Other Neurological Diseases Research Articles

Increased frequency of chromosome aberrations in long-term cultured cerebrospinal fluid lymphocytes of patients with multiple sclerosis

A method employing long-term lymphocyte culturing was developed to study chromosome aberrations in samples with very few cells. It was used to examine lymphocytes from the cerebrospinal fluid (CSF) and peripheral blood ( PB) in 23 patients with clinically definite multiple sclerosis (MS), nine patients with other neurological diseases (OND), and eight healthy individuals. MS patients had significantly more aberrations in CSF lymphocytes than in PB lymphocytes (6.4 vs 4.1; P = 0.003). In contrast, no such difference was noted among patients with OND (3.8 vs. 3.7; P = 0.89) or healthy controls (3.6 vs 3.5; P = 0.90). CSF lymphocytes from MS patients had more aberrations than CSF lymphocytes from healthy controls ( P = 0.012), but there was no difference between PB lymphocytes from MS patients and controls ( P = 0.58). The patients with OND were similar to healthy controls both in CSF (3.8 vs 3.6; P = 0.91) and PB lymphocytes (3.7 vs 3.5; P = 0.90).

Search for antibodies to neutral glycolipids in sera of patients with Guillain-Barré syndrome

Sera from 54 patients with Guillain-Barré syndrome (GBS), 34 patients with other neurological diseases (OND) and 32 healthy controls were tested for antibodies to total lipid fractions and higher neutral glycolipid fractions isolated from human and dog nerves, purified Forssman glycolipid and a panel of purified neutral glycolipids by both an enzyme-linked immunosorbent assay (ELISA) and a thin-layer chromatogram (TLC)-overlay technique. IgM and IgG antibodies to total lipid fractions, as well as to galactocerebroside, ceramide dihexoside, ceramide trihexoside, and globoside were not significantly elevated in the sera of GBS patients as compared to controls. High levels of anti-asialo-GM1 IgG antibodies, however, were detected in 6 of 54 (11%) GBS patients and 1 of 30 (3%) OND patients. Intense reactivity with purified Forssman glycolipid and a number of glycolipid antigens in higher neutral glycolipid enriched fractions of human cauda equina and dog sciatic nerves was noted by TLC-immunostaining in many GBS and control sera. Although the levels of anti-Forssman IgM were significantly decreased in GBS sera compared with normal sera ( P < 0.05) and OND sera ( P < 0.02), the levels of anti-Forssman IgG antibodies were not significantly different. With the possible exception of IgG antibodies to asialo-GM1, our results suggest that serum antibodies against Forssman glycolipid and neutral glycolipids are not significantly elevated in GBS patients and, thus, are unlikely to play an important role in the pathogenesis of this disease.

Antibodies to myelin-oligodendrocyte glycoprotein in cerebrospinal fluid from patients with multiple sclerosis and controls

Myelin-oligodendrocyte glycoprotein (MOG) has been implicated as a target for antibody-mediated immune attack in experimental autoimmune encephalomyelitis (EAE) which has been used extensively as an experimental model of multiple sclerosis (MS). We have screened cerebrospinal fluid (CSF) and plasma from 30 patients with MS, 30 with other neurological diseases (OND) and 30 with tension headache for anti-MOG antibodies of IgG isotype by enzyme-linked immunosorbent assay (ELISA). Such antibodies were detected in CSF from seven of the patients with MS, compared to two with OND and one with tension headache. No anti-MOG IgG antibodies were demonstrable in plasma. Antibody specificity was confirmed by Western blot immunostaining. Antibody levels were higher in MS compared to OND and tension headache. No correlation was observed between anti-MOG IgG antibodies and total IgG levels in CSF. The significance of anti-MOG antibodies demonstrated in MS CSF remains to be defined.

Serum and CSF levels of IL‐2, sIL‐2R, TNF‐α, and IL‐1β in chronic progressive multiple sclerosis

We measured interleukin-2 (IL-2), soluble IL-2 receptor (sIL-2R), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) by ELISA in paired sera and CSF from 50 chronic progressive multiple sclerosis (CPMS) patients during worsening disability, 19 patients with other neurologic diseases (OND), and in sera from 40 healthy volunteers. In the CPMS patients, 28% (14/50), 10% (5/50), 16% (8/50), and 6% (3/50) had elevated serum levels of IL-2, sIL-2R, TNF-alpha and IL-1 beta, respectively, compared with healthy controls. The only analyte we detected in the CSF was IL-2 in 1 CPMS patient (1/50, 2%). We also saw elevated serum sIL-2R in 16% (3/19) of OND patients. We found no significant difference in mean levels of serum sIL-2R between the 3 groups. Our study, the largest to date of CPMS patients, shows that serum and CSF levels of IL-2, sIL-2R, TNF-alpha, or IL-1 beta are not sensitive for, and the serum sIL-2R level is not specific for, CPMS. Therefore, measurement of these analytes will not be clinically useful for therapeutic or prognostic purposes in the majority of CPMS patients.

A comparison of regulatory cells in spinal fluid and blood in patients with multiple sclerosis and other neurologic diseases

Multiple sclerosis is a disease in which immune abnormalities are present both in the CNS and peripheral blood. Whether these changes are primary or secondary to the disease process is not known. We tested T-cell clones derived from activated lymphocytes in the blood and CSF of MS patients and controls for their capacity to regulate T-cell responses to alloantigens. A wide spectrum of regulatory functions were observed, ranging from marked enhancement to almost complete suppression. Clones from different patient populations and anatomic sites were equivalent in their regulatory functions with the net effect of clones in each compartment being suppression. However, certain clones from CSF and peripheral blood had the capacity to stimulate autologous T cells. Percentages of such clones in the peripheral blood of MS patients were significantly higher than in controls, while percentages in MS and other neurologic diseases (OND) CSF were equivalent. Our data suggest that (1) functional suppressor cells are not lost from the blood or CSF or MS and OND patients, (2) lymphocytes that have entered the CNS in patients with MS and other CNS diseases have equivalent regulatory functions, (3) MS may be an illness in which peripheral immunologic events are important in perpetuating the disease process, and (4) responses to autologous antigens may also play a role in this perpetuation.

Cytokine accumulations in CSF of multiple sclerosis patients

We identified the cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), and interleukin-6 (IL-6) by specific radioimmunoassays in the CSF of patients with multiple sclerosis (MS) and other neurologic diseases (OND). There was a high incidence of detectable IL-1 beta in patients with active MS compared with inactive MS or OND patients. TNF was also more frequently present in active MS than in OND CSF. By contrast, most MS CSF did not contain detectable IL-6. There was no correlation between the degree of CSF pleocytosis and the level of individual cytokines, suggesting that cytokine accumulations may be derived from CNS, and not CSF, cells. As IL-1 beta and TNF experimentally induce astrogliosis, demyelination, temperature elevation, lassitude, and sleep, and results raise the possibility that these cytokines may contribute to a variety of manifestations in MS and in other disease states.

Measles virus polypeptide‐specific antibody profile in multiple sclerosis

Elevated antibody (Ab) titers to measles virus (MV) is a frequent finding in MS. Although MV-Abs are synthesized intrathecally, it is not known whether this is due to polyclonal activation of B cells recruited from the blood, recognition of MV antigens within the CNS, or cross-reactivity with myelin antigens. This study examined these possibilities using purified MV polypeptides. We examined Ab reactivity to each polypeptide in serum and CSF from 21 MS patients, 5 with subacute sclerosing panencephalitis (SSPE), and 11 patients with other neurologic diseases (OND), and serum from 5 patients with acute MV infection and 11 normal controls. The serum of all subjects tested contained reactivity with MV and the 5 polypeptides. Of 21 MS patients, 20 had CSF reactivity with MV compared with 3/11 ONDs and 5/5 SSPE patients. Intrathecal MV-Ab synthesis was present in 11/21 MS patients, 5/5 SSPE, and in none of the ONDs. Nine of 21 MS patients had intrathecal synthesis of Ab to 2 MV polypeptides. Serum and CSF reactivity in MS patients was skewed towards the F polypeptide. The results are consistent with the concept of polyclonal B cell activation within the CNS, but the heightened response to F could also reflect cross-reactivity with a relevant antigen in MS.

Multiple Sclerosis: Cell-Mediated Immunity to Human Brain Gangliosides

Cell-mediated immunity (CMI) to myelin components has been implicated in Multiple Sclerosis (MS) pathogenesis: two targets were suggested, Myelin Basic Protein with controversial results and, more recently, gangliosides. In order to investigate their possible involvement, we have performed Leukocyte Migration inhibition (LMI) tests in the presence of human brain gangliosides. Thirty nine MS patients (twenty four being "definite", according to McDonald and Halliday's classification), twenty nine patients with Other Neurological Diseases (OND), thirty six patients with Inflammatory diseases (ID) and forty healthy controls were tested. MS patients were divided into two groups, depending on the clinical stage of the disease. The mean migration inhibition percentage of the MS-attack group was found to be significantly different from the four others (p less than 0.01) (24.4 +/- 16.2 versus 10.9 +/- 8.5 in MS without attack, 4.4 +/- 12.9 in OND, 3.9 +/- 13.9 in ID and 11.1 +/- 12.1 in healthy subjects). LMI to gangliosides is therefore significantly increased during the attack stage in MS. These results support the notion of a Delayed Type Hypersensitivity to these glycolipids during the active stage of the disease.

Absence of natural killer (NK) cell activity against oligodendrocytes in multiple sclerosis

The cytotoxicity of natural killer (NK) cells directed to enriched cultures of bovine oligodendrocytes was investigated in multiple sclerosis (MS) patients and controls. Macrophage-depleted peripheral blood lymphocytes were used as effector cells in a 4-h 51Cr release assay. No significant cytotoxic activity to oligodendrocytes was identified in either MS or control groups. In contrast, a definite cytotoxic activity directed toward K562 cells was observed in the study populations. No statistically significant difference was observed between chronic progressive or stable MS, other neurological diseases (OND), and normal controls. There results indicate that NK cell activity directed toward intact bovine oligodendrocytes is not significantly different between MS and control groups and question the significance of studies employing K562 cells as the target in NK assays in MS. Furthermore, these observations suggest that NK-mediated cytotoxicity against oligodendrocytes is unlikely to be a specific mechanism mediating demyelination in MS.

Activated T lymphocytes in cerebrospinal fluid of patients with HTLV-I-associated myelopathy (HAM/TSP)

In order to detect activated T lymphocytes in the cerebrospinal fluid (CSF) of patients with human T lymphotropic virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), we studied CSF lymphocytes in untreated patients with HAM/TSP and other neurological diseases (OND). Dual-immunofluorescence staining technique was performed using fluorescence microscopy. No significant difference in the CD4 +/CD8 + ratio of CSF lymphocytes was observed between HAM/TSP patients and patients with OND. However, both CD4 + and CD8 + CSF lymphocytes of HAM/TSP patients contained higher percentages of HLA-DR-positive cells than those of patients with OND ( P < 0.05). suggesting that the activated CSF T lymphocytes were composed of both CD4 + and CD8 + subsets in patients with HAM/TSP.

Search for a retrovirus in long-term cultured cerebrospinal fluid cells and peripheral blood mononuclear cells from patients with multiple sclerosis.

Long-term peripheral blood mononuclear cell (MNC) cultures stimulated with interleukin 2 (IL-2) or IL-2 + phytohemagglutinin were established from 33 multiple sclerosis (MS) patients, 9 with other neurological diseases (OND), and 24 normal controls (C). Cultures were analysed for growth characteristics, reverse transcriptase (RT) in the culture medium, 2'-5' oligoadenylate synthetase in the cells, and cell morphology. None of these parameters differed in the MS group compared with the OND and C groups. Furthermore, 11 cerebrospinal fluid cell cultures were established without feeder cells. Morphology studies of the cells and RT assays of the supernatants from these cultures were normal. Induction studies by dexamethasone and 2-bromo-5'-deoxyuridine in 2 of these cultures did not reveal any signs of a virus. The significance of these results for the retrovirus hypothesis is discussed.

Response of human T lymphocyte lines to myelin basic protein: Association of dominant epitopes with HLA class II restriction molecules

In animals, the selection in vitro of T cell lines to myelin basic protein (MBP) can define immunodominant and encephalitogenic epitopes which are preferentially associated with class II major histocompatibility (MHC) molecules. These principles were used to evaluate the specificity and MHC restriction of 14 human MBP-reactive T cell lines selected from normal individuals and patients with multiple sclerosis (MS) and other neurological diseases (OND). The four normal T cell lines recognized single, separate immunodominant MBP epitopes which were restricted by MHC molecules from the DR or in one case the DP class II locus. In contrast, the MS and OND T cell lines recognized multiple MBP epitopes, each in association with a discrete class II MHC molecule from the DR or DQ locus. Overall, HLA-DR molecules were used preferentially to associate with epitopes on human MBP, restricting 26/33 responses. As predicted from animal studies, T cells from genetically disparate individuals responded to different immunodominant epitopes on human MBP in association with distinct MHC class II molecules. HLA-DR2, which is overrepresented in MS patients, possessed an unusual capacity to restrict all eight epitopes identified on MBP in this study. These data provide the first evidence of genetically restricted human T cell recognition of potentially encephalitogenic epitopes of MBP.

Human T lymphocyte response to myelin basic protein: Selection of T lymphocyte lines from mbp‐responsive donors

The goal of this study was to delineate the importance of blood T lymphocyte responses to several myelin basic protein (MBP) preparations in the ultimate selection of MBP-specific T lymphocyte lines. Proliferation responses to human myelin basic protein (MBP) were assessed in blood samples from 27 multiple sclerosis (MS) patients, 20 patients with other neurologic diseases (OND), and 26 normal subjects, using five MBP preparations with different histories and electrophoretic characteristics to enhance the spectrum of epitopes represented. Substantial variations were observed in the ability of different MBP preparations to induce blood T cell proliferation in a given donor. However, four out of five of the MBPs induced modest but significant proliferation in the MS study population relative to normal individuals, with intermediate responses occurring in OND patients. Positive responses occurred more frequently in MS patients (78%) than in normal donors (31%), and were an important prerequisite for the successful selection of MBP-specific T cell lines.

Antibodies to Myelin-Associated Glycoprotein (MAG) in the cerebrospinal fluid of multiple sclerosis patients

Cerebrospinal fluids (CSF) and sera from patients with multiple sclerosis (MS), other neurological disease (ONDs) and healthy controls were tested for antibodies to myelin-associated glycoprotein (MAG) by several different assays. Using a very sensitive, solid-phase radioimmunoassay with radioiodinated protein A, a statistically significant elevation of anti-MAG antibodies was detected in MS CSFs in comparison to those from ONDs and healthy controls. The antibodies reacted with human MAG, but not with rat MAG, and appeared to be directed towards carbohydrate dterminants in the glycoprotein. The CSFs from high IgG producers had significantly greater anti-MAG antibody levels than those from low IgG producers, even though the assays were done on CSF samples that had been normalized to the same IgG concentration, the elevated antibodies were not detected when the same samples were tested with a liquid-phase radioimmunoassay or an enzyme-linked immunosorbent assay, and the antibodies in the MS CSF also could not be detected by Western blotting. An elevated level of antibodies was not found in sera from MS patients by any of the assays, possibly because these samples gave higher and more variable background. The results suggest that there is a low level of humoral immunity to MAG in MS patients that can only be detected by the most sensitive assays. This weak immune response to MAG may be secondary to the demyelinating process, but could play a role in the progression of the disease.

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45–89 peptide fragment, using a [ 3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15–31, 75–96, 83–96 and 131–141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131–141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the response in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.

Protein gradients in the cerebrospinal fluid and the calculation of intracerebral IgG synthesis

When six successive samples of 2.5 ml cerebrospinal fluid (CSF) were withdrawn from eight multiple sclerosis (MS) and 12 other neurological disease (OND) patients, the IgG index rose continuously between 2.9 and 24.0%, while both the Tourtellotte as well as the Reiber / Felgenhauer formulas were less affected.

T-cell subsets in the cerebrospinal fluid and blood of patients with multiple sclerosis

The absolute numbers and ratios of helper/inducer (T4) and cytotoxic/suppressor (T8) T-cells were determined in cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) and various other neurologic diseases (OND). In patients with MS, the T4:T8 ratio was higher in both blood and CSF, and the increase was significantly greater in CSF than in blood. These findings were due to an increased proportion of T4-lymphocytes in the CSF and to a decreased proportion of T8-cells in blood. These results indicate the need for additional studies of CSF lymphocytes in patients with MS.

Absence of antibodies to HTLV-1 in sera from Hungarian MS patients.

The aim of this study is to examine the association between multiple sclerosis (MS) and anti-human T-lymphotropic virus type I (HTLV-I) antibody. Serum samples from 16 Hungarian caucasians with MS, 2 gipsy patients with MS, 13 Hungarian caucasians with other neurological diseases (OND) and 2 gipsy patients with OND were tested by Western blot combined with autoradiography using disrupted virus from MT-2 cell line and recombinant p24 as antigens. Negative results were obtained in all samples except for 3 Hungarian OND which were reactive to disrupted virus, but not to recombinant p24.

Multiple sclerosis: effects of activated T-lymphocyte-derived products on organ cultures of nervous tissue

Supernatants from multiple sclerosis (MS) T-lymphocytes cause damage to both myelin and glial cells in cerebellar cultures assessed visually and by radiolabel release. Control T-lymphocytes, even after phytohaemagglutinin (PHA) stimulation, yielded supernatants which induced only slight damage, and at later times patients with other neurological diseases (OND) gave variable results. These differences suggest that MS T-lymphocytes are pre-activated in vivo to produce demyelinating factors while control T-lymphocytes are not pre-activated to the same extent. The visual evidence of activation of cerebellar macrophage-like cells was a common finding after MS T-lymphocyte supernatant treatment but there was no correlation with the severity of demyelination. There was a positive correlation between the percentage IL-2 receptor-bearing lymphocytes and the degree of supernatant-induced in vitro demyelination.

Interleukin-1 and interleukin-2 production by peripheral blood mononuclear cells in multiple sclerosis patients

The production of interleukin-1 (IL-1) and interleukin-2 (IL-2) by peripheral blood mononuclear cells (PBM) was assessed in multiple sclerosis (MS) patients in relapse, chronic progressive MS patients, patients with other neurological diseases (OND) and healthy subjects. Production was defined as the level of IL-1 and IL-2 in PBM supernatants. Neither spontaneous nor LPS-induced IL-1 production differed significantly in MS, OND patients or healthy individuals. On the other hand PHA-induced PBM IL-2 production was significantly less in MS patients in relapse (130 ± 10.0 U/ml) than in chronic progressive MS patients (172 ± 9.8 U/ml), OND patients (192 ± 11.5 U/ml) and healthy subjects (215 ± 13.8 U/ml) ( P < 0.02). Spontaneous IL-2 production was also diminished in MS patients in relapse (31 ± 7.2 U/ml) as compared to chronic progressive MS patients (46 ± 8.8 U/ml) and healthy subjects (49 ± 11.1 U/ml) ( P < 0.01). Anti-Tac monoclonal antibody was used to study IL-2 receptor expression on the same sample of PBM that was used for IL-2 study. MS patients in relapse had significantly higher levels of IL-2 receptor-positive unstimulated PBM (6.0 ± 2.2%) as compared to chronic progressive MS (2.0 ± 0.9%), OND (2.5 ± 1.1%) and healthy subjects (1.5 ± 0.7%) ( P < 0.002). We postulate that reduced apparent IL-2 production by PBM of MS patients in relapse may result from immediate IL-2 binding to receptor expressed on activated T lymphocytes and internalization of IL-2-receptor complex.