Protein biomarkers for multiple sclerosis: semi-quantitative analysis of cerebrospinal fluid candidate protein biomarkers in different forms of multiple sclerosis.

BACKGROUND: The complex pathogenesis of multiple sclerosis, combined with an unpredictable prognosis, requires identification of disease-specific diagnostic and prognostic biomarkers. OBJECTIVE: To determine whether inflammatory proteins, such as neurofilament light chain, myelin oligodendrocyte glycoprotein and myelin basic protein, and neurodegenerative proteins, such as tau and glial fibrillary acidic protein, can serve as biomarkers for predicting the clinical subtype and prognosis of MS. METHODS: Cerebrospinal fluid and serum samples were collected from patients with a diagnosis of clinically isolated syndrome (n = 46), relapsing-remitting MS (n = 67) or primary-progressive MS (n = 22) along with controls having other non-inflammatory neurological disease (n = 22). Western blot analyses were performed for the listed proteins. Protein levels were compared among different clinical subtypes using one-way analysis of variance analysis. The k-nearest neighbour algorithm was further used to assess the predictive use of these proteins for clinical subtype classification. RESULTS: The results showed that each of tau, GFAP, MOG and NFL protein concentrations differed significantly (p < 0.001) in multiple sclerosis clinical subtypes compared with the controls. Levels of the proteins also differed between the multiple sclerosis clinical subtypes, which may be associated with the underlying disease process. Classification studies revealed that these proteins might be useful for identifying multiple sclerosis clinical subtypes. CONCLUSIONS: We showed that select biomarkers may have potential in identifying multiple sclerosis clinical subtypes. We also showed that the predictive value of the prognosis increased when using a combination of the proteins versus using them individually.

Neutralization by human serum samples of a transmissible agent isolated from the cerebrospinal fluid of neurological patients.

A transmissible cytotoxic agent thought to be associated with one or more misfolded protein(s) was found in several cerebrospinal fluid (CSF) samples from neurological patients. Since some experiments carried out to identify this unusual infectious factor showed the block of its propagation by rabbit gammaglobulins (IgGs), the search for such an activity by human IgGs was programmed. Neutralizing assays carried out using human sera as IgGs source showed a blocking property displayed by: twenty serum samples from as many patients with a diagnosis of acute infection, two of ten sera from healthy subjects and four serum samples from patients with lupus erythematous (SLE). When neutralizing sera were tested on cell cultures in immunofluorescence assays for the serum ability to label specific protein( s), similar fluorescent pictures resulted in treated and control cells. On the other hand, the SLE serum samples disclosed a granulosity of the nuclear material of cytotoxic cells in accordance with the DNA apoptotic laddering reported in previous papers. Oxidative disorders, as suggested by the immunoblotting analysis of the antioxidant enzymes Mn-superoxide dismutase (SOD2) and heme-oxygenase 1 (HO-1), point to an alteration of the oxidative pathway among the causes of the DNA damage induced by the cytotoxic transmissible agent under study.

Determination of Cerebrospinal Fluid Proteome Variations by Isobaric Labeling Coupled with Strong Cation-Exchange Chromatography and Tandem Mass Spectrometry.

Cerebrospinal fluid (CSF) is in direct contact with the brain and represents a valuable source of mediators that reflect metabolic processes occurring in the central nervous system (CNS). In this sense, mass spectrometry (MS) methods have proven to be sensitive in quantifying the proteomic profiles of CSF, therefore being able to detect biomarker candidates for neurological disorders. In particular, a key development has been the use of multiplexing technologies to easily identify and quantify complex protein mixtures. This chapter describes a workflow suitable for the analysis of CSF proteome using isobaric labeling coupled to strong cation-exchange chromatography fractionation for its potential use as a biomarker discovery platform. In this case, the isobaric tags for relative and absolute quantitation (iTRAQ) label all proteins in a sample via free amines at the N-terminus and on the side chain of lysine residues. Then, the labeled samples are pooled and chromatographically fractionated. These fractions with the pooled samples are afterward analyzed by tandem mass spectrometry (MS/MS), and proteins are quantified by the relative intensities of the reporter ions in the MS/MS spectra, simultaneously obtaining the amino acid sequence. This method complements the neuroproteomic toolbox to identify new protein biomarkers not only for the early clinical diagnosis and disease staging of CNS-related disorders but also to elucidate the molecular mechanisms related to the pathophysiology of these symptoms.

Cystatin C and the risk of death and cardiovascular events among elderly persons.

BACKGROUND: Cystatin C is a serum measure of renal function that appears to be independent of age, sex, and lean muscle mass. We compared creatinine and cystatin C levels as predictors of mortality from cardiovascular causes and from all causes in the Cardiovascular Health Study, a cohort study of elderly persons living in the community. METHODS: Creatinine and cystatin C were measured in serum samples collected from 4637 participants at the study visit in 1992 or 1993; follow-up continued until June 30, 2001. For each measure, the study population was divided into quintiles, with the fifth quintile subdivided into thirds (designated 5a, 5b, and 5c). RESULTS: Higher cystatin C levels were directly associated, in a dose-response manner, with a higher risk of death from all causes. As compared with the first quintile, the hazard ratios (and 95 percent confidence intervals) for death were as follows: second quintile, 1.08 (0.86 to 1.35); third quintile, 1.23 (1.00 to 1.53); fourth quintile, 1.34 (1.09 to 1.66); quintile 5a, 1.77 (1.34 to 2.26); 5b, 2.18 (1.72 to 2.78); and 5c, 2.58 (2.03 to 3.27). In contrast, the association of creatinine categories with mortality from all causes appeared to be J-shaped. As compared with the two lowest quintiles combined (cystatin C level, < or =0.99 mg per liter), the highest quintile of cystatin C (> or =1.29 mg per liter) was associated with a significantly elevated risk of death from cardiovascular causes (hazard ratio, 2.27 [1.73 to 2.97]), myocardial infarction (hazard ratio, 1.48 [1.08 to 2.02]), and stroke (hazard ratio, 1.47 [ 1.09 to 1.96]) after multivariate adjustment. The fifth quintile of creatinine, as compared with the first quintile, was not independently associated with any of these three outcomes. CONCLUSIONS: Cystatin C, a serum measure of renal function, is a stronger predictor of the risk of death and cardiovascular events in elderly persons than is creatinine.

[Interest of cystatin C for individual dosing of anticancer drugs cleared by the kidneys]. / Intérêt de la cystatine C pour l'adaptation de posologie des médicaments anticancéreux à élimination rénale.

Cystatin C is a protein freely filtered in the renal glomerulus, then reabsorbed and completely metabolised within the tubular cells. The possibility to predict the clearance of compounds eliminated by the kidneys (and then to control their interindividual variability) was evaluated for two cytotoxic drugs (carboplatin and topotecan) in adults and EDTA (ethylene diamine tetraacetic acid), a compound used to determine the glomerular filtration rate in children. The population pharmacokinetic approach based on NONMEM program was used. For each of the three compounds, the cystatin C serum level was better predictive of clearance than that of creatinine. Moreover, for carboplatin and EDTA, the best equation between clearance and patients' characteristics included both cystatin C and creatinine level. A generalisation of cystatin C assay would contribute to standardise the clinical practices in Oncology.

High resolution protein electrophoresis of 100 paired canine cerebrospinal fluid and serum.

This study was performed to investigate the diagnostic relevance of cerebrospinal fluid (CSF) high resolution electrophoresis. The laboratory technique was applied to 100 paired samples of canine CSF and serum, with paired samples tested during the same analytical run, as recommended in human medicine. Ninety four of the dogs had a neurological disease and 6 healthy dogs served as a control group. A strong linear correlation between CSF total protein concentration and the albumin quota (AQ) was found in the control group and in the inflammatory (infectious or noninfectious), neoplastic, and miscellaneous groups: AQ = 0.015 CSF total protein--0.102, r = 0.990. This correlation suggests that an increased CSF total protein concentration can be an indicator of blood brain barrier dysfunction. The highest median AQ value was found in the aseptic suppurative meningitis group, but no statistical differences were found between this and the other groups. The AQ, calculated with this technique, did not provide any additional information. Moreover, although unexpected, the electrophoretic profiles were not characteristic of any particular disease. In conclusion, this study did not confirm high resolution electrophoresis of paired CSF and serum samples to be a valuable ancillary diagnostic tool for canine neurological diseases.

[Posttraumatic meningitis: incidence, bacteriology, and outcomes]. / La meningite post-traumatique: incidence, microbiologie et pronostic.

BACKGROUND AND PURPOSE: The aim of our study was to search for the incidence, the responsible organisms and the favoring causes of death of post-traumatic meningitis (PTM). METHODS: This retrospective study was conducted over a seven-year period (January 1st, 1996 - December 31, 2002) in the ICU and the neurosurgery department of the Habib-Bourguiba University Hospital, Sfax, Tunisia. RESULTS: Over the study period, 38 patients presented PTM (0.96% of patients hospitalized for head injury), 92% of them had received antibiotic prophylaxis on admission. Mean time between head injury and the diagnosis of PTM was 9+/- 8 days (range: 2-34 days). The most common isolated organisms were multidrug resistant A. baumanii, and K. pneumoniae and reduced susceptibility S. pneumoniae. Factors predictive of prognosis in the 14 days following the diagnosis of meningitis were Glasgow coma score (GCS) on the day of diagnosis of PTM, absence of nuchal rigidity, CSF protein, CSF/blood glucose ratio, and S. pneumoniae as the causal agent of PTM. CONCLUSIONS: Antibioprophylaxis in patients with head trauma must be avoided to prevent the emergence of multidrug resistant bacteria when PTM occurs. GCS on the day of diagnosis of PTM, CSF protein concentration, CSF/blood glucose ratio, and S. pneumoniae as the causal agent of PTM are predictive factors of mortality of patients with PTM.

Intrathecal synthesis of monocyte chemoattractant protein-1 (MCP-1) in amyotrophic lateral sclerosis: further evidence for microglial activation in neurodegeneration.

Autopsy studies and animal experiments suggest that microglial inflammation contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS). Monocyte-chemoattractant protein (MCP-1) might play an important role in microglial recruitment. We studied MCP-1 levels in sera and cerebrospinal fluid of 29 ALS patients and compared the results with 11 control patients with tension headache. The MCP-1 level was determined using enzyme-linked immunosorbent assays (ELISA). A significant increase in cerebrospinal fluid MCP-1 level but not serum level was seen in the patients with ALS compared to the control subjects. These results suggest that cerebrospinal fluid MCP-1 activity may be a sensitive marker for neuroinflammation in ALS useful for monitoring treatment trials in ALS.

Neuro-inflammatory risk factors for treatment failure in "early second stage" sleeping sickness patients treated with pentamidine.

In a clinical trial on efficacy of Pentamidine in second stage Trypanosoma brucei gambiense patients with /=4 and LATEX/T.b. gambiense positive CSF, were associated with treatment failure. Detection of intrathecal IgM synthesis is valuable for assessment of brain involvement and treatment decision.

Proteins in cerebrospinal fluid and blood: barriers, CSF flow rate and source-related dynamics.

Cerebrospinal fluid (CSF) routine analysis for diagnosis of neurological diseases is based on the concepts for discrimination of blood-derived and brain-derived immunoglobulin fractions in CSF. The actual molecular flux/CSF flow theory of the blood/CSF barrier function, which founded the hyperbolic discrimination lines in quotient diagrams, is derived from the laws of molecular diffusion combined with CSF flow rate. It emerged from this theory that the decrease of CSF flow rate is sufficient to explain quantitatively the increase of CSF protein concentrations as observed in many neurological diseases. With this concept of CSF flow rate as the modulator of the normal and pathological blood-CSF barrier function, we got for the first time a theoretical frame work to explain also quantitatively the dynamics of brain-derived proteins and their source related (neurons and glial cells or leptomeningal cells) differences. The review of the anatomical, physiological and biophysical knowledge points to the new interpretations: The changing albumin quotient is an indicator of changing CSF flow rate and not for a morphological "leakage" of the blood-brain barrier. As an application of these concepts the dynamics of brain-derived molecules in blood are discussed with two examples: beta trace protein, flowing with CSF into venous blood, and neuron-specific enolase, passing from tissue into blood the opposite direction of serum proteins, again a gradient-dependent protein diffusion across the intact blood vessel wall.

Inverse correlation between disappearance of intrathecally injected 111In-DTPA from CSF with CSF protein content and CSF-to-serum albumin ratio.

By means of cerebrospinal fluid (CSF) scintigraphy with 111In-DTPA injected following lumbar puncture in 18 patients after meningitis (12), with traumatic head injury (4), cholesteatoma (1) or a communicating hydrocephalus (1) the hypothesis of whether slow movement of CSF may contribute to the elevation of CSF protein and albumin content in neurological diseases other than spinal block was tested. The ratios of the count rates over the head (geometric mean of anterior and posterior view) at 23-25 h to 4-6 h after 111In-DTPA application (C24 h/C5 h) and the ratio 47-49 h to 23-25 h after injection (C48 h/24 h) were taken as measures of the velocity of 111In-DTPA disappearance from CSF. Both the CSF protein content and the CSF-to-serum albumin ratio correlated with C24 h/C5 h and C48 h/C24 h. Assuming log-linear elimination between 24 and 48 h the elimination half-life of 111In-DTPA was estimated to be 12.4-131.1 h (median = 31.7 h). It was concluded that slow CSF kinetics probably are involved in the elevation of CSF protein content in several neurological diseases.

Aquaporins AQP1 and AQP4 in the cerebrospinal fluid of bacterial meningitis patients.

Aquaporins facilitate water transport through cell membranes. Due to the localization of AQP1 and AQP4 in the brain, they might contribute to cerebral edema. Our study aimed to determine whether AQP1 and AQP4 can be measured in cerebrospinal fluid (CSF), and whether there is a difference in AQP1 and AQP4 concentration between patients with bacterial meningitis (BM) and healthy controls. AQP1 and AQP4 concentrations in CSF from 35 patients with BM and 27 controls were analyzed using a commercial ELISA. The mean concentration of AQP1 in CSF was significantly elevated in patients with BM (BM: 3.8±3.4ng/ml, controls: 0.8±0.5ng/ml; p<0.001). AQP4 had a tendency to be increased, however the difference was not significant (BM: 1.8±3.1ng/ml, controls: 0.1±0.2ng/ml; p=0.092). AQP1 and AQP4 in CSF of BM patients were inversely correlated (r=-0.47, p=0.004). We could not find any other correlation between concentration of AQP1 or AQP4 in CSF and CSF leukocytes, lactate, protein, albumin CSF/serum ratio, age, a prediction score, an outcome score or the Glasgow Coma Scale at admission in patients with BM. Control patients displayed a correlation between AQP1 and the albumin CSF/serum ratio (r=0.390, p=0.040). This is the first study that detected AQP1 and AQP4 in CSF. Whether the significant elevation of AQP1 is due to a higher expression and subsequent shedding into CSF or a BM-induced cell damage needs to be determined.

Cardiopulmonary bypass increases permeability of the blood-cerebrospinal fluid barrier.

BACKGROUND: The integrity of the blood-cerebrospinal fluid (CSF) barrier during cardiopulmonary bypass (CPB) with hypothermic circulatory arrest (HCA) has not been systematically studied, especially in children. We tested the hypothesis that the blood-CSF barrier is disrupted by CPB. METHODS: The study randomized 25 piglets (mean weight, 11 kg) to five groups (5 per group): anesthesia alone (control); CPB at 37 degrees C with full-flow (FF); CPB at 25 degrees C with very low flow (LF); and HCA at 15 degrees C and 25 degrees C. pH-stat strategy was applied during CPB. An epidural catheter was inserted into the cisterna magna for collection of CSF. CSF and blood samples were collected at seven points: after induction of anesthesia (baseline), at 10, 50 and 115 minutes after start of CPB, just before the end of CPB, and at 30 and 120 minutes after CPB. Albumin levels in CSF and plasma were measured to assess blood-CSF barrier integrity and the albumin ratio (CSF/plasma) was calculated (Q(Alb)). RESULTS: In both HCA groups, the Q(Alb) was significantly higher than in the control and 37 degrees C FF groups (all p < 0.05), whereas Q(Alb) in the 37 degrees C group was not significantly different vs control. CONCLUSIONS: The blood-CSF barrier is impaired by CPB with 1 hour of 15 degrees C or 25 degrees C HCA. Further investigations are needed to understand the behavior of the blood-CSF barrier during CPB and its role in neuroprotection.

Correlation between cystatin C- and renal scan-determined glomerular filtration rate in children with spina bifida.

We report on the relationships between serum cystatin C level, glomerular filtration rate (GFR) estimated from a cystatin C-based prediction equation (that of Filler and Lepage), GFR calculated by the Schwartz formula and technetium 99m-diethylene triamine penta-acetic acid ((99)Tc-DTPA)-determined GFR in 28 children with spina bifida. All children underwent measurement of height, weight, serum cystatin C level, and serum creatinine level at the time of their renal scan. The relationship between variables was assessed by Pearson correlation. Pearson correlation for the relationship between (99)Tc-DTPA GFR and GFR calculated by the cystatin C-based equation was significant and higher than that of the relationship between (99)Tc-DTPA GFR and GFR calculated by the Schwartz equation, which was not statistically significant. The correlation for Filler GFR was 0.42 (P = 0.03) and for Schwartz GFR was 0.21 (P = 0.28). Although we use renal scan determination of GFR as the best measure, and a creatinine-based formula as the most practical measure, perhaps a formula such as that published by Filler and Lepage, which is not dependent on anthropometric data, might be a more useful (and accurate) tool for establishing GFR in children with spina bifida.

Cystatin C in paediatric nephrology. Present situation and prospects.

Cystatin C is a small basic protein with a MW of 13,359 Daltons, consisting of a non-glycosylated polypeptide chain containing 120 amino-acid residues. Cystatin C is produced in all the nucleated cells of the human body and its output rate is constant. The kidney is the main catabolic site of cystatin C, since the protein, by virtue of its low MW and its positive charge at normal pH, is freely filtered by the glomerulus and almost completely reabsorbed, catabolised and broken down in the cells of the proximal convoluted tubule. It is practically entirely filtered via the glomerular membrane, without any significant tubular secretion. The constant production rate of cystatin C in all the tissues, its elimination via the glomerular filter and its non-dependence on many extrinsic factors, including sex, age, diet, inflammation, are potentially ideal conditions for an endogenous biochemical marker of glomerular filtration. A recent method for determining cystatin C, is based on an immune reaction, could increase its clinical application. Not many studies have been conducted to date on cystatin C in children. The cystatin C concentration was higher during the first few days of life (range: 1.64-2.59 mg/L) with a rapid reduction during the first 4 months. Beyond the first year of life, cystatin C concentration became constant, with a reference range of 0.7-1.38 mg/L. On the basis of the data currently available, neonatal serum cystatin C would appear to derive from the newborn itself. In fact no correlations were found between maternal and neonatal serum cystatin C values. Cystatin C determination appears to be at least equivalent to serum creatinine measurement for the assessment of glomerular filtration rate in children. Further extended studies are needed to investigate these aspects more thoroughly in neonates.

Quantitation of gamma-trace in human biological fluids: indications for production in the central nervous system.

Gamma-Trace was purified in large amounts from urine and used for the production of a specific rabbit antiserum. An enzyme immunoassay for quantitation of gamma-trace was developed using the pure protein as a primary standard. Its sensitivity was approximately 30 microgram/l. An enzyme amplified single radial immunodiffusion was developed as well. Its sensitivity was approximately 0.3 mg/l. These assays allowed quantitation of gamma-trace in normal human biological fluids. The following results were obtained (mean +/- SD): cerebrospinal fluid: 5.8 +/- 2.2 mg/l, plasma: 1.1 +/- 0.42 mg/l, saliva: 1.8 +/- 0.88 mg/l and urine: 0.095 +/- 0.057 mg/l. Plasma samples from patients with advanced renal failure revealed gamma-trace values up to 13 times the normal mean plasma value. The results indicate a production of gamma-trace in the central nervous system and that the protein is primarily catabolized by the kidney.

[The pattern of cerebrospinal fluid proteins in infants and children. Determination of normal values (author's transl)]. / Profil protéique du liquide céphalo-rachidien chez l'enfant. Détermination des limites de référence.

Cerebrospinal fluid (CSF) protein values were measured in 652 children between the ages of 1 day and 17 years, allowing the authors to define the dynamics of the blood-brain barrier under normal conditions and during inflammation of the nervous system. The ratio of CSF albumin/serum albumin (whose upper limit was 0.65 in the study) was the best sign of alteration of blood-brain barrier permeability. The CSF IgG level, whose upper limit was 0.85 (for serum IgG between 10 and 14 g/l) is the most useful criterion for detecting an intra-thecal synthesis of IgG. Six patterns of CSF proteins are defined on the basis of immunochemical and electrophoretic studies. The ratio of CSF albumin/serum albumin and the CSF IgG level must be compared to the electrophoretic pattern of CSF proteins in order to better characterize one aspect of the blood-brain barrier under normal and pathologic conditions of the central nervous system.

Cerebrospinal fluid 'neuronal thread protein' comes from serum by passage over the blood-brain barrier.

Cerebrospinal fluid (CSF) biochemical markers for Alzheimer's disease (AD) would be of great value, both to improve clinical diagnostic accuracy and to increase our knowledge of the pathogenesis of the disorder. An increase in the CSF-level of 'neuronal thread protein' (pancreatic thread protein (PTP) immunoreactive material in the brain) has been suggested to be just such a biochemical marker. We have studied CSF 'neuronal thread protein'-like immunoreactivity (NTPLI) using a microparticle enzyme immunoassay. CSF-NTPLI did not differ significantly between AD type I (pure AD) and controls, but was significantly higher in AD type II (senile dementia) and vascular dementia (VAD) as compared with controls. Signs of blood-brain barrier (BBB) damage (elevated CSF/S albumin ratio) were found in both AD type II and in VAD, but not in AD type I. In a multiple ANOVA, with age and CSF/S albumin ratio as covariates, no significant difference in CSF-NTPLI between diagnostic groups was noted though both CSF/S albumin ratio and age (P < 0.0001 and P < 0.001 respectively) were found to influence the CSF-NTPLI level. Since BBB function was found to influence the CSF-NTPLI level, we examined whether NTPLI was present in serum. Indeed, serum NTPLI was about 40 times higher than CSF-NTPLI in neurological patients. Moreover, there was a statistically significant correlation between S-NTPLI and CSF-NTPLI. Taken together, present findings suggest that most of NTPLI in CSF comes from the serum, by passage over the BBB.(ABSTRACT TRUNCATED AT 250 WORDS)