In vivo relationship of interleukin-2 and soluble IL-2 receptor to blood-brain barrier impairment in patients with active multiple sclerosis

Interleukin (IL)-2 has well-recognized effects on cerebral endothelial cells and, therefore, may mediate disruption of the blood-brain barrier in patients with multiple sclerosis (MS). To evaluate the in vivo relationship of the IL-2 system to blood-brain barrier impairment in MS, levels of IL-2 and soluble IL-2 receptors (sIL-2R) in cerebrospinal fluid (CSF) and serum samples from 50 patients with active MS and 49 controls were correlated with values of the CSF to serum albumin ratio. Intrathecal levels of IL-2 and sIL-2R were significantly higher in MS compared with the control groups and correlated with albumin ratios in MS patients. Intrathecal levels of IL-2 and sIL-2R also correlated with the degree of barrier damage in these patients. It is suggested that intrathecal levels of IL-2 and sIL-2R are related to barrier impairment in MS and may be important in understanding some of the pathological changes of this condition.     

Alpha 1-antitrypsin in cerebrospinal fluid of patients with neurologic diseases

Proteases and their inhibitors have been implicated in the pathogenesis of neuroimmunologic diseases, particularly multiple sclerosis (MS). We measured the immunochemical level and functional activity of alpha 1-antitrypsin (AAT) in cerebrospinal fluid (CSF) and serum in patients with MS and other neurologic diseases. Increases in the immunochemical level of AAT in CSF correlated directly with disturbances in the blood-brain barrier, as reflected by the ratio of albumin in CSF to that in serum. The AAT activity in CSF directly correlated with the immunochemical level when all patients were compared. However, the AAT activity in patients with inflammatory diseases tended to be decreased relative to the immunochemical level, suggesting inactivation of AAT in these disorders. The AAT activity was not significantly altered in patients with MS, despite reports of increased protease activity in active MS.     

Antibodies against oligodendrocytes in serum and CSF in multiple sclerosis and other neurological diseases: 125I-protein A studies

Antibodies against oligodendrocytes were determined in pairs of unconcentrated CSF serum from 12 patients with multiple sclerosis (MS) and 25 control patients including 10 with aseptic meningoencephalitis (AM), using a 125I-protein A microassay. Antibody levels in serum and in CSF did not differ between MS and controls. Calculating the antibody index equal to (CSF/serum antibodies against oligodendrocytes):(CSF/serum albumin) in analogy to the CSF IgG index, thereby compensating for influence of serum antibody concentration as well as altered blood-brain barrier, no evidence was obtained for intrathecal antibody production in the patients with MS. Those with AM had higher antibody index values, probably reflecting intrathecal synthesis. Antibodies against oligodendrocytes seem to be a regular component of CSF and serum in neurological diseases; intrathecal antibody production is less frequent in MS than in AM.     

多发性硬化和格林-巴利综合征患者体液中TNF及IL-1水平与临床关系的研究

测定了多发性硬化(MS)和格林-巴利综合征(GBS)患者的细胞因子，发现活动的MS血清和脑脊液(CSF)TNF水平显著高于稳定的MS和对照组。GBS患者急性期CSF和血清TNF水平显著高于对照组及治疗后水平。另外，MS和GBS患者CSF的TNF水平均高于相应的血清水平。通过研究还发现MS和GBS组CSF的蛋白含量显著增高。此外，MS组CSF白细胞敷与CSF的TNF水平及CSF蛋白含量相关，且CSF蛋白含量与血清、CSF的TNF水平相关。这表明TNF一方面可能来源干鞘内的单个核细胞，另一方面由于血脑屏障受损可能来源于血液，另外还可能来源于神经肢质细胞及血管内皮细胞。TNF可能在炎性脱鞘病发病初期起作用。     

Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG

Abstract. Recently it has been shown that axonal damage occurs in all stages of multiple sclerosis (MS) and can be detected very early in the course of the disease. Axonal pathology has been related to the inflammatory demyelinating environment, but its dependence on inflammation is still unknown. We measured tau protein and 14-3-3, two intracellular proteins expressed in neurons and glial cells, in the cerebrospinal fluid (CSF) of 114 patients with MS, in 79 patients with other inflammatory neurological diseases (IND) and in the CSF of 60 patients with non-inflammatory neurological diseases (NIND) as controls. Concentrations of tau protein and 14-3-3 were measured by enzymelinked immunoassay and were correlated to the following immune parameters in the CSF: leukocyte cell count, total protein, albumin CSF/serum ratio as a marker of disruption of the blood-brain barrier, immunoglobulin (IgG concentrations and IgG index as an indicator for intrathecal synthesis of IgG in the CSF). Both in MS and IND tau protein levels were significantly higher than in NIND (p < 0.05). In MS patients levels of tau protein were positively correlated with the IgG index (p < 0.05) and this association was present in both relapsing remitting MS (RRMS) (p < 0.05) and progressive MS (p < 0.05). Tau and 14-3-3 were also correlated with the IgG index in patients with IND (p < 0.05). These findings strengthen the hypothesis that inflammation may be at least in part responsible for the axonal damage observed in MS patients.     

Quantitation of IgG and albumin in CSF and serum from multiple sclerosis patients by enzyme-linked immunosorbent assay

Immunoglobulin G (IgG) and albumin from unconcentrated cerebrospinal fluid (CSF) and serum of patients with multiple sclerosis (MS) and non-MS controls were quantitated using enzyme-linked immunosorbent assay (ELISA). The IgG levels, IgG/albumin ratios and IgG indexes were significantly increased in CSF of MS patients compared to those of non-MS controls. The method is sensitive, rapid and reproducible and can be applied to routine laboratory use for quantitation of IgG and albumin in unconcentrated CSF from humans as well as in experimental animals used as models for demyelinating diseases.     

Approach to discriminate subgroups in multiple sclerosis with cerebrospinal fluid (CSF) basic inflammation indices and TNF-alpha,IL-1beta,IL-6, IL-8

Lumbar CSF and serum pairs of untreated multiple sclerosis patients (MS; n=47) were analyzed on admission. On average, higher CSF leukocyte (lymphocyte and monocyte) counts, IgG index, CSF IgG contents, but not of TNF-alpha, IL-1beta, IL-6, IL-8 in CSF and serum, were revealed in all MS or patients with long disease course (LO-MS) compared with controls. In primary progressive MS (PP-MS) cell counts were low, but IgG contents were high, when compared to relapsing-remitting MS (RR-MS). In clinically probable MS (CP-MS) both contents were low, in clinically definite MS (CD-MS) high. Spearman's correlation with the four monokines and the basic indices in CSF revealed activation patterns known for microglia/macrophages in the four MS subgroups, for astrocytes in CP-MS and RR-MS, for CSF lymphocytes in CP-MS and PP-MS, for cells of blood-brain barrier (BBB) in CP-MS, for intrathecal IgG synthesis in PP-MS and for lymphocyte transfer in CD-MS. Correlations between CSF and serum parameters indicated CNS disease processes to be associated with systemic processes of inflammation (acute, chronic) in CD-MS, RR-MS, and PP-MS in different ways. CSF IgG content, IgG index and systemic markers of inflammation correlated with overall disability scores in LO-MS; increasing levels may indicate a bad outcome.     

Intrathecal synthesis of matrix metalloproteinase-9 in patients with multiple sclerosis: implication for pathogenesis

Matrix metalloproteinase-9 (MMP-9) was detected by zymography and enzyme-linked immunosorbent assay (ELISA) in matched serum and cerebrospinal fluid (CSF) samples from patients with neurological diseases. Patients with relapsing-remitting multiple sclerosis (RR-MS) had serum and CSF MMP-9 levels comparable to those from patients with inflammatory neurological diseases (INDs), but higher than patients with non-inflammatory neurological diseases (NINDs) and healthy donors (HDs). MMP-9 increased in active RR-MS in comparison with inactive RR-MS implying that MMP-9 in MS is related with clinical disease activity. A correlation between the CSF/serum albumin (Q(AIb)) and CSF/serum MMP-9 (Q(MMP-9)) was observed in IND and NIND but not in RR-MS patients, indicating that CSF MMP-9 levels in NIND and IND patents could be influenced by serum MMP-9 and blood-brain barrier (BBB) permeability properties. MS patients had higher values of Q(MMP-9):Q(Alb)(MMP-9 index) than IND and NIND patients suggesting that in MS the increase in CSF MMP-9 could be due to intrathecal synthesis of MMP-9. A significant inverse correlation was found between MMP-9 and its endogenous inhibitor TIMP-1 in RR-MS indicating that in MS patients both the increase in MMP-9 and the decrease in TIMP-1 serum levels could contribute to BBB disruption and T-lymphocyte entry into the CNS.     

CSF and serum levels of soluble fractalkine (CX3CL1) in inflammatory diseases of the nervous system

The new CX(3)C-chemokine fractalkine (CX(3)CL1) was measured by Western blot in the cerebrospinal fluid (CSF) and serum of patients with inflammatory diseases of the peripheral and central nervous system (Bell's palsy, BP; Guillain-Barré Syndrome, GBS; multiple sclerosis, MS; viral meningitis, VM; bacterial meningitis, BM) and patients with noninflammatory neurological diseases (controls). In controls, fractalkine was detectable at low concentrations in the CSF and, at much higher levels, in serum. In all inflammatory neurological diseases under study, CSF fractalkine levels were significantly (p<0.01) increased vs. controls (BM>GBS>VM>MS>BP>controls). In serum, fractalkine levels were significantly increased only in MS patients. The fractalkine CSF/serum ratios (a measure of the chemotactic gradient) were significantly elevated in BM, VM and GBS; furthermore, they tended to be increased in BP and to be decreased in MS. The elevated fractalkine CSF/serum ratios in diseases without CSF pleocytosis (GBS, BP) and a lack of correlation between fractalkine levels and CSF leukocyte counts suggested that soluble fractalkine is not a major chemokine in the CSF. There was no evidence of significant intrathecal production of fractalkine as the mean fractalkine indices (fractalkine CSF/serum ratio:albumin CSF/serum ratio) were <1 in all inflammatory diseases and not significantly elevated vs. controls.     

Intrathecal synthesis of autoantibodies to myelin basic protein in multiple sclerosis

A solid phase radioimmunoassay was used to detect anti-myelin basic protein (MBP) antibodies in the CSF and serum of multiple sclerosis (MS) patients and controls. CSF and serum samples were assayed prior to acid hydrolysis in order to detect free anti-MBP as well as after acid hydrolysis to measure the total (free and bound) amount of antibody. An anti-MBP index controlling for serum levels as well as the degree of breakdown of the blood brain barrier was used to estimate intrathecal synthesis of anti-MBP. MS patients with acute exacerbations or chronically progressive disease have significantly elevated levels of both free and total CSF anti-MBP. The anti-MBP index is also significantly increased in MS patients with both forms of active disease. Anti-MBP antibodies are intrathecally produced in MS patients with active disease.     

Soluble CD8 and ICAM-1 in serum and CSF of MS patients treated with 6-methylprednisolone

Objective - We studied the effects of large doses of 6-methylprednisolone (6-MP) on serum and cerebrospinal fluid (CSF) soluble CD8 (sCD8) and intercellular adhesion molecule-1 (sICAM-1) levels in clinically active multiple sclerosis (MS) patients.
Material and methods - Paired serum and CSF samples were from 16 patients with definite MS, treated with 6-MP (1 g daily for 6 d) during an active phase of the disease. sCD8 and sICAM-1 levels were determined with ELISA before and after the therapy.
Results - Before 6-MP treatment, sCD8 levels in CSF were higher in MS patients than in patients with noninflammatory neurological disease and in healthy controls; sICAM-1 levels in serum and in CSF were higher in MS patients than in the two control groups. Ten of the 16 patients showed clinical improvement at the end of the treatment. After the therapy, serum and CSF sCD8 levels increased, whereas serum and CSF sICAM-1 levels decreased. There was no correlation between clinical improvement and laboratory parameters. We evaluated sCD8 and sICAM-1 in serum samples from 10 patients 6 months after the 6-MP treatment, when the disease was clinically silent. Neither sCD8 nor sICAM-1 levels differed from those of the control groups.
Conclusions - Our results suggest that high doses of 6-MP can influence serum and CSF sCD8 and sICAM-1 levels in active MS. At least part of the efficacy of corticosteroid treatment in MS might be ascribed to its effect both on the suppressive circuits of immune response, and on the expression of an adhesion molecule that favours lymphocyte trafficking across the blood-brain barrier.     

Cerebrospinal fluid insulin-like growth factor-1, insulin growth factor binding protein-2 or nitric oxide are not increased in MS or ALS

OBJECTIVES: Many studies have shown that nitric oxide (NO) and growth factors including insulin growth factors (IGFs) may be involved in the pathogenesis of multiple sclerosis (MS) and neurodegenerative diseases. Our previous studies suggested a relationship between cerebrospinal fluid (CSF) NO metabolites (nitrates and nitrites, NN(x)) and IGF-1 in patients with progressive encephalopathy, hypsarrhythmia and optic atrophy syndrome. MATERIAL AND METHODS: We examined CSF concentrations of NN(x), IGF-1 and IGF binding protein-2 (IGFBP-2) in 25 controls, 14 patients with MS and 14 patients with amyotrophic lateralis sclerosis (ALS). RESULTS: There were no significant differences in CSF levels of NN(x), IGF-1 or IGFBP-2 between the groups. CSF IGFBP-2 concentrations correlated significantly with age in controls, which may reflect age-related changes in the blood-brain barrier function. CONCLUSION: Upregulation of the production of NO and IGF-1 in the brain or spinal cord does not influence CSF levels of these molecules in MS or ALS.     

Prednisone effects on blood-brain barrier permeability and CNS IgG synthesis in healthy humans

Corticosteroids reportedly decrease blood-brain barrier (BBB) permeability and/or IgG synthesis in patients with multiple sclerosis or brain tumors. However, these effects have not been studied in healthy humans. We investigated the effects of prednisone, 80 mg/day for five days, on the ratio of cerebrospinal fluid (CSF) albumin/serum albumin, a measure of blood-brain barrier (BBB) permeability, and on CSF and serum IgG levels in six healthy, normal volunteers. We found significant steroid-induced decreases in serum and CSF albumin levels and in serum IgG levels. However, we found only a nonsignificant decrease in BBB permeability and no significant change in CNS IgG synthesis. These findings, based on a small number of volunteers, suggest that it may be difficult to further decrease BBB permeability and CNS IgG synthesis in medically healthy subjects.     

VIRUS ANTIBODY LEVELS IN THE CEREBROSPINAL FLUID FROM PATIENTS WITH OPTIC NEURITIS

Virus antibody levels were studied in the cerebrospinal fluid (CSF) of 58 patients with optic neuritis and 58 control patients with no indication of multiple sclerosis (MS) or infectious disorders of the central nervous system (CNS). The specimens were tested against three different structural components of measles virus with measles hemagglutination inhibition (HI), measles hemolysis inhibition (HLI) and gel precipitation (GP) tests. Measles antibodies occurred in 62 per cent of CSF specimens from patients with optic neuritis, and 21 per cent of the controls. In the specimens from patients with optic neuritis, the positive rate figures were: for rubella HI test 35, parainfluenza-1 HI 16, and Epstein-Barr virus immunofuorescence (IF) 53 per cent. The frequencies in the control group were 10, 10 and 26 per cent, respectively. Serum/CSF antibody ratios below 80 occurred in measles tests in 45 per cent of patients with optic neuritis and 16 per cent of the control group. Some patients with optic neuritis (but none from the control group) had a reduced serum/CSF antibody ratio in more than one measles antibody test, The patients with optic neuritis had a higher frequency of low serum/CSF albumin ratios indicating blood brain barrier damage, There were, however, several patients with a normal serum/CSF albumin ratio but low serum/CSF immunoglobulin G and measles antibody ratios. This supports the hypothesis that local production of measles antibodies takes place in CNS in some patients with optic neuritis as well as in MS patients. The CSF specimens were further tested against 12 other viruses and mycoplasma pneumoniae complement fixation, but there were no positive specimens. New CSF specimens were taken from five patients during optic neuritis, and from seven patients later on during the follow-up because of the appearance of new neurological symptoms. There were no changes in virus antibody levels, except for two patients with an increase of measles virus antibody titres.     

CSF in Alzheimer's disease. Studies on blood-brain barrier function and intrathecal protein synthesis

Serum and cerebrospinal fluid (CSF) from 22 ambulatory and 10 institutionalised patients with Alzheimer's disease (AD) and 22 age-matched controls were assayed nephelometrically for concentrations of IgG, IgA, IgM, haptoglobin, transferrin, prealbumin and albumin. The CSF/serum ratio and index were calculated for each protein. In the CSF of ambulatory patients IgG, transferrin and albumin were elevated while the institutionalised patients had higher IgG and IgA levels compared to the controls. The CSF haptoglobin was elevated in institutionalised AD patients compared to those who were ambulatory. The CSF/serum ratio for albumin was elevated in both groups. An increase in the IgG ratio was also found in both groups. The ratios for haptoglobin and prealbumin were markedly increased in the institutionalised patients. CSF indices gave no evidence for increased intrathecal synthesis of any of the proteins investigated. The increased CSF/serum ratios for IgG and albumin and also the higher CSF albumin in patients with AD suggest an increased blood-brain barrier permeability in this disease. The high prealbumin ratio may be related to amyloidogenesis often present in AD.     

IgM-class rheumatoid factor in serum and cerebrospinal fluid of multiple sclerosis and matched neurological control patients

Paired serum and cerebrospinal fluid (CSF) specimens from 30 multiple sclerosis (MS) patients and 30 matched neurological control (NC) patients were quantitatively tested for IgM-class rheumatoid factor (RF). Significantly elevated RF levels were found in serum from 6 MS patients and 12 NC patients. Seven of the latter 12 patients had a diagnosis of recurrent head pain. RF was detected in CSF from 2 MS patients and 2 NC patients. In 3 of the 4 cases, this could be explained by blood-brain barrier damage or normal diffusion of RF into CSF. Intrathecal RF synthesis was found in the remaining MS patient. These results suggest that RF production may be related to underlying pathological mechanisms in at least some MS and NC patients. They also confirm that RF must be carefully accounted for in etiological studies designed to demonstrate pathogen-specific IgM antibodies in, for example, MS patients.     

IgG production within the central nervous system: a critical review of proposed formulae

Demonstration of intrathecal IgG production is employed in the diagnosis of various neurological disorders. This pathological IgG fraction in cerebrospinal fluid (CSF) can be visualized directly as oligoclonal bands by electrophoresis or isoelectric focusing or can be calculated as "excess" or "synthesized" IgG according to different formulae. A comparison of the results obtained with isoelectric focusing and with five formulae showed that even though three of the formulae discriminated well between a reference population and patients with multiple sclerosis, all five gave wrong and misleading results in the presence of blood-brain barrier damage, as defined by an abnormally raised CSF/serum albumin ratio. A mathematical and statistical evaluation of the different formulae showed only those based on covariance between CSF/serum IgG and CSF/serum albumin to be valid, and these only when values of CSF/serum albumin were normal. Among the five formulae the IgG index (equal to CSF/serum IgG:CSF/serum albumin) is unique in having a comparatively small and constant maximal relative error resulting from the variation coefficients of the IgG and albumin assays. In the case of blood-brain barrier damage, there exists currently no valid procedure to calculate intrathecally produced IgG; in such instances sensitive electrophoretic or isoelectric focusing methods demonstrating oligoclonal IgG bands are most appropriate to demonstrate intrathecal IgG production.     

Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase (TIMP-1) in patients with relapsing-remitting multiple sclerosis treated with interferon beta

OBJECTIVES: Matrix metalloproteinases (MMPs), particularly MMP-9, facilitate T-cell migration into the central nervous system. They play a key role in the disruption of the blood-brain barrier (BBB) and thus in the pathogenesis of multiple sclerosis. Interferon beta's (IFNbeta) ability to alter the balance between MMP-9 and MMP-9s natural inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), may play a role in stabilizing the BBB. The aim of this study, was to evaluate serum MMP-9 and TIMP-1 and cerebrospinal fluid (CSF) TIMP-1 levels in patients with relapsing-remitting multiple sclerosis (RRMS) treated with IFNbeta-1a. PATIENTS AND METHODS: Blood and CSF samples from 14 patients with RRMS before and 6 months after IFNbeta therapy and 14 age and sex-matched controls were obtained. Levels of MMP-9 and TIMP-1 were measured using ELISA. RESULTS: Before treatment, patients with MS had higher levels of serum MMP-9 and a higher MMP-9/TIMP-1 ratio than the controls. Although serum levels of TIMP-1 were lower in RRMS patients than in the controls, the differences did not reach statistical significance. CSF levels of TIMP-1 were significantly lower in RRMS patients. In the sixth month of IFNbeta therapy serum MMP-9 and the MMP-9/TIMP-1 ratio were significantly decreased, whereas the changes in serum TIMP-1 were not statistically significant. There was a significant increase in CSF TIMP-1 levels in the sixth month of IFNbeta therapy. CONCLUSIONS: Our result shows that RRMS patients have an impaired MMP-9 and TIMP-1 balance, and that 6 months of IFNbeta therapy is beneficial in restoring this balance.     

Soluble complement receptor type 1 in serum and cerebrospinal fluid of patients with Guillain-Barré syndrome and multiple sclerosis

Activation of complement is critically involved in inflammatory reactions in both Guillain-Barré syndrome (GBS) and multiple sclerosis (MS). Soluble human complement receptor 1 (sCR1) blocks complement activation by both classical and alternative pathways. We studied serum and cerebrospinal fluid (CSF) concentrations of sCR1 in 23 patients with GBS, 27 patients with MS and 30 controls. No significant differences were found between patients and controls. Transient liver affection probably caused high serum sCR1 levels in two patients with GBS. The serum and CSF sCR1 levels were not correlated to the disease activity of GBS and MS, nor to the relapsing-remitting or chronic-progressive forms of MS. In GBS the CSF sCR1 levels correlated with the CSF total protein concentrations (r = 0.9, P < 0.01), suggesting that sCR leaks from serum into CSF via a damaged blood-nerve barrier. The serum sCRl levels in GBS were slightly higher than in MS (P < 0.05). Whether this reflects changes in the release or consumption of sCR in these patients is at present unknown.     

Thrombomodulin in the sera of patients with multiple sclerosis and human lymphotropic virus type-1-associated myelopathy

Damage to the blood-brain barrier, which mainly consists of cerebral endothelial cells, has been demonstrated in multiple sclerosis (MS) clinically and histochemically. To investigate the endothelial cell damage, we evaluated the presence of soluble thrombomodulin in the sera of patients with MS and human T lymphotropic virus type-1-associated myelopathy (HAM) using an enzyme-linked immunosorbent assay. Serum thrombomodulin levels were significantly increased in patients with acute relapsing MS during an exacerbation and chronic progressive MS as compared with those of controls (P < 0.001, respectively). Patients with HAM also had higher serum levels of thrombomodulin than did controls (P < 0.001). There was significant difference between patients with HAM and seropositive non-HAM carriers (P < 0.01). These results suggest that the detection of serum thrombomodulin could be used as a marker of endothelial cell damage in inflammatory diseases such as MS and HAM.