Clinical and Immunological Features of Opsoclonus-Myoclonus Syndrome in the Era of Neuronal Cell Surface Antibodies.

IMPORTANCE: Most studies on opsoclonus-myoclonus syndrome (OMS) in adults are based on small case series before the era of neuronal cell surface antibody discovery. OBJECTIVE: To report the clinical and immunological features of idiopathic OMS (I-OMS) and paraneoplastic OMS (P-OMS), the occurrence of antibodies to cell surface antigens, and the discovery of a novel cell surface epitope. DESIGN, SETTING, AND PARTICIPANTS: Retrospective cohort study and laboratory investigations of 114 adult patients with OMS at a center for autoimmune neurological disorders done between January 2013 and September 2015. MAIN OUTCOMES AND MEASURES: Review of clinical records. Immunohistochemistry on rat brain and cultured neurons as well as cell-based assays were used to identify known autoantibodies. Immunoprecipitation and mass spectrometry were used to characterize novel antigens. RESULTS: Of the 114 patients (62 [54%] female; median age, 45 years; interquartile range, 32-60 years), 45 (39%) had P-OMS and 69 (61%) had I-OMS. In patients with P-OMS, the associated tumors included lung cancer (n = 19), breast cancer (n = 10), other cancers (n = 5), and ovarian teratoma (n = 8); 3 additional patients without detectable cancer were considered to have P-OMS because they had positive results for onconeuronal antibodies. Patients with I-OMS, compared with those who had P-OMS, were younger (median age, 38 [interquartile range, 31-50] vs 54 [interquartile range, 45-65] years; P < .001), presented more often with prodromal symptoms or active infection (33% vs 13%; P = .02), less frequently had encephalopathy (10% vs 29%; P = .01), and had better outcome (defined by a modified Rankin Scale score ≤ 2 at last visit; 84% vs 39%; P < .001) with fewer relapses (7% vs 24%; P= .04). Onconeuronal antibodies occurred in 13 patients (11%), mostly Ri/ANNA2 antibodies, which were detected in 7 of 10 patients (70%) with breast cancer. Neuronal surface antibodies were identified in 12 patients (11%), mainly glycine receptor antibodies (9 cases), which predominated in P-OMS with lung cancer (21% vs 5% in patients with OMS without lung cancer; P = .02); however, a similar frequency of glycine receptor antibodies was found in patients with lung cancer without OMS (13 of 65 patients [20%]). A novel cell surface epitope, human natural killer 1 (HNK-1), was the target of the antibodies in 3 patients with lung cancer and P-OMS. CONCLUSIONS AND RELEVANCE: Patients with I-OMS responded better to treatment and had fewer relapses than those with P-OMS. Older age and encephalopathy, significantly associated with P-OMS, are clinical clues suggesting an underlying tumor. Glycine receptor antibodies occur frequently in P-OMS with lung cancer, but the sensitivity and specificity are low. The HNK-1 epitope is a novel epitope in a subset of patients with P-OMS and lung cancer.

Determination of neuronal antibodies in suspected and definite Creutzfeldt-Jakob disease.

IMPORTANCE: Creutzfeldt-Jakob disease (CJD) and autoimmune encephalitis with antibodies against neuronal surface antigens (NSA-abs) may present with similar clinical features. Establishing the correct diagnosis has practical implications in the management of care for these patients. OBJECTIVE: To determine the frequency of NSA-abs in the cerebrospinal fluid of patients with suspected CJD and in patients with pathologically confirmed (ie, definite) CJD. DESIGN, SETTING, AND PARTICIPANTS: A mixed prospective (suspected) and retrospective (definite) CJD cohort study was conducted in a reference center for detection of NSA-abs. The population included 346 patients with suspected CJD and 49 patients with definite CJD. MAIN OUTCOMES AND MEASURES: Analysis of NSA-abs in cerebrospinal fluid with brain immunohistochemistry optimized for cell-surface antigens was performed. Positive cases in the suspected CJD group were further studied for antigen specificity using cell-based assays. All definite CJD cases were comprehensively tested for NSA-abs, with cell-based assays used for leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein-like 2 (CASPR2), N-methyl-d-aspartate (NMDA), and glycine (GlY) receptors. RESULTS: Neuronal surface antigens were detected in 6 of 346 patients (1.7%) with rapid neurologic deterioration suggestive of CJD. None of these 6 patients fulfilled the diagnostic criteria for probable or possible CJD. The target antigens included CASPR2, LGI1, NMDAR, aquaporin 4, Tr (DNER [δ/notch-like epidermal growth factor-related receptor]), and an unknown protein. Four of the patients developed rapidly progressive dementia, and the other 2 patients had cerebellar ataxia or seizures that were initially considered to be myoclonus without cognitive decline. The patient with Tr-abs had a positive 14-3-3 test result. Small cell lung carcinoma was diagnosed in the patient with antibodies against an unknown antigen. All patients improved or stabilized after appropriate treatment. None of the 49 patients with definite CJD had NSA-abs. CONCLUSIONS AND RELEVANCE: A low, but clinically relevant, number of patients with suspected CJD had potentially treatable disorders associated with NSA-abs. In contrast, none of 49 patients with definite CJD had NSA-abs, including NMDAR-abs, GlyR-abs, LGI1-abs, or CASPR2-abs. These findings suggest that cerebrospinal fluid NSA-abs analysis should be included in the diagnostic workup of patients with rapidly progressive central nervous system syndromes, particularly when they do not fulfill the diagnostic criteria of probable or possible CJD.

Biomarkers of inflammatory and auto-immune central nervous system disorders.

PURPOSE OF REVIEW: Inflammatory and auto-immune disorders of the central nervous system are a heterogeneous group of disorders. Many of these disorders are potentially treatable with immune therapies that can reduce disability or prevent death. We review the clinical value of biomarkers which can aid in the diagnosis of paediatric inflammatory and auto-immune central nervous system (CNS) disorders. RECENT FINDINGS: This review will first describe the clinical usefulness of nonspecific biomarkers of CNS inflammation such as cerebrospinal fluid neopterin and oligoclonal bands. Neopterin is produced by immune and neuronal cells after stimulation by interferon species and is increased in a broad range of inflammatory and auto-immune CNS disorders. Oligoclonal bands represent clonal production of immunoglobulin G in the CNS and are present in demyelinating, auto-immune, and infectious CNS disorders. In addition, we will review new advances in the immunogenetic investigation of familial auto-inflammatory disorders such as Aicardi-Goutières syndrome and Chronic Infantile Neurologic Cutaneous Articular syndrome. Finally, we will review the clinical utility of auto-antibodies in CNS disorders, with specific focus on auto-antibodies that bind to cell surface proteins such as N-methyl-D-asparate receptor, voltage-gated potassium channels, myelin oligodendrocyte glycoprotein, and aquaporin-4. SUMMARY: These biomarkers are increasingly important in the recognition and treatment of inflammatory and auto-immune CNS disorders. Like many biomarkers in paediatric practice, it is essential to interpret the findings in the context of the patient history and examination.

Cell surface bound and soluble adhesion molecules in CSF and blood in multiple sclerosis: correlation with MRI-measures of subclinical disease severity and activity.

BACKGROUND: The expression of soluble cell adhesion molecules (AM) in cerebrospinal fluid (CSF) and blood and their significance as measures of disease activity has been extensively studied in patients with multiple sclerosis (MS). In previous studies, we found that cell surface bound AM on mononuclear cells (MNC) in CSF and blood might be useful markers of clinical disease activity in MS patients. OBJECTIVE: To analyze the correlation of cell surface bound and soluble AM in CSF and blood with magnetic resonance imaging (MRI) markers of subclinical disease severity and activity in patients with MS. METHODS: Expression levels of cell surface bound AM on peripheral blood and CSF MNC were determined by flow cytometry analysis in 77 (CSF: 33) MS patients. Concentration levels of the soluble forms of AM were measured by enzyme-linked immunosorbent assay (ELISA). In corresponding cerebral gadolinium (Gd)-enhanced MRI scans, we determined both measures of subclinical disease severity and subclinical disease activity. RESULTS: The expression levels of cell surface bound AM in peripheral blood correlated inversely with parameters for subclinical disease severity and activity on cerebral MRI scans as well as with the disease duration. Furthermore, we found significant correlations between serum levels of soluble AM and patient age but not with disease duration. CONCLUSIONS: Our results suggest that subclinical disease progression may be associated with a decrease of the expression of cell surface bound AM on peripheral blood MNC. This might be a result of activated MNC migration into the CNS.

More laboratory testing: greater cost but not necessarily better.

BACKGROUND: The bacterial latex agglutination assay is ordered predominantly on the pediatric population, for rapid screening for bacterial surface antigens in cerebrospinal fluid (CSF) or urine specimens. The high cost of this assay and questions raised in the literature regarding its accuracy led to a retrospective review of the use of this assay at a medium-sized midwest teaching hospital. The results of 6,370 bacterial latex agglutination tests performed between May, 1995, and November, 1996, and charts of patients being tested were reviewed. RESULTS: This study demonstrated a sensitivity and specificity of 28.6% and 86.7% for urine specimens and 70.0% and 99.4% for CSF specimens. A total of 11 pathogens were accurately detected (7 CSF and 4 urine). There were 13 false negatives and 59 false positives. None of the true positives had a discernible effect on either treatment or hospital course; however, several of the erroneous tests resulted in delayed or unnecessary treatment and workup of the involved patients. The annual billed cost of this test at this institution (fiscal years 1995 to 1997) averaged $167,000 per annum. This does not include indirect costs associated with increased length of hospital stay, overutilization of antibiotics and excess laboratory tests ordered as a result of false positives. CONCLUSIONS: Bacterial antigen latex agglutination testing is neither sufficiently sensitive nor specific to be used as a screening test. Accurate results have no demonstrable clinical impact, whereas numerous inaccurate results are often generated at great cost. The continued use of the latex agglutination assay should be seriously questioned in an era when cost containment and clinical efficiency are becoming increasingly important.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45 of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19 of the CSF samples and 62 were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals.

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Simultaneous expression of Borrelia OspA and OspC and IgM response in cerebrospinal fluid in early neurologic Lyme disease.

Lyme disease is the major tick-borne disease, caused by Borrelia burgdorferi (Bb). Neurological involvement is common in all stages. In vivo expression of Bb antigens (Ags) and the immune response to them has not been well investigated in the cerebrospinal fluid (CSF). Upregulation of outer surface protein (Osp) C and concomitant downregulation of OspA before tick inoculation of the spirochete has been reported in skin and blood in animals. CSF OspA Ag in early disease suggests otherwise in CSF. Early Ag expression and IgM response in human CSF was investigated here. Paired CSF and serum was collected from 16 early, predominantly erythema migrans Lyme disease patients with neurologic problems, 13 late Lyme disease patients, and 19 other neurologic disease (OND) controls. Samples were examined for IgM reactivity to recombinant Bb-specific Osps using ELISA and immunoblot. Of 12 early Lyme disease patients with neurologic involvement with both CSF and serum IgM against OspC, 7 (58%) had IgM to OspA (n = 5) or OspB (n = 2) that was restricted to the CSF, not serum. Overall, 12 of 16 (75%) of these early Lyme disease patients with neurologic involvement had CSF and serum IgM against OspC. Only 3 of 13 (23%) late Lyme disease patients and none of 19 OND controls had CSF IgM directed against OspC. In conclusion, in CSF, OspC and OspA can be coexpressed, and IgM response to them occurs in early Lyme disease patients with neurologic involvement. This biologic finding may also provide a discriminating marker for CNS infection in Lyme disease.

Diversity of OspA and OspC among cerebrospinal fluid isolates of Borrelia burgdorferi sensu lato from patients with neuroborreliosis in Germany.

Neuroborreliosis is the most frequent manifestation of the second stage of Lyme borreliosis in Europe. However, only few isolates from the cerebrospinal fluid (CSF) have been characterized with controversial results. A large panel of 36 CSF isolates isolated over a 10-year period in Munich has now been analyzed for their OspA and OspC type, resulting in at least eight different types, respectively. Representatives of the different types cultivated from CSF in Munich have also been isolated from other geographical regions in Europe from CSF or ticks, suggesting a widespread distribution of pathogenic strains. A certain OspA type (type 4) was frequently observed in adults but rarely in children or ticks. Since OspA and OspC are the most promising candidates for a Borrelia vaccine, the considerable heterogeneity found among CSF isolates has important implications for development of a vaccine in Europe.

Detection of Borrelia burgdorferi-specific antigen in antibody-negative cerebrospinal fluid in neurologic Lyme disease.

OBJECTIVE: To determine the potential of detection in CSF of specific Borrelia burgdorferi antigen, OspA, as a marker of infection in neurologic Lyme disease and compare this with the detection of antibody. DESIGN: CSF from 83 neurologic patients in an area highly endemic for Lyme disease was examined prospectively for (1) OspA by antigen capture ELISA and Western blot employing monoclonal antibodies, and for (2) B burgdorferi antibodies by ELISA. RESULTS: Of the 35 of 83 (42%) patients who were positive for OspA antigen in their CSF, 15 (43%) were antigen positive despite being antibody-negative in CSF. Seven of these 15 (47%) had otherwise normal routine CSF analyses. Six of these 15 (40%) patients met strict CDC surveillance criteria for Lyme disease; four (27%) patients had seroconversion coincident with new neurologic problems; and three (20%) with characteristic syndromes for Lyme disease were seronegative, but had complexed antibody to B burgdorferi. The final two patients (13%) were seropositive and had unexplained neurologic problems not characteristic of Lyme disease. CONCLUSIONS: B burgdorferi antigen can be detected in CSF that is otherwise normal by conventional methodology, and can be present without positive CSF antibody. Since CSF antigen implies intrathecal seeding of the infection, the diagnosis of neurologic infection by B burgdorferi should not be excluded solely on the basis of normal routine CSF or negative CSF antibody analyses.

Embryonic neural cell adhesion molecule in cerebrospinal fluid of younger children: age-dependent decrease during the first year.

Poly-alpha-2,8-N-acetylneuraminic acid (poly-alpha-2,8-NeuAc) is developmentally expressed in neural tissue of higher animals, where it is covalently attached to the neural cell adhesion molecule (NCAM), a large integral membrane glycoprotein mediating cell-cell adhesion during neuronal development. NCAM exists in several molecular forms, of which only embryonic NCAM carries lengthy chains (n greater than 5) of poly-alpha-2,8-NeuAc. Chemically identical poly-alpha-2,8-NeuAc of bacterial origin is an important virulence factor in infections caused by Neisseria meningitidis group B and Escherichia coli K1, the predominant pathogens of bacterial meningitis. A quantitative enzyme-linked immunoassay was developed using monoclonal antibody (MAb) 735, an MAb specifically recognizing poly-alpha-2,8-NeuAc, and applied to CSF specimens from younger children. Poly-alpha-2,8-NeuAc contents were within the range of 20-0.2 micrograms/ml, decreasing from day 1 to day 300. Immunoprecipitation, immunoblot with a rabbit anti-mouse NCAM serum recognizing the protein part of human NCAM by cross-reactivity, affinity enrichment using immobilized MAb 735, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that poly-alpha-2,8-NeuAc in CSF is bound to human NCAM, probably NCAM-120.

Differential spectrum of expression of neural cell adhesion molecule isoforms and L1 adhesion molecules on human neuroectodermal tumors.

A series of four medulloblastomas, seven neuroblastomas (Nb), two ependymomas, and three gliomas, human neuroectodermal tumors, were screened for their expression of adhesion molecules L1, carcinoembryonic antigen, neural cell adhesion molecule isoforms (N-CAM) and HNK1 epitope by Western blotting and double immunofluorescence labeling. All seven neuroblastomas, whatever their differentiated state, expressed L1, a neural cell surface developmental antigen, whereas all other tumors tested were negative. All tumors expressed N-CAM; however, a large diversity was observed among the isoforms. Low sialylated N-CAM 140 was present, with different intensity, in ependymomas and astrocytomas. High sialylated isoforms were detected by a monoclonal antibody (anti-MenB) specifically recognizing high polymers of alpha 2-8 linked neuraminic acid. They were expressed in all medulloblastomas studied (4 of 4), and in 4 of 7 Nbs examined. Negative cases corresponded to tumors having undergone chemotherapeutic treatment or to ganglioneuroma. The interconversion from high to low sialylated forms might reflect changes which are critical for the conversion of Nbs into benign ganglioneuromas. HNK1 epitope was expressed on a large diversity of molecules by nearly all tumors except two Nbs which were also negative with anti-MenB antibody. This simultaneous loss of carbohydrate epitopes could correlate with higher maturation states of the tumors. None of the tumors expressed carcinoembryonic antigen. Therefore, anti-L1 and anti-MenB antibodies define differentiation-related antigens that could differentiate between Nbs and other tumors and may prove helpful in diagnosis and understanding of the biological nature of neuroectodermal tumors. An immunodot assay was set up and allowed to titrate the presence of polysialic acid units in cerebrospinal fluid from patients presenting meningeal spread of medulloblastomas. It could help to assess metastasis and to monitor the effects of chemotherapeutic treatment on polysialylated N-CAM positive tumors.

Neuropeptides and neural cell adhesion molecule (NCAM) in CSF from patients with ALS.

In 10 patients with amyotrophic lateral sclerosis (ALS), the CSF content of the neuropeptides vasoactive intestinal polypeptide (VIP) and cholecystokinin (CCK) as well as neural cell adhesion molecule (NCAM) was investigated. Compared with normal controls, no deviations were found in CCK or NCAM, while the values of VIP were significantly lower in ALS patients. This finding may reflect a loss of motor neurons.

Soluble neural cell adhesion molecule in brain, cerebrospinal fluid and plasma in the developing rat.

Neural cell adhesion molecule (NCAM) is a glycoprotein found in neurons, glial cells and muscle cells. In this report we describe the polypeptide composition and amount of soluble NCAM in brain, cerebrospinal fluid (CSF) and plasma in rats of various ages. One-two percent of total NCAM in rat brain was shown to be buffer-soluble. Soluble NCAM in brain, extracted at pH 7.2, constituted an increasing relative proportion of total NCAM during development, whereas the concentration of soluble NCAM in both CSF and plasma decreased in the same period. By radioiodination of brain extracts, CSF, and plasma, soluble NCAM was found to be composed of up to five polypeptides with molecular weights (Mr) of 200,000, 170,000, 150,000, 115,000 and 80,000. The relative composition did not vary significantly with age. The major forms in brain were the polypeptides with Mr of 200,000 and 115,000, whereas in CSF all forms seemed to appear in equal amounts. In plasma the 200,000 and the 170,000 Mr polypeptides were not observed and here the major forms were the 150,000 and 115,000 Mr polypeptides.

Characterization of a hydrophilic form of Thy-1 purified from human cerebrospinal fluid.

Thy-1 is a developmentally regulated cell surface glycoprotein in nervous tissue. An inositol-containing glycolipid structure is covalently attached to its carboxyl terminus, which anchors the protein to the cell membrane. In the present paper we report the characterization of a water-soluble form of Thy-1, purified from human cerebrospinal fluid (CSF). In contrast to the membrane-bound form of Thy-1 (M-Thy-1) isolated from human brain cerebral cortex, CSF-Thy-1 behaved like a completely hydrophilic glycoprotein, as analyzed by charge-shift electrophoresis in the presence of detergents and by liposome incorporation experiments. CSF-Thy-1 displayed a slightly higher apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than M-Thy-1. Digestions with endoglycosidases demonstrated that this difference in size was correlated to different processing of the three N-linked oligosaccharides, and the mobilities of the deglycosylated molecules were indistinguishable in sodium dodecyl sulfate gels. A Pronase-resistant carboxyl-terminal fragment was isolated from the CSF-Thy-1 after trypsin digestion and compared with the corresponding structure of M-Thy-1, obtained by treatment either with bacterial phosphatidylinositol-specific phospholipase C or with human serum (as a source of phosphatidylinositol-specific phospholipase D). The major fragment from CSF-Thy-1 behaved identically, with respect to size and charge, to the carboxyl-terminal fragment from M-Thy-1 solubilized by phospholipase D. These findings suggest an in vivo release of phosphatidylinositol-anchored Thy-1 glycoprotein from brain cells by the action of an endogenous phospholipase D.

Neural cell adhesion molecule (NCAM) and prealbumin in cerebrospinal fluid from depressed patients.

The size of the soluble form of the human cerebrospinal fluid (CSF) neural cell adhesion molecule, NCAM-sol, was by gel permeation chromatography estimated to 160-250 kDa. Within the CSF the concentration of NCAM-sol was found about 15-25% increased in lumbar fluid and 25% increased in ventricular fluid, both compared to cisternal fluid. Whereas prealbumin was found evenly distributed in CSF, albumin was relatively enriched in lumbar fluid. The concentrations of NCAM-sol and prealbumin were measured in lumbar CSF from psychiatric patients. Prealbumin was increased 7.2% and NCAM-sol was decreased 15.1% in depressed patients. The changes were partially normalized during recovery from the depression. The findings can be explained by hypothesizing that endogenous depression is associated with an increased choroid plexus activity and CSF production.

Shedding of leukemia-associated P24 antigen by lymphoblastoid cell lines.

We report the development of a unique enzyme-linked immunosorbent assay (ELISA) which makes possible the detection of leukemia-associated P24 antigen, utilizing its ability to bind the Ricinus communis agglutinin (RCA1) and a monoclonal antibody, SJ-9A4 simultaneously. Using the RCA1/SJ-9A4-ELISA, P24 antigen, as few as 50 X 10(3) cells from a common acute lymphoblastic leukemia (C-ALL) cell line could be detected. The presence of D-galactose gave complete and specific inhibition of P24 antigen binding to RCA1. Matched concentrations of D-glucose and D-sucrose had no effect on binding. The release of the P24 antigen into the culture medium by a C-ALL cell line maintained at 37 degrees C could be detected; however, no P24 antigen was present in the culture medium when the cells were maintained at 4 degrees C. Sequential analysis of the culture medium for soluble P24 antigen revealed that release of the P24 antigen associated with cell growth. Molecular sieve chromatography of concentrated culture medium indicated that shed P24 antigen was eluted in the macromolecule fraction. P24 antigen was detected in the cerebrospinal fluid (CSF) of four patients with P24 positive ALL at the time of relapse of the central nervous system (CNS) and was undetectable while in complete remission. The CSF from three patients with P24 negative ALL and three patients with aseptic meningitis had no detectable activity.

N-CAM in cerebrospinal fluid: a marker of synaptic remodelling after acute phases of multiple sclerosis?

We made a longitudinal study of the cerebrospinal fluid (CSF) neural cell adhesion molecule (N-CAM) content of 20 multiple sclerosis (MS) patients in an acute phase, and 10 in a non-acute phase. Both groups were compared with a control group of 23 subjects without neurological diseases. All MS patients were subjected to two or three lumbar punctures for collection of CSF samples, once a week. N-CAM analysis was performed by an enzyme-linked immunosorbent assay (ELISA) method. Comparison of N-CAM concentrations of the first CSF samples from each MS group with the control group values showed statistically different (lower) levels in the non-acute phase MS patients. Furthermore, a statistically significant increase of CSF N-CAM emerged from a comparison of the values of the first, second, and third samples of the acute phase MS patients group. This increase paralleled the patients' clinical improvement.