvenile forms by age of onset, rate of disease progression, and age of death. There are no approved treatments for the gangliosidoses. In search of potential biomarkers of disease, we quantified 188 analytes in CSF and serum from living human patients with longitudinal (serial) measurements. Notably, several associated with inflammation were elevated in the CSF of infantile gangliosidosis patients, and less so in more slowly progressing forms of juvenile gangliosidosis, but not in MPS disease. Thirteen CSF and two serum biomarker candidates were identified. Five candidate biomarkers were distinguished by persistent elevation in the CSF of patients with the severe infantile phenotype: ENA-78, MCP-1, MIP-1α, MIP-1ß, and TNFR2. Correspondence of abnormal elevation with other variables of disease-i.e., severity of clinical phenotype, differentiation from changes in serum, and lack of abnormality in other neurodegenerative lysosomal diseases-identifies these analytes as biomarkers of neuropathology specific to the gangliosidosis diseases.

CSF-biomarkers in Olympic boxing: diagnosis and effects of repetitive head trauma.

BACKGROUND: Sports-related head trauma is common but still there is no established laboratory test used in the diagnostics of minimal or mild traumatic brain injuries. Further the effects of recurrent head trauma on brain injury markers are unknown. The purpose of this study was to investigate the relationship between Olympic (amateur) boxing and cerebrospinal fluid (CSF) brain injury biomarkers. METHODS: The study was designed as a prospective cohort study. Thirty Olympic boxers with a minimum of 45 bouts and 25 non-boxing matched controls were included in the study. CSF samples were collected by lumbar puncture 1-6 days after a bout and after a rest period for at least 14 days. The controls were tested once. Biomarkers for acute and chronic brain injury were analysed. RESULTS: NFL (mean ± SD, 532±553 vs 135±51 ng/L p = 0.001), GFAP (496±238 vs 247±147 ng/L p<0.001), T-tau (58±26 vs 49±21 ng/L p<0.025) and S-100B (0.76±0.29 vs 0.60±0.23 ng/L p = 0.03) concentrations were significantly increased after boxing compared to controls. NFL (402±434 ng/L p = 0.004) and GFAP (369±113 ng/L p = 0.001) concentrations remained elevated after the rest period. CONCLUSION: Increased CSF levels of T-tau, NFL, GFAP, and S-100B in >80% of the boxers demonstrate that both the acute and the cumulative effect of head trauma in Olympic boxing may induce CSF biomarker changes that suggest minor central nervous injuries. The lack of normalization of NFL and GFAP after the rest period in a subgroup of boxers may indicate ongoing degeneration. The recurrent head trauma in boxing may be associated with increased risk of chronic traumatic brain injury.

Anti-myelin antibodies modulate clinical expression of childhood multiple sclerosis.

Anti-myelin basic protein (MBP) antibodies in pediatric-onset MS and controls were characterized. Serum samples were obtained from 94 children with MS and 106 controls. Paired CSF and serum were obtained from 25 children with MS at time of their initial episode of acute demyelinating syndrome (ADS). Complementary assays were applied across samples to evaluate the presence, and the physical binding properties, of anti-MBP antibodies. While the prevalence and titers of serum anti-MBP antibodies against both immature and mature forms of MBP were similar in children with MS and in controls, binding characteristics and formal Surface Plasmon Resonance (SPR) studies indicated surprisingly high binding affinities of all pediatric anti-MBP antibodies. Serum levels of anti-MBP antibodies correlated significantly with their CSF levels, and their presence in children with MS was associated with significantly increased risk of an acute disseminated encephalomyelitis-like initial clinical presentation. While antibodies to both immature and mature forms of MBP can be present as part of the normal pediatric humoral repertoire, these anti-myelin antibodies are of surprisingly high affinity, can access the CNS during inflammation, and have the capacity to modulate disease expression. Our findings identify an immune mechanism that could contribute to the observed heterogeneity in spectrum of clinical presentations in early-onset MS.

Gene expression profiling in multiple sclerosis: a disease of the central nervous system, but with relapses triggered in the periphery?

The aetiology of multiple sclerosis (MS), an autoimmune demyelinating disease of the central nervous system (CNS), includes both genetic and environmental factors, but the pathogenesis is still incompletely known. We performed gene expression profiling on paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) samples from 26 MS patients without immunomodulatory treatment, sampled in relapse or remission, and 18 controls using Human Genome U133 plus 2.0 arrays (Affymetrix). In the CSF, 939 probe sets detected differential expression in MS patients compared to controls, but none in PBMCs, confirming that CSF cells might mirror the disease processes. The regulation of selected transcripts in CSF of MS patients was confirmed by quantitative PCR. Unexpectedly however, when comparing MS patients in relapse to those in remission, 266 probe sets detected differential expression in PBMCs, but not in CSF cells, indicating the importance of events outside of the CNS in the triggering of relapse.

[Elimination of NSE and MBP into cerebrospinal fluid and blood in acute experimental autoimmune encephalomyelitis].

An immuno-enzyme assay of NSE and MBP in the cerebrospinal fluid (CFS) and blood serum of rats during the development of acute experimental autoimmune encephalomyelitis revealed the increase of NSE, a neuronal marker, and MBP, a marker for myelin. The investigation has shown that the increased level of NSE in CSF was prior to the increased concentration of MBP. The changes of these neurospecific proteins (NSP) in the serum were not the same as in the CSF. The analysis of the ratio between NSP concentrations in CSF to serum revealed the initial marked decrease of BBB permeability to MBP but not to NSE.

[Study on immunological pathogenesis of 59 patients with multiple sclerosis of different TCM syndrome types].

OBJECTIVE: To explore the immunological pathogenesis of multiple sclerosis (MS) patients of different TCM syndrome types. METHODS: Fifty-nine MS patients were assigned to two types by syndrome typing according to their clinical manifestations, the Gan-Shen yin-deficiency (GSYD, 40 cases) type and the both yin-yang deficiency (YYD, 19 cases) type. Difference of patients' age of first attack, times of relapsing, duration of disease, MRI finding and evoked potential between the two groups were compared. The immunology indexes were also compared in part of the patients (26 cases in GSYD type and 12 cases in YYD type). RESULTS: The age of first attack was later (P < 0.01), level of myelin basic protein in cerebrospinal fluid was higher (P < 0.05), in the YYD type than those in the GSYD type. Besides, the relapsing time in GSYD type, and the blood-brain barrier index and level of myelin basic protein in YYD type showed an ascending trend (P = 0.056, 0.074, 0.093, respectively). CONCLUSION: Immunological difference exists between the MS patients of GSYD type and those of YYD type.

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000.

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.