Axon Reactive B Cells Clonally Expanded in the Cerebrospinal Fluid of Patients with Multiple Sclerosis

Demyelination and axonal loss have been described as the histological hallmarks of inflammatory lesions of multiple sclerosis (MS) and are the pathological correlates of persistent disability. However, the immune mechanisms underlying axonal damage in MS remain unknown. Here, we report the use of single chain-variable domain fragments (scFv) from clonally expanded cerebrospinal fluid (CSF) B cells to show the role of an anti-axon immune response in the central nervous system (CNS) in MS. The cellular and subcellular distribution of the antigen(s) recognized by these CSF-derived clonal scFv antibodies (CSFC-scFv Abs) was studied by immunochemical staining of brain tissues obtained at autopsy from patients with MS. Immunochemistry showed specific binding of CSFC-scFv Abs to axons in acute MS lesions. The stained axons showed three major types of axonal pathological changes: 1) linear axons, axonal ovoid formation, and axonal transection were seen in the myelinated white matter adjacent to the lesion; 2) accumulation of axonal ovoid formations and Wallerian degeneration were seen at the border between demyelinated lesions and the adjacent white matter; and 3) Wallerian degeneration occurred at the center and edge of acute demyelinated lesions. These findings suggest a B cell axonal specific immune response in the CNS in MS.     

多发性硬化患者血清和脑脊液中S-100b蛋白测定及临床意义

为探讨S 10 0b蛋白与多发性硬化 (MS )临床的关系 ,采用ELISA方法检测 6 0例急性活动期MS ,2 6例对照组血清及脑脊液中S 10 0b含量 ,并进行比较分析。结果发现 ,血清及脑脊液中S 10 0b水平 ,急性活动期MS高于对照组 (P均 <0 0 1) ,MS组血清和脑脊液中S 10 0b含量异常增高者分别为 2 0 %和 96 6 % ;脑脊液S 10 0b水平MS急性活动 7d内高于急性活动 7d以上者 (P <0 0 5 ) ,两者又均高于对照组 (P均 <0 0 1) ;血清S 10 0b水平MS急性活动 7d内高于急性活动 7d以上者及对照组 (P均 <0 0 1) ,急性活动 7d后与对照组比较无差异 (P >0 0 5 ) ;脑脊液S 10 0b水平不同病残程度之间比较无显著性差异 (P >0 0 5 ) ,血清S 10 0b水平在中、重度病残者高于轻度病残者 (P均 <0 0 1) ,重度病残者虽高于中度病残者 ,但差异无显著性意义 (P >0 0 5 )。提示S 10 0b蛋白可作为判断MS疾病活动性及病情严重程度的重要生化标志物之一。     

多发性硬化患者淋巴细胞亚群的研究

目的 观察多发性硬化患者外周血及脑脊液中淋巴细胞亚群的水平。方法 用碱性磷酸酶抗磷酸酶法检测了56例多发性硬化（MS）活动期患者外周血和脑脊液（CSF）的淋巴细胞亚群。结果：活动期MS患者外周血CD4^ 、CD8^ 细胞较对照组减少，CD25^ 细胞、CD4^ /CD8^ 比值较对照组升高（P＜0．05）。CSF中CD4^ 、CD25^ 细胞、CD4^ ／CD8^ 比值较对照组升高，CD8^ 细胞降低（P＜0．05＜0．05），且CSF中淋巴细胞亚群均高于自身外周血中的相应细胞（P＜0．05）。经肾上腺皮质类固醇激素治疗后，随病情缓解，外周血、CSF中的淋巴细胞亚群均有不同程度的改善。结论 淋巴细胞亚群可能参与MS的发病，并与MS的缓解－复发有关。     

Immunoglobulin Synthesis In Vitro by Cerebrospinal Fluid Cells in Patients with Multiple Sclerosis

Incubated cerebrospinal fluid (CSF) cells from patients with multiple sclerosis synthesized IgG and IgA in vitro. The synthesized IgG had an oligoclonal distribution and showed the same elcctrophoretic pattern as the IgG of the CSF. The amount synthesized was greater during exacerbations than during remissions. Blood lymphocytes from the same patients synthesized an IgG in vitro that showed a completely different electrophoretic pattern The amount IgG synthesized by the blood lymphocytes was less than the amount synthesized by the CSF cells. The results demonstrate that at least part of the oligoclonal IgG of the CSF of patients with multiple sclerosis is synthesized intrathecally and suggest that the CSF cells are antigenically stimulated in vivo.     

多发性硬化症发作期外周血和脑脊液淋巴细胞亚群的分析

为分析多发性硬化症 (MS )患者发作期淋巴细胞亚群及给予甲基强的松龙 (MP )治疗后的变化 ,流式细胞仪测定 2 6例处于复发期MS患者外周血 (PB )和脑脊液 (CSF )及 8例MS患者予MP治疗后PB淋巴细胞CD3+ 、CD4 + 、CD8+ 、CD4 5RA+ 、CD4 + /CD4 5RA+ 、CD4 + /CD2 9+ 、CD19+ 、CD5 + /CD19+ 的百分率。结果发现MS患者PB中CD8+ 、CD4 5RA+ 和CD4 + /CD4 5RA+ 百分率降低 ,CD4 + /CD2 9+ 百分率和CD4 + /CD8+ 比值升高 ;CSF中CD3+ 、CD4 + 、CD4 + /CD2 9+ 百分率和CD4 + /CD8+ 比值高于PB ;淋巴细胞亚群与临床伤残程度和距此次发作的时间无关 ;MP治疗不影响PB淋巴细胞亚群变化。表明MS患者淋巴细胞通过血脑屏障有选择性 ,淋巴细胞亚群的变化在MS发病机制中起作用     

Thiol-Selective Mechanism of HIV Antigen Conjugation with Serum IgM, IgG, and IgA

The effects of HIV antigen glycoproteins gp160, gp41, and gp36 on thiol-dependent specific (affinity) binding of serum IgM, IgG, and IgA with the corresponding antigens were determined by the formation of signal thiol-containing analytes (free nonprotein SH groups). Free nonprotein SH groups were not found in the reaction mixture, which indicated that HIV antigen glycoproteins blocked this process. The results suggest that the thiol-selective mechanism underlies in vitro conjugation of HIV antigen glycoproteins gp160, gp41, and gp36 with serum immunoglobulins. Previous studies showed that this mechanism of conjugation with immunoglobulins is not characteristic of other lymphotropic viruses (e.g., hepatitis B virus).     

Virus-specific serum IgG, IgM, and IgA antibodies in cytomegalovirus mononucleosis patients as determined by immunoblotting technique

The immune response to individual human cytomegalovirus (CMV) structural polypeptides was studied in paired sera from 15 adult CMV mononucleosis (CMV-MN) patients and healthy controls by immunoblotting technique (IB). IgM and IgG antibodies to at least 11 structural polypeptides with molecular weights of 28K, 49K, 55K, 57K, 66-70K, 82K, 87K, 110K, 150K, 205K, and 235K were detected in the patients' sera in the serum sample obtained in the acute phase of the disease. IgA antibodies to polypeptides with molecular weights of 66-70K, 82K, 110K, and 150K were also detected in these sera. In healthy seropositive adults, IgG antibodies with the same molecular weight polypeptides, excluding the 205K and 235K polypeptides, were detected as in convalescent CMV-MN patients. A prominent reactivity of IgM and IgA antibodies to the 66-70K and 150K polypeptides was noted in the acute sera from all the CMV-MN patients examined, but not in a number of late convalescent sera. The potential implications of these findings in the development of specific serological tests are discussed.     

Oligoclonal Measles Virus-Specific IgG Antibodies Isolated from Cerebrospinal Fluids, Brain Extracts, and Sera from Patients with Subacute Sclerosing Panencephalitis and Multiple Sclerosis

Measles virus-specific antibodies were isolated from sera, cerebrospinal fluids (CSF), and brain extracts of patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) by absorption with measles antigens and subsequent acid elution of the antigen–antibody precipitates. Electrophoretically homogeneous measles antibodies were isolated from CSF or brain extracts in five patients with SSPE and in five out of seven patients with MS. Homogeneous IgG antibodies were also demonstrated in the sera from all SSPE patients and from three of the MS patients. The antibodies isolated from various control sera and from pooled CSF were electrophoretically heterogeneous. The results support the concept of a local synthesis in the nervous system of oligoclonal IgG antibodies to measles virus in all patients with SSPH and in some patients with MS. In SSPE, most or all oligoclonal IgG proteins of the CSF or brain carry measles antibody activities. In MS, only part of the oligoclonal IgG appears to be associated with measles antibody activity     

In Vitro Effects of Sodium Benzoate on Th1/Th2 Deviation in Patients with Multiple Sclerosis

Interleukin 4 (IL-4) can improve the clinical manifestations in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Sodium benzoate (NaB) deviates the cytokine profile to Th2 (or IL-4 producing) cells in EAE and thus might be effective in the treatment of MS. Therefore, in this study the effect of different concentrations of NaB on the percentage and mRNA levels of IL-4 and interferon gamma (IFN-γ)-producing peripheral blood mononuclear cells (PBMCs) of 20 Relapsing-remitting multiple sclerosis (RR-MS) patients and eight healthy controls was evaluated in the presence of mitogen (phytohemagglutinin, PHA) or specific antigen (myelin basic protein, MBP). Our results showed that in the patient’s group the percentage of CD4⁺IL-4⁺ cells was significantly increased in the presence of all concentrations of NaB when PBMCs were stimulated by MBP (p = 0.001) or PHA (p < 0.03). The same results were obtained for normal donors in the highest concentration of NaB, 1000 µg/ml (p = 0.02). Moreover, in the patient’s group the percentage of CD4⁺IFN-γ⁺ cells was decreased significantly when the PBMCs were stimulated by PHA and NaB (p < 0.004) or by MBP and 1000 µg/ml of NaB (p < 0.03). The effect of NaB on IL-4 and IFN-γ production was also documented at the mRNA levels. In conclusion, our data suggest that NaB is able to induce IL-4 production by human PBMCs and therefore might be a useful candidate for conjunctive therapy in RR-MS.     

Lymphocyte Subpopulations in the Cerebrospinal Fluid and Peripheral Blood in Patients with Multiple Sclerosis

The cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) were studied with respect to the frequency of lymphocytes with intra-cellular immunoglobulins of different Ig classes as well as the relative frequency of B and T lymphocytes. An increased number of Ig-positive cells were found in CSF (mean, 0.52%) when compared with blood (mean, 0.18%). In CSF there was a striking dominance of IgG-positive cells, very few IgA-positive cells, and almost no IgM-positive cells. The distribution in blood was approximately normal. The ratios between x ⁻ and λ-positive cells in CSF were all outside the range in blood. In CSF there were fewer B cells (mean, 4.7%) and more T cells (mean, 74.2%) when compared with blood (mean, 11.5% and 61.8%, respectively). The values for MS blood were approximately the same as for normal controls. The increased number of IgG-containing cells in the CSF are in agreement with earlier studies, which showed a local immunoglobulin synthesis. The increased proportion of T lymphocytes in CSF of MS patients may indicate that these cells play a role in the pathogenesis of MS.     

Lymphocyte Subpopulations in the Cerebrospinal Fluid and Peripheral Blood in Patients with Multiple Sclerosis

The cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) were studied with respect to the frequency of lymphocytes with intracellular immunoglobulins of different Ig classes as well as the relative frequency of B and T lymphocytes. An increased number of Ig-positive cells were found in CSF (mean, 0.52%) when compared with blood (mean, 0.18%). In CSF there was a striking dominance of IgG-positive cells, very few IgA-positive cells, and almost no IgM-positive cells. The distribution in blood was approximately normal. The ratios between χ- and λ-positive cells in CSF were all outside the range in blood. In CSF there were fewer B cells (mean, 4.7%) and more T cells (mean, 74.2%) when compared with blood (mean, 11.5% and 61.8%, respectively). The values for MS blood were approximately the same as for normal controls. The increased number of IgG-containing cells in the CSF are in agreement with earlier studies, which showed a local immunoglobulin synthesis. The increased proportion of T lymphocytes in CSF of MS patients may indicate that these cells play a role in the pathogenesis of MS.     

Cerebrospinal Fluid Cytokine Expression Profile in Multiple Sclerosis and Chronic Inflammatory Demyelinating Polyneuropathy

Background: Cerebrospinal fluid (CSF) analysis in patients with particular neurologic disorders is a powerful tool to evaluate specific central nervous system inflammatory markers for diagnostic needs, because CSF represents the specific immune micro-environment to the central nervous system. Methods: CSF samples from 49 patients with multiple sclerosis (MS), chronic inflammatory demyelinating polyneuropathy (CIDP), and non-inflammatory neurologic disorders (NIND) as controls were submitted to protein expression profiles of 47 inflammatory biomarkers by multiplex Luminex bead assay to investigate possible differences in the inflammatory process for MS and CIDP. Results: Our results showed differences in CSF cytokine levels in MS and CIDP; in particular, IL12 (p40) was significantly highly expressed in MS in comparison with CIDP and NIND, while SDF-1α and SCGF-β were significantly highly expressed in CIDP cohort when compared to MS and NIND. IL-9, IL-13, and IL-17 had higher expression levels in NIND if compared with the other groups. Conclusions: Our study showed that, despite some common pathogenic mechanisms, central and peripheral nervous system demyelinating diseases, such as MS and CIDP, differ in some specific inflammatory soluble proteins in CSF, underlining differences in the immune response involved in those autoimmune diseases.     

多发性硬化患者淋巴细胞亚群的研究

目的观察多发性硬化患者外周血及脑脊液中淋巴细胞亚群的水平. 方法用碱性磷酸酶抗磷酸酶法检测了56例多发性硬化(MS)活动期患者外周血和脑脊液(CSF)的淋巴细胞亚群.结果:活动期MS患者外周血CD4+、CD8+细胞较对照组减少,CD25+细胞、CD4+/CD8+比值较对照组升高(p<0.05).CSF中CD4+、CD25+细胞、CD4+/CD8+比值较对照组升高,CD8+细胞降低(p<0.05<0.05),且CSF中淋巴细胞亚群均高于自身外周血中的相应细胞(p<0.05).经肾上腺皮质类固醇激素治疗后,随病情缓解,外周血、CSF中的淋巴细胞亚群均有不同程度的改善. 结论淋巴细胞亚群可能参与MS的发病,并与MS的缓解-复发有关.     

Cells of Cerebrospinal Fluid of Multiple Sclerosis Patients Secrete Antibodies to Myelin Basic Protein In Vitro

To characterize the role of B lymphocytes in the pathogenesis of Multiple Sclerosis (MS), we have isolated mononuclear cells from cerebrospinal fluid (CSF) and stimulated them with a polyclonal B-cell mitogen (pokeweed mitogen). This study has been done with MS patients selected for the occurrence of an acute attack in the course of the disease and with patients hospitalized for other neurological diseases. Five of the 11 MS patients had B lymphocytes producing in vitro antibodies (Abs) directed against purified human myelin basic protein (hMBP), as revealed by Western blot analysis. None of the 20 patients with other neurological diseases showed such a reactivity. The produced Abs recognized only 1 or 2 hMBP peptides without dominance for a certain peptide. This result emphasizes the presence of B cells producing Abs against MBP in CSF of MS patients and shows the interest of studying mononuclear cells of CSF as a good marker of the pathogenesis.     

Tetanus Toxoid Reactive T Lymphocytes in the Cerebrospinal Fluid of Multiple Sclerosis Patients

Cerebrospinal fluid (CSF) lymphocytes of 6 multiple sclerosis (MS) patients were cultured with tetanus toxoid (TT) and irradiated autologous antigen presenting cells (APC) followed by propagation of the responding T-cells in Interleukin-2 containing medium. TT-reactive cell lines were recovered from 4 of the 6 CSF samples, even though the patients had not been TT booster immunized in recent years. These findings suggest an active circulation of antigen reactive lymphocytes from the systemic immune compartment(s) into the CSF even without recent activation by booster immunization. Since immune reactions to TT are very unlikely to be pathogenic in MS, these findings also indicate that the presence of CSF lymphocytes reactive to a particular antigen does not necessarily imply a causal role.     

Oligoclonal IgG and Free Light Chains in the Cerebrospinal Fluid of Patients with Multiple Sclerosis and Infectious Diseases of the Central Nervous System

Conventional and crossed immunoelectrophoresis were used to characterize oligoclonal gamma-globulin bands of the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) or subacute sclerosing panencephalitis (SSPE) or other infections of the central nervous system. Most gamma-globulin bands were identified as IgG, but some bands were identified as kappa or lambda, or both, free light chains, Bands of IgG showed various degrees of light-chain diversity, and individual bands appeared in many instances to be derived from more than one clone of cells. Sequential changes of the oligoclonal IgG were observed in SSPE but not in MS. Oligoclonal IgG was detected in sera from most patients with SSPE and some patients with MS.     

Kappa/lambda ratios in IgG, IgA and IgM of cerebrospinal fluid and of sera in patients with multiple sclerosis

Kappa/lambda (kappa/lambda) ratios of each immunoglobulin, i.e. IgG, IgA and IgM in cerebrospinal fluid (CSF) and in sera of patients with multiple sclerosis (MS) were analysed by sandwich enzyme linked immunosorbent assay. In CSF kappa/lambda ratios of IgG of MS patients were significantly (p less than 0.05) higher than those of normal controls, whereas the values of IgA and IgM did not differ significantly from those of normal controls. In sera kappa/lambda ratios of IgG, IgA and IgM did not differ significantly from those of normal controls. Our results suggest that in MS patients abnormal kappa/lambda ratios are also restricted to IgG components in CSF, as oligoclonal IgG bands are.     

Lymphocyte Populations in the Cerebrospinal Fluid and Peripheral Blood of Patients with Multiple Sclerosis and Optic Neuritis

The cerebrospinal fluid (CSF) and peripheral blood (PB) of patients with multiple sclerosis (MS), optic neuritis (ON), and aseptic meningitis (AM) were studied with respect to the percentage of B cells (using membrane Ig fluorescence), T cells, and T-cell subpopulations (using a rosetting technique or monoclonal antibodies). In the PB of all three patient groups there were normal B-cell levels but a significant decrease in T cells compared with PB of normal individuals. In the CSF the B cells were reduced but the T cells elevated when compared with the PB of the patients, and these differences were statistically significant. The level of total T cells was not influenced by disease activity in MS or ON, but there was a significant reduction of suppressor cells in PB during disease activity in MS patients. In CSF there were also fewer suppressor cells during active disease, but the reduction was not statistically significant. Differences in B and T cells in CSF and PB indicate that the immune reactions within the central nervous system are at least partially isolated from the rest of the immune system. The changes in the T-cell subpopulations in MS support the evidence for an immunoregulatory defect in this disease.