Specificity of autoantibodies to epitopes of myelin proteins in multiple sclerosis

An autoimmune response to one or more myelin-protein components is thought to be part of the pathogenesis of multiple sclerosis (MS). The immunodominant-autoantibody epitope may be localized on a linear peptide segment, on a conformation-sensitive epitope, or on an epitope resulting from post-translational modifications. Primary, secondary, and tertiary structures of myelin proteins may determine the specific site for binding of autoantibodies. A myelin protein-specific autoantibody can bind to either a linear or conformational epitope, whereas all of the T cell epitopes are linear. At present, the conformational epitopes of myelin proteins have not been identified; most of the methods used to identify the myelin-protein epitopes corresponding to the pathogenesis of multiple sclerosis are involved in the linear epitope mapping. Polymorphism or mutations may cause inappropriate expression of the myelin proteins with alterations to their linear and/or conformational epitopes, and make them susceptible to autoantibody binding, especially if these changes occur at the surface of the protein. This review focuses on the specificity of autoantibodies to the epitopes of myelin proteins and correlates this to the structures of proteins. Factors that influence the expression of myelin-protein epitopes such as the alpha-helical or beta-sheet structure of the protein, the tri-proline site, and the post-translational modifications as well as physicochemical properties of amino acid changed are included.     

Longitudinal spinal cord sections as substratum for anti-neurofilament antibody detection

A rapid and technically simple method for demonstrating anti-neurofilament antibodies using longitudinal sections of rat spinal cord as substratum and indirect immunofluorescent technique is reported. The results compare well with those obtained by the technically more difficult and time-consuming methods using as substratum central neurons cultivated in vitro. A total of 195 serum specimens from different neurological disorders and healthy subjects were studied. Immunofluorescent autoantibodies to neurofilaments were found in specimens of serum from patients with Creutzfeldt-Jakob disease (CJD), kuru, amyotrophic lateral sclerosis, parkinsonism dementia (Guam), Alzheimer's disease, and multiple sclerosis but in higher frequency in CJD and kuru than in the other disease or in healthy control subjects.     

Use of anti-neurofilament antibody to identify paired-helical filaments in inclusion-body myositis

Paired-helical filaments (PHF_s) are an important diagnostic criterion of the inclusion-body myositis (IBM) muscle biopsy; but, until now, their presence could be identified only by electronmicroscopy. In this report, we describe an easy immunocytochemical procedure, utilizing commercially available antibody, that enables reliable identification of muscle PHF_s by light microscopy. This procedure greatly facilitates diagnosis of IBM.     

Immunohistochemistry with anti-calbindin and anti-neurofilament antibodies in the cerebellum of methylazoxymethanol-treated mice

Mice pups were injected with methylazoxymethanol at birth (MAM0) or on the fifth postnatal day (MAM5) and their cerebella were examined when adult. Immunohistochemistry with an antiserum directed against calbindin, a protein specific for Purkinje cells, was used to survey more easily Purkinje cell position and orientation. For a general view of basket cell axon distribution, we used a monoclonal antibody that recognized the phosphorylated form of the 200 kD constituent protein of neurofilaments, which is axon specific. The present results confirm that in MAM5 the cytoarchitecture was preserved, some Purkinje cells degenerated, and the pericellular basket around the Purkinje cells was apparently normal. In MAM0 animals, the Purkinje cells appeared malpositioned and disoriented, the pinceau around the Purkinje cell hillock was absent, but basket cell axons were present. This indicated that the absence of pinceau was not due to the absence of basket cells, but probably to alterations of cell interactions, which hindered the proper pericellular basket formation.     

Circulating human pituitary prolactin cell autoantibodies and Alzheimer's disease

Prolactin cell autoantibodies (PRL cells Ab) were detected in 96 p. cent of 27 cases of Alzheimer's presenile dementia and senile dementia, as defined by clinical criteria and data from CT scans. The very high frequency of these autoantibodies appears to be even more significant of Alzheimer's disease in that they were found with a similar frequency in patients with Down's syndrome aged from 13 to 33 years. The frequency of PRL cells Ab is very low in the general population including patients with endocrine disorders. Current evidence indicates that higher levels exist only in cases of arteriopathic dementia, in subjects over 80 years and in subjects with isolated organic memory disturbances, though their frequency may be such that it suggests the probability of Alzheimer's disease. The etiopathogenic significance of these autoantibodies is not clear, but they support a role of autoimmune factors in Alzheimer's disease.     

Alzheimer's disease: Immunoreactivity of neurofibrillary tangles with anti-neurofilament and anti-paired helical filament antibodies

The origin of the paired helical filaments (PHP) that accumulate in human neurons during aging and in Alzheimer's disease and their relationship to normal neurofilaments remain unclear. The observation that a rabbit antiserum to highly enriched PHF fractions specifically labeled PHF in Alzheimer neurofibrillary tangles but showed no reaction with neurofilaments or other normal cytoskeletal proteins led us to compare this antiserum to two monoclonal antibodies, RT97 and BF10, previously found to cross-react with tangles and with the 210, 000 and 155, 000 mol. wt. neurofilament proteins, respectively. Both α-PHF serum and the neurofilament monoclonals strongly immunolabel almost all neurofibrillary tangles in Alzheimer cortical sections. Double-immunolabeling studies show that both reagents recognize the same tangles and usually show identical patterns of staining of intraneuronal fibrous material. Following prolonged extraction of cortex in sodium dodecyl sulfate, a step which removes normal neurofilaments but leaves PHF intact, almost all isolated tangles retain strong immunoreactivity with α-PHF serum at an intensity which is slightly reduced from that in cortical sections. In contrast, only a small number of isolated tangles are stained strongly by RT97 and BF10; most show much decreased or no reactivity with these monoclonal neurofilament antibodies. This differential immunoreactivity was confirmed by double-labeling studies. Tangles prepared under gentle extraction conditions show strong reactivity with α-PHF antibodies but again only a small number are strongly labeled by RT97 and BF10. We conclude that neurofibrillary tangles in Alzheimer's disease are heterogeneous as regards their filamentous content and contain both antigens cross-reacting with neurofilaments and antigens which are apparently unique to PHF and not shared with normal neurofilaments.     

Specificity of human IgM monoclonal antibodies from patients with peripheral neuropathy

Some patients with peripheral neuropathy and gammopathy have IgM monoclonal antibodies that react with the myelin-associated glycoprotein (MAG), some 20-26 kDa glycoproteins present only in the peripheral nervous system (PNS), and some acidic glycolipids that are also PNS-specific. This communication describes an investigation of 18 patients with IgM paraproteinemia and neuropathy to test for the presence of antibodies that react with each of these components. Eleven patients had IgM that reacted with MAG, and in all cases the IgM also reacted with the lower Mr glycoproteins and the acidic glycolipids that are specific for the PNS. With respect to the other 7 patients that did not react with MAG, in no instance did immune-staining of electroblots reveal the presence of reactivity with the 20-26 kDa glycoproteins of the PNS or with any other protein antigen in the PNS or central nervous system (CNS). However, these 7 patients fell into 3 categories with regard to reactivity with acidic glycolipids: three reacted with the acidic glycolipid fraction of both PNS and CNS tissue; two reacted with the acidic glycolipid fraction of the PNS but not the CNS; and two showed no reactivity with the acidic glycolipids from either PNS or CNS.     

Differential effects of prednisone and cyclophosphamide on autoantibodies in human neuromuscular disorders

We compared the effects of treatment of patients with prednisone or cyclophosphamide on a series of different types of autoantibodies. Levels of antiacetylcholine receptor (anti-AChR) antibodies and of antibodies to GM1 and GD1a gangliosides were measured in patients with a variety of neuromuscular disorders before and after treatment. Most patients had several autoantibodies present. We showed that prednisone treatment resulted in a reduction in titers of anti-AChR but not antiganglioside antibodies. Cyclophosphamide treatment produced a reduction of antiganglioside antibody titers. An intravenous and oral regimen was more effective than a single intravenous course of cyclophosphamide. We conclude that an immunosuppressive medication such as prednisone may reduce levels of some autoantibodies while producing no change in others, even in an individual patient. In addition, cyclophosphamide can suppress autoantibodies that prednisone does not. These differences in immunopharmacologic responses suggest that there are several distinct mechanisms of autoantibody production in humans. The utility of immunosuppressive medications in specific disease processes may be related in part to the mechanism of production of pathogenic antibodies.     

Immunohistochemical screening for autoantibodies against lateral hypothalamic neurons in human narcolepsy

Most human patients with narcolepsy have no detectable hypocretin-1 in their cerebrospinal fluid. The cause of this hypocretin deficiency is unknown, but the prevailing hypothesis states that an autoimmune-mediated mechanism is responsible. We screened for the presence of autoantibodies against neurons in the lateral hypothalamus in 76 patients and 63 controls, using immunohistochemistry. Autoantibodies were present in two patients, but also in two controls. However, one of the patients had a clearly different staining pattern and nerve endings of immunolabeled cells were found to project onto hypocretin-producing neurons, suggesting a possible pathophysiological role. Humoral immune mechanisms appear not to play a role in the pathogenesis of narcolepsy, at least not in the clinically overt stage of the disease.     

Specificity of action of human brain alanyl aminopeptidase on Leu-enkephalin and dynorphin-related peptides

The major cystosolic aminopeptidase (alanylaminopeptidase) was purified to homogeneity from human cerebral cortex and the specificity of its actions on a series of Leu-enkephalin-related peptides of increasing chain length was determined. In each case, only the N-terminal Tyr-Gly bond was hydrolysed. Kinetic analysis of the data revealed that the specificity constant (kcat/Km;s-1M-1) falls with increasing chain length from a maximum of 13.6 x 10(4) for Leu-enkephalin (5 residues) to 5.8 x 10(2) for dynorphin (1-13). Dynorphin 1-17, while not being degraded itself acted as a competitive inhibitor (Ki = 2.7 microM) of the degradation of smaller peptides. Beta-endorphin was not hydrolysed by analylaminopeptidase, nor did it act as an inhibitor of the enzyme.     

Canine and human myasthenia gravis autoantibodies recognize similar regions on the acetylcholine receptor

Serum from 35 cases of naturally occurring acquired canine myasthenia gravis (MG) were assayed for patterns of autoantibody specificities against canine acetylcholine receptor (AChR) using monoclonal antibodies (mAbs) and antiserum against defined regions of the AChR as competitive inhibitors of autoantibody binding. In human MG patients and in animals immunized with AChR purified from fish electric organs or mammalian muscle, most of the antibodies are directed against the main immunogenic region (MIR), a conformationally dependent region located on the extracellular surface of the alpha subunit away from the ACh binding site. In our studies using canine MG serum, we found that, as in human MG and in animals immunized with AChR, the antibody response is heterogeneous and predominantly IgG, with a large proportion of the autoantibodies directed against the MIR. The mAbs to the MIR blocked an average of 68% of serum antibody binding. A mAb to the beta subunit and polyclonal antiserum to the gamma subunit blocked an average of 34% and 39% of serum antibody binding, respectively, indicating that these subunits also contain relevant antigenic determinants, a pattern that has also been observed in human MG serum. Anti-alpha bungarotoxin binding site antibodies made up only a small fraction of the autoantibody population in canine MG as in human MG. These and other features described here suggest that canine MG is a useful model of human MG.     

Specificity of the thrombin-induced release of tissue plasminogen activator from cultured human endothelial cells

The addition of thrombin (9 nM) to primary cultures of human endothelial cells induces a 6- to 7-fold increase in the rate of release of tissue plasminogen activator (tPA). Several other serine proteases which specifically interact with endothelial cells were also analyzed for their effect on tPA release. Gamma-thrombin, an autocatalytic product of alpha-thrombin, promoted tPA release but was less effective than alpha-thrombin. A maximum increase of 5.5-fold was observed, although a concentration of gamma-thrombin 20 times greater than alpha-thrombin was required. The response to Factor Xa was similar to alpha-thrombin, although the stimulation was significantly reduced by the addition of hirudin or DAPA suggesting that prothrombin activation was occurring. The simultaneous addition of prothrombin with Factor Xa resulted in enhanced tPA release equal to that observed with an equimolar concentration of active alpha-thrombin. Thus, under these conditions, Factor Xa-cell surface mediated activation of prothrombin can lead to a secondary effect resulting from cell-thrombin interaction. Activated protein C, which has been implicated as a profibrinolytic agent, was also tested. No change in tPA release occurred after the addition of up to 325 nM activated protein C in the presence or absence of proteins. Factor IXa and plasmin were also ineffective. The effect of thrombin on the endothelial cell derived plasminogen activator specific inhibitor was also studied. Thrombin produced a small but variable release of the inhibitor with an increase of less than twice that of non-thrombin treated controls.     

Specificity of antisera to human fibrinopeptide A used in clinical fibrinopeptide A assays.

Distinction between fibrinopeptide A (FPA) and larger polypeptides containing the FPA sequence is critical for the interpretation of clinical results with FPA immunoassay methods. Therefore, the immunochemical reactivity of 14 rabbit anti-FPA sera with six different FPA containing antigens was studied in detail. Antigens tested included: fibrinogen; fragment E of fibrinogen; the amino-terminal disulfide knot of fibrinogen; Aalpha 1(Ala)-51(Met); Aalpha 1(Ala)-23(Arg); and, FPA. Synthetic partial sequences of FPA were also tested. The 14 FPA-specific antisera were divided into 3 distinct categories with: I, FPA immunoreactivity of larger polypeptides containing FPA approximately 1/100 of FPA on a molar basis, II, FPA immunoreactivity of the larger polypeptides intermediate between I and III; and III, FPA immunoreactivity of the larger polypeptides approximately equal to that of FPA on a molar basis. The antigenic determinants of a category I antiserum (R 2) are included in Aalpha 7(Asp)-16(Arg) with Asp(7), Phe(8) and Arg(16) being essential. When attached to FPA, the sequence Gly(17)-Arg(23) decreases the immunoreactivity of FPA with category I antisera 100-fold. The practical consequence of these findings is that, when category III antisera are employed, both FPA and larger FPA-containing polypeptides are equally immunoreactive. Since thrombin treatment of the larger polypeptides does not alter their immunoreactivity, category III antisera cannot discriminate between FPA and the larger polypeptides. On the other hand, with category I antisera, although the immunoreactivity of FPA itself is unaltered by thrombin treatment, larger polypeptides [e.g., Aalpha 1(Ala)-23tArg)] show a 100-fold increase in immunoreactivity following thrombin treatment and thus can readily be identified and separately quantitated. It is concluded that antisera with the specificity of category I are essential for the specific and accurate measurement of FPA, and for its distinction from larger FPA-containing polypeptides, in clinical plasma samples.     

Specificity of human T cell clones reactive to immunodominant epitopes of myelin basic protein.

Several recently discovered lines of evidence support the involvement of myelin basic protein (BP)-specific T cells in multiple sclerosis (MS). To identify potentially relevant immunodominant T cell epitopes, human BP (Hu-BP)-reactive T cell lines were selected from MS and normal donors and tested for reactivity to cleavage fragments and synthetic peptides of Hu-BP. The MS T cell lines responded to more Hu-BP epitopes than did normal lines, showing biased recognition of the N terminal half of the molecule, and one region in the C terminal half, suggesting increased sensitization to BP. The MS lines also differed from normal lines in their decreased percentage of CD8+ T cells. One hundred nine T cell clones isolated from these lines confirmed the reactivity pattern of the lines but did not reflect the mixed phenotype, since all but three clones tested were CD4+. T cell clones from HLA-DR2 homozygous donors responded to a variety of epitopes, indicating that this molecule was permissive in its ability to restrict T cell responses. Other epitopes, including the immunodominant 149-170 sequence, were restricted by several different major histocompatibility complex (MHC) molecules from both MS and normal donors. T cell receptor (TCR) V gene products could be identified on six of 38 clones tested using monoclonal antibodies. From one HLA-DR2 homozygous donor, four of eight clones utilized V beta 5.2 in response to different BP epitopes, providing initial support for the preferential use of a limited set of V region genes in the human response to BP. Preferential TCR V gene use in MS patients would provide the rationale to regulate selectively BP-reactive T cells through immunity directed at the TCR and thus test for the first time the hypothesis that BP-reactive T cells play a critical role in the pathogenesis of MS.     

Cross-reactivity of anti-acetylcholine receptor autoantibodies

The heterogeneity of the specificities of anti-acetylcholine receptor (anti-AChR) antibodies of myasthenia gravis (MG) patients has been demonstrated by comparing reactions against a panel of xenogeneic AChR. For each patient there was a more or less unique cross-reactivity profile. Such heterogeneity emphasizes the need to use human AChR for the routine detection of anti-AChR. In vitro cross-reactivity was important in predicting the effect of anti-AChR after passive transfer to rats. Specificity may influence the outcome in human neonates receiving maternal anti-AChR via the placenta. In contrast to the extreme heterogeneity seen in spontaneous MG, the antibodies associated with D-penicillamine–induced MG were more homogeneous.     

精神分裂症与自身抗体的观察

目的探讨精神分裂症与自身抗体的关系。方法测定了精神分裂症１００例,正常人６１人的血清抗甲状腺球蛋白抗体（ＴＧＡ）、抗甲状腺微粒体抗体（ＴＭＡ）、抗心磷脂抗体（ＡＣＡ）,抗脑抗体（ＡＢＡｂ）和抗胰岛素抗体。结果精神分裂症患者血清自身抗体阳性率均偏高;总阳性率为６６％,有２种以上自身抗体者占４６％,均明显高于正常人（Ｐ＜００５）。结论精神分裂症存在自身免疫倾向;自身抗体在精神分裂症的发病机制中,可能起重要作用。     

Association of polyclonal anti-GM1 IgM and anti-neurofilament antibodies with CSF oligoclonal bands in a young with amyotrophic lateral sclerosis

Introduction – The significance of the association of motor neuron syndromes with anti-GM1 antibodies remains unclear. We report the immunological study of a juvenile case of amyotrophic lateral sclerosis (ALS). Material and methods - Serum anti-GM1 and anti-neurofilament antibodies were assayed by ELISA and western blotting and cerebrospinal fluid (CSF) isoelectrofocusing was performed. Immunocytochemical studies were carried out with the patient's serum and CSF on human brain and spinal cord sections. Results - Serum polyclonal IgM anti-GM1, anti-neurofilament antibodies and CSF oligoclonal bands were detected. Furthermore, an in vitro production of anti-GM1 IgM was demonstrated. Immunocytochemical studies showed cytoplasm motor neuron immunostaining, due to both IgG and IgM, that substantially decreased after immunoabsorption of the serum with bovine neurofilament proteins but not with GM1-containing liposomes. No immunostaining was obtained with CSF. Immunosuppressive treatment with cyclophosphamide and two cycles of plasma exchanges lowered anti-GM1 antibody levels, but did not determine any clinical improvement. Conclusion - To our knowledge, this is the first report of ALS, associated with circulating levels and in vitro production of polyclonal IgM anti-GM1, anti-neurofilament antibodies and CSF oligoclonal bands. These findings suggest the occurrence in our patient of an autoimmune process that could be involved in the pathogenesis of ALS.