Persistent anti-myelin basic protein IgG antibody response in multiple sclerosis cerebrospinal fluid

Antibodies to myelin components, such as myelin basic protein (MBP), may play a role in pathogenesis of multiple sclerosis (MS) but results from determinations of anti-MBP antibodies are inconsistent. Enumeration of cells secreting antibodies represents a new approach to evaluate a specific antibody response regarding extent and localization, and reduces effects of e.g. antibody binding to target. Anti-MBP IgG antibody secreting cells were present in MS patients' cerebrospinal fluid (CSF) at a mean value of 1 per 833 cells, and they amounted to a mean value of about 2454 in the whole CSF compartment. Similar numbers were encountered in patients with other inflammatory neurological diseases (OIND). During follow-up, anti-MBP IgG antibody secreting cells persisted regarding frequency and numbers in MS, but decreased in OIND. Such cells were rarely detected in patients with tension headache. No correlations to clinical exacerbation of MS, disability or duration were discernable. In blood from MS and OIND patients, anti-MBP IgG antibody secreting cells were detected infrequently and at low numbers. The anti-MBP antibody response is strongly restricted to the IgG isotype. The anti-MBP IgG antibody response which is persistent and compartmentalized to the diseased organ, may be important for the development of MS.     

Anti-MOG and anti-MBP antibody subclasses in multiple sclerosis.

In a subset of multiple sclerosis (MS) patients antibodies against myelin antigens seem to be important in the demyelinating process. In this study we investigated IgM, IgA and IgG serum antibodies against the myelin oligodendrocyte glycoprotein (MOG) and the myelin basic protein (MBP) in 261 MS patients. Seventy-two per cent had anti-MOG antibodies, 59% were anti-MBP seropositive. The dominating antibody was anti-MOG IgM. A significant relationship between IgA and a progressive disease course was found. The predominance of IgGI together with the significantly associated occurrence of IgG3 against MOG corresponds to the prevailing IgGI and IgG3 isotypes in other autoimmune diseases. Patients who actually suffered from a relapse were significant more often anti-MOG and anti-MBP IgG3 seropositive than those in remission. However, patients treated either with intravenous immunoglobulins or interferon-beta showed a significant reduction of anti-MOG IgG3 antibodies.     

多发性硬化症髓鞘素组分特异性抗体分泌细胞

本文用酶联免疫斑点法（Ｅｌｉｓｐｏｔ）检测了２３例临床确诊多发性硬化症（ＭＳ）和１２例无菌性脑膜炎（ＡＭ）患者外周血（ＰＢ）和脑脊液（ＣＳＦ）中髓鞘素碱性蛋白（ＭＢＰ）、髓鞘素结合糖蛋白（ＭＡＧ）和含脂质蛋白（ＰＬＰ）特异性ＩｇＧ抗体分泌细胞。两组患者ＣＳＦ中该３种抗体分泌细胞均呈明显增多趋势，ＭＳ组尤著，但两组ＰＢ中该类细胞数均很少。指示对髓鞘素组分的Ｂ细胞免疫应答主要局限于与中枢神经系统（ＣＮ     

Estradiol potentiates poke-weed mitogen-induced B cell stimulation in multiple sclerosis and healthy subjects

Female preponderance in many diseases suggested with autoimmune pathogenesis, multiple sclerosis (MS) being classified as one of them, indicates a role for hormonal factors such as estrogen in disease development. To bypass monthly hormonal fluctuations in females, we evaluated in male patients with MS and male blood donors the effect of 17-beta-estradiol on numbers of IgG, IgA and IgM producing cells in cultures of peripheral blood lymphocytes. While estradiol alone had no effect, estradiol in combination with poke-weed mitogen (PWM) yielded in both groups higher numbers of IgG and IgA producing cells when compared with numbers obtained by PWM stimulation alone, indicating an additory effect of estradiol to that of PWM on B cell maturation. This effect was less pronounced in MS than in blood donors, especially for IgG producing cells, probably reflecting higher B cell activation in vivo taking place in MS. On the contrary, cells producing IgG, IgA and IgM antibodies against myelin, myelin basic protein and measles virus were not detectable after stimulation with PWM, nor with PWM and estradiol. Estradiol can in many patients with MS and in blood donors be considered a potent co-activator of B cells in presence of B cell stimulating factor in the form of PWM.     

亚低温治疗对实验性脑出血大鼠死亡率及脑组织钙含量的影响

目的本研究观察３２℃亚低温对实验性脑出血大鼠２４ｈ内死亡率和脑组织钙含量的影响。方法１３４只大鼠分成两部分：（１）６８只大鼠用于死亡率观察;（２）６６只大鼠用于脑组织钙含量测定。每一部分分成假手术对照组、常温脑出血组及亚低温脑出血组。结果常温组２４ｈ内死亡率为３６．６７％,亚低温组为４．５５％;脑组织钙含量常温组较对照组和亚低温组为高。结论亚低温治疗能显著减少实验性脑出血大鼠２４ｈ内死亡率,减少脑出血后脑组织钙含量。     

Antimyelin basic protein and antimyelin antibody-producing cells in multiple sclerosis.

The B-cell response to myelin and myelin basic protein was studied in patients with multiple sclerosis and in patients with acute aseptic meningoencephalitis by using a nitrocellulose immunospot assay. This method allows detection of single cells producing antibodies. Twenty-seven (79%) of 34 patients with multiple sclerosis had cells producing IgG antibodies against myelin, and 11 (57%) of 19 had cells producing IgG antibodies against myelin basic protein in cerebrospinal fluid (CSF), with mean values of 30 and 14 per 10(4) mononuclear cells, respectively. Total numbers of IgG-producing cells occurred at a mean number of 75 per 10(4) CSF cells. Cells producing antimyelin or anti-myelin basic protein antibodies of IgA or IgM isotypes were rarely found in CSF. Patients with acute aseptic meningoencephalitis less frequently showed CSF cells producing IgG antibodies against myelin and myelin basic protein. No cells producing antibodies against myelin or myelin basic protein were detected in peripheral blood of patients with multiple sclerosis or meningoencephalitis. Thus, a majority of patients with multiple sclerosis had CSF cells that produced IgG antibodies against myelin and myelin basic protein. These cells comprised a large proportion of the total IgG-producing cells. A pronounced B-cell response against autoantigens produced at the target for immune attack might be important in the pathogenesis of multiple sclerosis.     

Immunoglobulin-producing cells in CSF and blood from patients with multiple sclerosis and other inflammatory neurological diseases enumerated by Protein-A plaque assay

Using the Protein-A plaque assay, numbers of IgG + IgA + IgM producing cells determined in patients with multiple sclerosis (MS) were 0.1–5% in CSF and 0.1–0.7% in peripheral blood; interestingly, 7 of 11 MS patients had IgM producing cells in CSF. In patients with aseptic meningitis (AM), the corresponding values were 0.04–7.5% in CSF and 0.4–2.4% in peripheral blood. There were more Ig producing cells in peripheral blood from patients with AM and MS than in healthy subjects. cocorrelation between numbers of IgG producing cells in CSF and the concentrations of intrathecally produced IgG (CSF IgG index) was registered in patients with AM: the same was true for IgA. The Protein-A plaque method, adopted for 20 × 10³ lymphocytes, makes possible enumeration of Ig-producing cells in CSF and discrimination among cells secreting different Ig classes, thereby being a powerful tool for studying immune reactions in the CNS-CSF compartment.     

Lyme neuroborreliosis: cerebrospinal fluid contains myelin protein-reactive cells secreting interferon-γ

The mechanisms causing neurological manifestations and influencing the outcome in patients infected with the spirochete Borrelia burgdorferi are unknown. To study the involvement of autoimmune mechanisms in patients with Lyme neuroborreliosis (LN), ELIspot assays were used to determine the numbers of T lymphocytes which, upon stimulation with the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) and two MBP peptides, responded by the secretion of interferon-γ (IFN-γ). LN patients had compared to patients with other neurological diseases and tension headache in CSF elevated numbers of MBP- and PLP-reactive T helper type 1 (Th1) like IFN-γ secreting cells at mean frequencies of about 1 per 1300 and 1 per 1500 CSF cells, respectively. Numbers were elevated up to 5 months after successful treatment of LN, and they did not correlate with clinical findings. Autoreactive T cells were not elevated in the LN patients' blood. These findings match the previously reported elevation of anti-MBP IgG antibody secreting cells exclusively in CSF in LN. The IFN-γ release by autoreactive cells and the secretion of autoantibodies in the CSF may have relevance for development and outcome of LN.     

Intrathecal production of Chlamydia pneumoniae-specific high-affinity antibodies is significantly associated to a subset of multiple sclerosis patients with progressive forms

The purpose of this study was to provide further insight into the effective relevance of the association between Chlamydia pneumoniae and MS. We evaluated by ELISA technique cerebrospinal fluid (CSF) and serum levels of anti-C. pneumoniae IgG in 46 relapsing-remitting (RR), 14 secondary progressive (SP) and 11 primary progressive (PP) MS patients grouped according to clinical and Magnetic Resonance Imaging (MRI) evidence of disease activity. Fifty-one patients with other inflammatory neurological disorders (OIND) and 52 with non-inflammatory neurological disorders (NIND) were used as controls. A C. pneumoniae-specific intrathecal IgG synthesis as detected by the relative specific index was present in a small proportion of MS (17%), OIND (22%) and NIND (2%) patients and was significantly more frequent in MS and in OIND than in NIND (p<0.001) and in SP and PP MS than in RR MS patients (p<0.02). Among the patients with C. pneumoniae-specific intratecally produced antibodies, CSF high-affinity anti-C. pneumoniae IgG were found in the majority of SP or PP MS, occasionally in OIND, but not in RR MS and NIND patients. These findings confirm that the presence of a humoral immune response to C. pneumoniae within the central nervous system (CNS) is not selectively restricted to MS, but is shared by several inflammatory neurological conditions. In addition, our results suggest that an intrathecal production of C. pneumoniae-specific high-affinity IgG can occur in a subset of patients with MS progressive forms in which a C. pneumoniae brain chronic persistent infection may play an important pathogenetic role.     

Virus-reactive and autoreactive T cells are accumulated in cerebrospinal fluid in multiple sclerosis

Elevated numbers of B cells--plasma cells secreting antibodies to measles and mumps virus, and to myelin associated glycoprotein (MAG), one of several putative myelin autoantigens--have previously been reported in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS), while it is unknown if corresponding T cell reactivities occur. We have defined the T cell reactivities to measles and mumps virus and to MAG in an immunospot assay which is based on the detection of secretion of interferon-gamma (IFN-gamma) by single cells upon stimulation with specific antigen in short term cultures. Patients with MS had higher numbers of MAG-reactive T cells in blood compared to controls, while no differences were observed for measles or mumps virus-reactive T cells. In CSF, elevated numbers of MAG-reactive T cells and also of measles- and mumps-reactive T cells were found in patients with MS compared to other neurological diseases. A strong accumulation of antigen-reactive T cells was observed in the MS patients' CSF compared to blood. The magnitude of these T cell reactivities did not correlate with clinical MS variables. The T cell repertoire in MS thus includes, besides myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein, also MAG and, in addition, measles and mumps virus. It is not clear whether these T cell reactivities accumulated in the CSF have importance for the pathogenesis of MS or reflect phenomena secondary to myelin damage, or result from both these alternatives.     

Absence of oligoclonally restricted immunoglobulins in tears from multiple sclerosis patients

To study the extent of systemic immunodysregulation in multiple sclerosis (MS) we measured immunoglobulin (Ig)G, A, and M levels and studied their migrational properties after agarose isoelectric focusing in serum, cerebrospinal fluid (CSF) and tear samples from 18 MS patients with other neurological diseases (OND), and tears and serum samples from ten normal controls (NC). A slight elevation of total IgG, IgM and IgA levels was detected in tears from patients with MS and OND compared to NC. Of the five patients (two MS, three OND) that showed IgG oligoclonal bands (OCB) in tears, only one MS patient showed unique bands in tears not seen in the paired CSF and serum. We never found IgA, and IgM OCB in serum, CSF or tear samples. Our results suggest that polyclonal Igs are systemically elevated during chornic neurological inflammatory diseases. Oligoclonal Ig in MS, although ocassionally detectable in tears, is mainly confined to the central nervous system and appears restricted to class G.     

Autoantigen-induced IL-13 mRNA expression is increased in blood mononuclear cells in myasthenia gravis and multiple sclerosis

Evidence has been presented for the involvement of immune mechanisms in the pathogenesis of myasthenia gravis (MG) and multiple sclerosis (MS). The production of autoantibodies in both diseases is regulated by T-cells by means of cytokines. Interleukin-13 (IL-13) is mainly produced by T-helper type 2 cells and induces B-cell proliferation and antibody class switch. The role of IL-13 in MG and MS is not known. We employed in situ hybridization with synthetic radiolabelled oligonucleotide probes to detect and enumerate blood and cerebrospinal fluid (CSF) mononuclear cells (MNC) expressing IL-13 mRNA from patients with MG, MS, optic neuritis (ON), other inflammatory neurological diseases (OIND) and healthy controls. MG is associated with elevated levels of acetylcholine receptor (AChR) reactive IL-13 mRNA expressing blood MNC compared to control patients. In MS, numbers of MBP-reactive IL-13 mRNA expressing MNC were higher compared to cultures without antigen stimulation. The levels of MBP-reactive IL-13 mRNA positive MNC were higher in MS compared to MG, but not other controls. There were no differences in spontaneous IL-13 mRNA expressing blood MNC numbers between MG, MS, ON and control patients. The data suggest the involvement of IL-13 in both MG and MS.     

A myelin basic protein antibody cascade in purified IgG from cerebrospinal fluid of multiple sclerosis patients.

Immunoglobulin G (IgG) was purified by affinity chromatography from the CSF of multiple sclerosis (MS) patients and controls. In MS patients, the IgG fraction contains anti-myelin basic protein (anti-MBP), anti-MBP neutralizing antibody and an antibody which inhibits neutralization of anti-MBP. Anti-MBP was detected in patients with acute relapses, anti-MBP neutralizing antibody was present in patients in clinical remission and the inhibiting antibody was detected in patients with chronically progressing MS. A myelin basic protein antibody cascade could be involved in the mechanism of MS.     

Characterization of the local and systemic B cell response of normal and athymic nude mice with Sindbis virus encephalitis

During Sindbis virus (SV) encephalitis in mice B cells are an important component of the mononuclear inflammatory response and recovery depends primarily on the development of antiviral antibody. To begin to characterize various parameters of the local B cell response during SV encephalitis we have defined B cell isotype expression in brain sections, splenocytes and peripheral blood mononuclear cells (PBMC) in normal and athymic nude mice using an immunoperoxidase technique. Early (days 3-5) in SV encephalitis brain perivascular B cells are IgM or IgM/IgD-bearing lymphocytes, later (days 10-14) most B cells express one of the IgG isotypes or IgA. The pattern of isotype expression seen in the brain during the course of the encephalitis is reflected in the spleen and blood. The data suggest that progressive isotype switching results in an increasingly higher percentage of certain isotypes, especially IgG2a. Isotype switching of most B cells may occur outside of the brain, or may arise in situ from the IgM/IgD-bearing B cells found in the brain throughout the course of encephalitis. In athymic nude mice numbers of B cells in brain were markedly decreased and the cells present were primarily IgM-bearing, although IgG isotypes and IgA did appear late (day 14). The data suggest that T cells are required for recruitment of B cells into the inflammatory response as well as for normal isotype switching and peripheral B cell maturation during SV encephalitis.     

Adhesion of mononuclear cells from multiple sclerosis patients to cerebral vessels in cryostat sections of normal human brain

Leukocyte extravasation across the blood-brain barrier is a critical event in the pathogenesis of multiple sclerosis (MS). This complex multistep process includes the adhesion of leukocytes to the endothelial cells of the central nervous system microvasculature. To investigate this phenomenon in MS, we developed a modified version of the frozen-section assay. Peripheral blood mononuclear cells (PBM) from 26 MS patients, 26 healthy controls and 10 patients with other inflammatory non- neurological diseases (OIND) were co-incubated with cryostat sections of normal brain white matter, immunohistochemically labelled with anti-CD45 antibody and counterstained with Giemsa stain. CD45-positive PBM adherent to transected microvasculature were counted with an automated image analyzer. MS patients showed an increased number of vessel-bound PBM (48.8 +/- 36.4) with respect to healthy controls (27.4 +/- 20.7, P = 0.01) and OIND patients (22.6 +/- 7.8, P = 0.01). Significant differences were also obtained counting the number of vessel-bound PBM as a percent of total vascular cells between MS patients (12.7 +/- 7.2%) and healthy controls (6.9 +/- 5.4%, P = 0.002) or OIND patients (7.4 +/- 4.4%, P = 0.03). We confirm that PBM from MS patients show an increased potential of binding to cerebral vessels. The frozen-section assay provides a unique tool to study in situ the molecular interactions of leukocytes with brain vascular structures. Copyright Lippincott Williams & Wilkins     

Oral tolerance in experimental autoimmune encephalomyelitis: serum and salivary antibody responses.

We have recently reported that the oral administration of myelin basic protein (MBP) prior to encephalitogenic challenge results in suppression of experimental autoimmune encephalomyelitis (EAE). We examined the serum and salivary antibody responses to MBP in orally tolerant rats using an avidin-biotin enzyme-linked immunosorbent assay. Serum anti-MBP IgA and IgG, but not IgM levels are suppressed in orally tolerant versus control rats. This suppression is time dependent and is confined to the period when animals would otherwise be manifesting EAE clinical signs. In contrast, there is an increase in salivary anti-MBP IgA levels in MBP-fed rats relative to vehicle-fed controls. Thus, MBP-induced unresponsiveness is demonstrable at the humoral level, and moreover, a discrete compartmentalization between the serum and salivary anti-MBP responses exists.     

髓鞘少突胶质细胞糖蛋白抗体在多发性硬化中的意义

目的探讨髓鞘少突胶质细胞糖蛋白(MOG)抗体与多发性硬化(MS)临床表现及复发缓解型MS(RRMS)复发的关系。方法采用酶联免疫吸附法(ELISA)对60例MS、23例其他炎性神经疾病(OIND)、29例非炎性神经疾病(NIND)以及50例神经系统正常的对照(NC)患者血清及脑脊液(CSF)MOG抗体进行检测。结果MS患者CSFMOG抗体阳性率为28.3%(17/60),明显高于NC组[2%(1/50)]和NIND组[0%(0/29)],但与OIND组[21.7%(5/23)]比较差异无统计学意义。各组血清MOG抗体均为阴性。抗体阳性率急性活动期为31.8%(14/44),与稳定期[18.75%(3/16)]比较差异无统计学意义。CSFMOG抗体阳性的RRMS患者复发时间早于阴性患者,且其第1、2年复发率均高于阴性患者。结论在部分MS患者中枢神经系统内存在异常的MOG特异性B细胞免疫应答,且CSFMOG抗体对RRMS的复发有一定预测作用。     

Serum and cerebrospinal fluid antibodies to Nogo-A in patients with multiple sclerosis and acute neurological disorders

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large N-terminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing-remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.     

Myasthenia gravis: T and B cell reactivities to the beta-bungarotoxin binding protein presynaptic membrane receptor.

Antibodies against acetylcholine receptor (AChR) can be detected in most patients with myasthenia gravis (MG) and are considered to be involved in the immunopathogenesis of this disease. AChR are isolated from crude receptor preparations by binding to alpha-bungarotoxin (alpha-BuTx). Patients with MG have also antibodies against a second protein tentatively named presynaptic membrane receptor (PsmR), which has been isolated from crude receptor utilizing beta-bungarotoxin (beta-BuTx). PsmR could represent another antigen besides AChR relevant for the development of MG. We have now evaluated the T cell reactivity to PsmR in MG and controls by analysing the frequencies of cells which in response to PsmR in short-term cultures secreted interferon-gamma (IFN-gamma). The B cell response to PsmR was analysed in parallel by counting cells secreting anti-PsmR antibodies. Most patients with MG had PsmR reactive T cell in blood with a median number of about 1 per 44,000 mononuclear cells. Cells secreting anti-PsmR antibodies belonging to the IgG and IgA isotypes, less frequently of the IgM isotype were detected in most MG patients. A positive correlation was found between T cells reactive with PsmR and anti-PsmR IgG antibody secreting cells. PsmR reactive T and B cells were also detected in control patients, but at much lower numbers. Our results indicate that MG is accompanied by T as well as B cell responses to PsmR, in addition to the previously recognized responses to AChR. It remains to be shown whether these PsmR reactivities are of pathogenetic importance in MG.