Serum matrix metalloproteinase-2 levels indicate blood-CSF barrier damage in patients with infectious meningitis.

OBJECTIVE: Protein components in cerebrospinal fluid (CSF) are maintained at a specific concentration by a dynamic gradient between the capillary and intrathecal spaces via the blood-cerebrospinal fluid barrier (BCB) in the brain and spinal cord. Permeability to proteins increases when there is structural damage to the BCB. Matrix metalloproteinase-2 (MMP-2; gelatinase A) has been shown to degrade type IV collagen, a major component of the cellular basement membrane. We analyzed alpha2 macroglobulin (alpha2M) indices and evaluated the relationship between alpha2M, as an indicator of BCB permeability, and MMP-2, which degrades the extra-cellular matrix in patients with infectious meningitis. MATERIALS AND METHODS: Albumin levels in CSF or serum were determined by turbidimetric immunoassay, or bromcresol green assay, respectively. alpha2M levels in CSF or serum were measured with enzyme-linked immunosorbent assay, or laser-nephelometry, respectively. Serum MMP-2 levels were determined by enzyme immuno assay. We calculated the alpha2M index, i.e. the ratio of alpha2M (CSF / serum) to albumin (CSF / serum; alpha2M in CSF / alpha2M in serum x albumin in serum / albumin in CSF). RESULTS: alpha2M indices were significantly increased in infectious meningitis compared to healthy controls (p < 0.05). They were highest in bacterial meningitis, and there was a significant difference between viral or mycotic and bacterial meningitis (p < 0.05). Serum MMP-2 levels were increased in infectious meningitis, being highest in bacterial meningitis, where they were significantly different from healthy controls (p < 0.05). There was a significant positive correlation between serum MMP-2 levels and alpha2M indices (r = 0.64, p < 0.0001). CONCLUSION: Markedly increased levels of serum MMP-2 in infectious, especially bacterial, meningitis may reflect the degree of damage to the BCB.

Evaluation of blood-brain barrier function by quotient alpha2 macroglobulin and its relationship with interleukin-6 and complement component 3 levels in neuropsychiatric systemic lupus erythematosus.

Although quotient of alpha2 macroglobulin (Qα2MG) was previously reported to be useful for the evaluation of blood-brain barrier (BBB) function, it is not commonly used. We therefore evaluated BBB function among the various subsets of neuropsychiatric systemic lupus erythematosus (NPSLE) using quotient Q α2MG. Furthermore, we determined the correlation between Q α2MG and cerebrospinal (CSF) interleukin (IL)-6 level and quotient complement component 3 (Q C3). To determine intrathecal production of C3, the C3 index (Q C3/Q α2MG) was also calculated. Fifty-six patients with SLE were included in this study. Of these, 48 were diagnosed with NPSLE, consisting of 30 diffuse NPSLE patients (acute confusional state (ACS): n = 14, non-ACS: n = 16) and 18 patients with focal NPSLE. CSF IL-6 concentration, and paired serum and CSF levels of α2MG and C3, were measured by enzyme-linked immuno solvent assay (ELISA). The Q α2MG, Q C3, and C3 index were then calculated. Q α2MG, Q C3, and IL-6 concentrations in the CSF were significantly elevated in NPSLE compared with non-NPSLE. Among the subsets of NPSLE, significant increases in Q α2MG, CSF IL-6, and Q C3 were observed in ACS compared with non-ACS or focal NPSLE. There was a positive correlation between CSF IL-6 level and Q α2MG, as well as between Q C3 and Q α2MG, in diffuse NPSLE. There were no significant differences in C3 index between NPSLE and non-NPSLE, as well as among the subgroups of NPSLE. Our study suggests that BBB disruption is present in ACS, and elevated levels of IL-6 and C3 in CSF in diffuse NPSLE, especially in ACS, might result from their entry to the CSF from the systemic circulation through the damaged BBB, as well as increased intrathecal production. Furthermore, Q α2MG might be useful for the evaluation of BBB integrity.

Quantification of hepcidin-25 in human cerebrospinal fluid using LC-MS/MS.

AIM: Hepcidin, the main iron metabolism regulator, can be detected in various biological fluids. Here, we describe a quantitative method of LC-MS/MS to quantify the 25 amino acid isoform of hepcidin (hepcidin-25) in human cerebrospinal fluid (CSF). Results & methodology: Samples were prepared through protein precipitation followed by solid phase extraction (SPE) and injected into a triple-quadrupole mass spectrometer. Validation of our method included determination of LOQ (0.1 ng/ml), repeatability, intermediate precision, recovery and linearity (up to 25 ng/ml). Hepcidin-25 was subsequently quantified in 36 human CSF samples and its concentration ranged from 0.21 to 3.54 ng/ml. CONCLUSION: This is the first time that hepcidin-25 can be reliably quantified in human CSF. This may open interesting perspectives for the management of iron-related neurological disorders.

Idiopathic normal pressure hydrocephalus has a different cerebrospinal fluid biomarker profile from Alzheimer's disease.

The diagnosis of idiopathic normal pressure hydrocephalus (iNPH) is sometimes complicated by concomitant Alzheimer's disease (AD) pathology. The purpose of the present study is to identify an iNPH-specific cerebrospinal fluid (CSF) biomarker dynamics and to assess its ability to differentiate iNPH from AD. Total tau (t-tau), tau phosphorylated at threonine 181 (p-tau), amyloid-ß (Aß) 42 and 40, and leucine-rich α-2-glycoprotein (LRG) were measured in 93 consecutive CSF samples consisting of 55 iNPH (46 tap test responders), 20 AD, 11 corticobasal syndrome, and 7 spinocerebeller disease. Levels of t-tau and p-tau were significantly decreased in iNPH patients especially in tap test responders compared to AD. Correlation was observed between Mini-Mental State Examination scores and Aß42 in AD (R = 0.44) and mildly in iNPH (R = 0.28). Although Aß42/40 ratio showed no significant difference between iNPH and AD (p = 0.08), the levels of Aß40 and Aß42 correlated positively with each other in iNPH (R = 0.73) but much less in AD (R = 0.26), suggesting that they have discrete amyloid clearance and pathology. LRG levels did not differ between the two. Thus, our study shows that although CSF biomarkers of iNPH patients can be affected by concomitant tau and/or amyloid pathology, CSF t-tau and p-tau are highly useful for differentiation of iNPH and AD.

Function of the blood-cerebrospinal fluid barrier in human cerebral malaria: rejection of the permeability hypothesis.

We tested the hypothesis that cerebral malaria is caused by blood-brain barrier inflammation and cerebral edema. In a group of 157 Thai patients with strictly defined cerebral malaria, cerebrospinal fluid (CSF) opening pressures were normal in 79% and were lower in fatal cases than in survivors (means +/- 1 SD, 144 +/- 58 and 167 +/- 51 mm CSF, respectively, P = 0.051). CSF: serum albumin ratios (X 10(3)) in 39 of them were significantly higher than in 61 British controls (medians 8.5 and 5.5, respectively, P = 0.04), but were no higher in 7 fatal cases. In a group of 12 patients this ratio was not significantly higher during coma than after full recovery (means +/- 1 SD, 9.0 +/- 6.2 and 6.7 +/- 4.2, respectively, P greater than 0.1). CSF alpha 2-macroglobulin concentrations were always normal. CSF : serum 77Br- ratios were elevated in 11/19 comatose cases but fell to normal 4 to 9 days later in 11/11 cases. Dexamethasone treatment had no significant effect on bromide partition. The percentage of an intravenously administered dose of 125I-human serum albumin detectable per ml of CSF 6 hr after intravenous injection was 2.4 +/- 1.3 X 10(-5) in 14 comatose patients and 4.4 +/- 4.0 X 10(-5) in 9 of them during convalescence (P greater than 0.1). These results demonstrate that the blood-CSF barrier is essentially intact in patients with cerebral malaria and give no support to the idea that cerebral edema is the cause of coma.

Correlation between protein data in normal lumbar CSF and morphological findings of choroid plexus epithelium: a biochemical corroboration of barrier transport via tight junction pores.

Specific "reference areas" were derived from relationships between the proteins prealbumin, albumin, alpha 1-acid glycoprotein, alpha 2-macroglobulin, total transferrin, IgG, IgA, IgM, and the corresponding total protein in normal lumbar CSF samples. The procedure for calculating the boundary lines of these reference areas was carried out on the basis of double standard deviations in subgroups with total protein differences of 50 ml/l within the whole range of 150-400 ml/l CSF. The resulting biochemical data, hydrodynamic radii of the individual proteins investigated, and van Deurs' and Koehler's morphological findings on the existence of pores in the barrier-forming tight junctions of the choroid plexus epithelium could be surprisingly well correlated with one another, although these morphological findings were obtained in choroid plexus of the rat brain. The correlation allowed the conclusion that proteins undergo ultrafiltration via a pattern of tight junction pores with various diameters. However, the molecular mechanism seems to include an additional facilitating component.

Third complement component in cerebrospinal fluid in neuro-Behçet's syndrome. Conversion patterns by crossed immunoelectrophoresis.

Thirteen cerebrospinal fluid specimens (CSFs) from 10 patients with neuro-Behçet's syndrome (nB) were examined for the presence of conversion products of C3 protein by means of crossed immunoelectrophoresis. C3 conversion products were found in 4 CSFs from nB patients, and the proportions of the split products to the total immunoprecipitate zones were 17, 34, 61 and 63%, respectively. CSFs with C3 conversion patterns showed higher C3 protein (beta 1C/A) levels compared with those with non-conversion, whereas the differences of C4 protein (beta 1E) levels were unremarkable. CSF with C3 conversion patterns also showed apparent inflammatory characteristics indicating blood-CSF barrier involvement which would be deduced from increased alpha 2-macroglobulin and IgA, both being regarded as sensitive internal barrier parameters: but IgA measurement was much higher than usually expected values. It is suggested that the increase in CSF C3 might be initiated by accelerated consumption of complement through the alternative pathway, leading to hypersynthetic or hyperinflux response in CSF.

Heterogeneous models for blood-cerebrospinal fluid barrier permeability to serum proteins in normal and abnormal cerebrospinal fluid/serum protein concentration gradients.

Cerebrospinal fluid (CSF)/serum concentration gradients (Q) of individual proteins (albumin, IgG, alpha 2-macroglobulin) have been studied in controls and in patients in whom the lumbar CSF flow is altered (medullary compression) or the blood-CSF barrier (BCB) function impaired (acute idiopathic polyneuropathy and acute meningoencephalitis). The analysis of relationships among protein Q has been performed by total and multiple regressions and the actual BCB permeability to individual proteins has been interpreted according to the accepted theoretical porous or vesicular BCB models. The exponential Q-IgG vs. Q-albumin total regression, and the poor Q-alpha 2-macroglobulin vs. Q-albumin regression found in controls, together with the different multiple regressions among proteins and the high Q-IgG vs. Q-albumin partial regression coefficients found in medullary compression, acute idiopathic polyneuropathy and acute meningoencephalitis, indicated that different permeability mechanisms can be postulated. Heterogeneous, fairly independent permeability BCB mechanisms maintain the normal CSF/serum protein concentration gradient. Pinocytotic vesicles or pores of radius exceeding 1000-1500 A, probably located at the capillary endothelium, account for the main serum-derived CSF protein fraction(s) with large hydrodynamic radius (R). A more selective endothelial vesicular transport with a radius of 250 A transfers a negligible amount of protein from serum into CSF. Proteins with small R also enter the CSF through a set of selective pores of radius 120 A, probably at the level of the choroidal epithelium. Pinocytotic vesicles with a radius of 250 A and increased rate of formation induce the accumulation of proteins below an obstruction of lumbar CSF flow. An increased formation rate of vesicles with a radius of 450 A can explain the increased capillary permeability in nerve roots in acute idiopathic polyneuropathy. Loss of selectivity was the main feature of BCB in acute meningoencephalitis, and it seemed to be due to pores or vesicles with a radius larger than 1000-1500 A. The heterogeneity of BCB mechanisms must be taken into account when the intrathecal synthesis of a protein, also derived from serum (for example IgG), has to be measured.

The normal cerebrospinal fluid proteins identified by means of thin-layer isoelectric focusing and crossed immunoelectrofocusing.

The cerebrospinal fluid and serum proteins from 32 healthy individuals have been examined by isoelectric focusing in a pH-gradient from 3.5--11.0. Seventeen normal proteins have been identified by means of crossed immunoelectrofocusing. The pI-values of the main fractions of these proteins have been determined. On crossed immunoelectrofocusing microheterogeneity was observed for all of the proteins except haemopexin, gamma-trace protein of CSF and prealbumin of serum. Differences between serum and CSF were observed for 6 of the proteins. Prealbumin of CSF had a lower pI-value than in serum and showed partial immunological identity with prealbumin of serum. Transferrin of CSF included several components with lower sialic acid contents than in serum. The conversion of C'3-complement from beta-1-C to beta 1-A was slower in CSF than in serum. Qualitative differences between CSF and serum were also noticed for alpha2-macroglobulin and haptoglobin. IgM was not detected in CSF. In CSF the gamma-trace protein was found on isoelectric focusing in one third of the subjects. Two subjects had 2 close tau-fractions and another two had one unidentified band in the anodal IgG-area. The gamma-trace protein did not show any proteolytic activity, nor any resemblance in charge to 4 basic enzymes or with the histones tested.

Proteinase inhibitors in cerebrospinal fluid in multiple sclerosis.

Levels of the proteinase inhibitors alpha 2-macroglobulin (alpha 2-m) and alpha 1-antitrypsin (alpha 1-at), and total protein, IgG and transferrin were measured in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurological diseases. All groups except that termed "meningitis" had similar alpha 2-m levels, but alpha 1-at and transferrin were significantly depressed in MS. Total protein levels were normal and IgG levels were elevated in MS. Serum levels of alpha 1-at were normal so the decreases observed in the CSF in MS were not due to impaired systemic production. In view of previous reports that proteinase activity is high in MS plaques and CSF, the inhibitory capacity of alpha 2-m and alpha 1-at in CSF was measured. As any decreases in inhibitory capacity noted in MS were slight, they could only be important in the local environment of a plaque where enzyme levels may be critically high.

Age-related changes in cerebrospinal fluid protein concentrations.

The blood-brain barrier (BBB) has a key role in CSF homeostasis and preservation of normal neuronal function. There has been little investigation of changes in barrier function in elderly subjects without evidence of neurological disease. The ageing brain demonstrates increased vulnerability to a variety of insults, which may reflect a deterioration in barrier performance. Direct measurement of BBB function is difficult but the CSF/serum quotients of individual proteins is the most widely used parameter. The present study sought age related changes in the CSF/serum quotients of prealbumin, albumin, immunoglobulin G and alpha 2-macroglobulin in adults with no neurological deficit. A prospective study was established, and 107 subjects (aged 18-89 yrs) were recruited from patients undergoing spinal anaesthesia or diagnostic myelography. A weak but significant positive correlation was found between the CSF/serum quotients and age for all the proteins studied, but with the exception of alpha 2-macroglobulin, these changes probably reflect age related falls in serum protein concentrations found in this study. alpha 2-Macroglobulin appears to be a sensitive indicator of barrier function for large populations, suggesting a subtle decline of BBB integrity with increasing age. This may be a true age-related change or could reflect an increased incidence of subclinical disease in an ageing population.

Laser nephelometric evaluation of albumin, IgG and alpha 2-macroglobulin: applications to the study of alterations of the blood-brain barrier.

Cerebrospinal fluid and serum levels of albumin, immunoglobulin G and alpha 2-macroglobulin were determined by laser nephelometer in various pathological conditions: Guillain-Barré syndrome amyotrophic lateral sclerosis, lumbar disc herniation. These proteins are different due to their molecular weight, spacial conformation, CSF and serum concentrations and their CSF/serum quotient reflects the status of the blood-brain barrier. In the subjects group comparisons, albumin quotient and IgG quotient show a significant differentiations. The linear regression of albumin quotient compared to alpha 2-macroglobulin quotient behaves differently in the individual groups. The protein CSF/serum quotients are helpful in distinguishing the causes of alterations in the blood-brain barrier.

Evaluation of the blood-CSF barrier by protein gradients and the humoral immune response within the central nervous system.

A linear correlation was found between the serum/cerebrospinal fluid (CSF) concentration ratios of albumin, caeruloplasmin and alpha2-macroglobulin and their hydrodynamic radii in a semilogarithmic plot. This protein gradient is used as a parameter to evaluate the blood-CSF barrier under normal and pathological conditions. Irrespective of vastly different transfer rates, the ratio/size permeation curves of proteins at the blood-CSF barrier and the blood-lymph barrier have comparable characteristics. Therefore the protein gradients found in various disease states are interpreted by means of Renkin's general law of lymph formation. Declined gradients are caused either by an increased permeability of the barrier sites or by a decreased turnover rate of the CSF within the compartment punctured. The concentration ratios of immunoglobulins are related to the gradient that is constructed with the ratios of the barrier-indicative marker proteins. As judged by comparative disc electrophoresis of serum and CSF, those disease states that are dominated by barrier impairment are used to establish the range of concentration ratios, compatible with a passive immunoglobulin transfer in any condition in which the barrier is disordered. A mathematical approach is described, which allows the quantitative evaluation of the minimal immunoglobulin portion that is synthesized within the central nervous system.

[Determination of proteins in cerebrospinal fluid of adults using two immunochemical methods (author's transl)]. / Dosage des fractions protéiques du liquide céphalorachidien de l'adulte par deux méthodes immunochimiques.

A study of CSF proteins by electrophoretical and immunological techniques suggests the Laser immunonephelemetrical method is suitable as a "Standard method". The average levels of albumin, IgG, IgM, IgA and alpha-2-macroglobulin have been determined and the correlations between these levels have been calculated. The values of albumin and IgG allow a classification of neurologic diseases according to a diagram. The variations of IgM, IgA and alpha-2-macroglobulin levels are not yet explained.

CSF alpha 2-macroglobulin and C-reactive protein as aids to rapid diagnosis of acute bacterial meningitis.

alpha 2-Macroglobulin (AMG) and C-reactive protein (CRP) levels in cerebrospinal fluid (CSF) of patients with bacterial and aseptic meningitis have been analyzed by a rate nephelometric method to determine if these acute phase proteins can aid in differentiation of bacterial from aseptic meningitis. The mean CSF concentrations of AMG and CRP were 15 and 3.5 times greater, respectively, in the bacterial compared to the aseptic meningitis group. Also, the range of AMG levels showed minimal overlap between the two groups. The elevated levels of the proteins persisted after CSF cultures became negative. Quantitation of specific acute phase proteins in CSF may assist the differentiation of bacterial from aseptic meningitis.

Sodium dodecylsulfate capillary gel electrophoretic measurement of the concentration ratios of albumin and alpha2-macroglobulin in cerebrospinal fluid and serum of patients with neurological disorders.

Sodium dodecylsulfate capillary gel electrophoresis (SDS-CGE) was applied to measure the concentration ratios of albumin (Alb) and alpha2-macroglobulin (alphaMG) in the cerebrospinal fluid (CSF) and concurrent serum samples from patients with various neurological disorders. The values of the alphaMG index in individual patients were calculated on the basis of the peak area ratios of Alb and an alphaMG subunit on the CSF and serum electropherograms. The alphaMG index value thus obtained was most prominently raised in patients with inflammatory diseases of the brain and/or meninges, suggesting that the function of the blood-brain barrier (BBB) was disturbed under the pathological conditions in the central nervous system. The measurement of the concentration ratios of Alb and alphaMG in CSF and the concurrent serum samples by the present SDS-CGE system seems to be useful as an aid in the biochemical examination of the BBB function in patients with neurological disorders.

Cerebrospinal fluid proteins in muscular dystrophy patients.

We analyzed and quantified the microcomponents of protein fractions in the cerebrospinal fluid of patients with various types of muscular dystrophy. The degenerative pattern is characterized by an increase in the prealbumin fraction and a decrease in the gamma-globulin fraction as shown in the Duchenne and congenital muscular dystrophy. The increase in CSF IgG, gamma-globulin fraction, and myelin basic protein is shown in the myotonic dystrophy. In addition to the lowness of IQ, and the abnormality of EEG and brain CT, abnormal CSF proteins obviously suggest the presence of CNS involvement in muscular dystrophy.

Microheterogeneity of CSF alpha 2-macroglobulin in multiple sclerosis and other neurological diseases. A study performed by direct immunofixation after isoelectric focusing.

Cerebrospinal Fluid (CSF) and serum alpha 2-Macroglobulin (alpha 2M) was studied in patients affected by Multiple Sclerosis (MS) and other neurological diseases (OND) using Isoelectric Focusing followed by direct immunofixation. In the serum a simple broad band has always been observed; inside it several more stained subfractions were sometimes detected. In 74.3% of MS patients and in 65.7% of OND patients the CSF alpha 2-M was divided in two major bands. One corresponded to that observed in serum and the other was more anodal. The significance of this finding is discussed.

[Determination of immunoglobulins G, A, M, alpha-2-macroglobulin and ceruloplasmin levels in cerebrospinal fluid of children and adults by a nephelometric laser method (author's transl)]. / Dosage immunonéphélémétrique des immunoglobulines G, A, M, de l'alpha-2-macroglobuline et de la céruloplasmine : concentrations dans le liquide céphalorachidien de l'enfant et de l'adulte.

Cerebrospinal fluid, G, A, M, immunoglobulins, alpha-2-macroglobulin and ceruloplasmin levels were determined by a nephelometric Laser method of a control group of 21 adult patients strictly suffering from a "slipped disc" disease. The same CSF parameters were studied on a second group of 90 patients including adults and children with normal CSF proteins levels (< 500 mg per liter). Normal values were established according to the different age groups. Results were given in milligrams per liter and nanomols per liter.

[Quantitative levels of serum amyloid A protein and other proteins in cerebrospinal fluid and serum of patients with meningitis].

Serum amyloid A protein (SAA), a putative precursor of the AA protein which constitutes amyloid fibrils in secondary amyloidosis, is evaluated as a sensitive acute-phase reactant in serum. At present, SAA concentration in serum is determined by latex nephelometry, but this assay cannot detect SAA in the low concentration range lower than 10 ng/ml. We have developed a sensitive enzyme immunoassay (EIA) for determining low concentrations of SAA. The assay is reproducible, reliable and requires no pretreatment of specimen prior to assay. We measured levels of SAA by this EIA in both cerebrospinal fluid (CSF) and serum of patients with suspected meningitis, measured also levels of albumin, alpha 2 macroglobulin and C-reactive protein (CRP) to investigate if these protein levels are useful for differential diagnoses of meningitis and for indicators damage of blood-CSF barrier. The SAA reference value in CSF is 3.99 +/- 1.74 ng/ml (mean +/- SD for nonmeningitis patients). The CRP concentration in CSF in bacterial meningitis was much higher than in viral meningitis, but CRP in CSF was also high in bacterial infection other than meningitis. On the other hand, SAA concentration in CSF in these patients with any meningitis are significantly higher than the reference values of SAA (p < 0.001). However, the differences in SAA concentrations among the three types (bacterial, viral and mycotic meningitis) of meningitis were not significant.