Biomedical subjects
Publications and source records attributed to A J Steck.
We discuss current approaches in the treatment of immune mediated neuropathies and review recent progress in management. Intensive evaluation has led to improved diagnosis of the cause of neuropathies. Because immunosuppressive drugs are potentially hazardous, strict guidelines for their clinical use, including clinical immunological, neurophysiological and histological investigations, should be followed.
A 74-year-old woman with a sensory neuropathy and IgM M-protein monoclonal gammopathy of undetermined significance developed a fatal B-cell cerebral lymphoma. CSF protein immunofixation revealed intrathecal secretion of a paraprotein of the same heavy- and light-chain isotypes as the serum monoclonal component (IgM-lambda). Reactivation of Epstein-Barr virus was present in the lymphoma cells. Different factors may be involved in the preferential malignant development of the monoclonal B-cell clone within the CNS.
Paraneoplastic syndromes, or the remote non metastatic effects of cancer, are wellknown entities that may affect any organ or tissue in the body, including the central nervous system. Some have been shown to be associated with autoantibodies against the nervous system. One typical example ist the subacute sensory neuronopathy of Denny-Brown, classically associated with small cell lung cancer. These patients present with the subacute onset of a sensory neuronopathy which may appear as much as one year before the primary tumour is detected. An antibody called anti-Hu has been found in the serum in these patients, which recognizes a neuronal protein called Hu (M.W. 30 to 40 kD). This antibody can currently be detected by a combination of immunological methods such as Western blot analysis or immunohistochemistry.
Immunostaining of human, bovine and rodent unfixed nervous tissue sections was performed in order to characterize the structures recognized by anti-glycolipid antibodies. Four human sera from patients, two with M-IgM and motor neuron syndrome or motor neuropathy and two with motor neuropathy and polyclonal IgG antibody activity against gangliosides (GL; i.e. GM1, GD1b, GD1a), were utilized. Serum from a patient with sensory neuropathy and M-IgM immunoglobulins with antibody activity against sulfatide (SUL) was included in this series. This study shows that polyclonal and monoclonal anti-glycolipid antibodies give three different patterns of staining. The first is cholera toxin-like showing a more restricted neuronal pattern of staining. The second is peanut agglutinin-like, which includes the carbohydrate epitope shared by a group of glycoproteins in the gray and white matter. The third (anti-SUL) gives a preferential myelin staining. However, sera with anti-GM1 and anti-SUL antibodies recognize a number of closely situated determinants in the gray matter of the spinal cord and in the granule cells, while in peripheral nerves or in neuronal cells in culture their binding produces a different pattern (nodes of Ranvier for anti-GL; myelin for anti-SUL). These findings indicate that immunohistochemistry with anti-GL and anti-SUL antibodies may provide information regarding the glycolipid-bearing anatomical structures as target antigens and further substantiate the role of these molecules in the pathogenesis of autoimmune neurological disorders.
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Paraneoplastic syndromes such as the subacute sensory neuronopathy (SSN) and paraneoplastic cerebellar degeneration (PCD) are associated with autoantibodies directed against various neural antigenic structures. SSN is characterised by autoantibodies against a neuronal intranuclear component called Hu (M. W. 35 to 40 kD), whereas in PCD these antibodies are directed against a Purkinje cell cytoplasmic component called (M. W. 34 and 65 kD). Neuroblastoma cell lines maintained in culture have been shown to contain neuronal antigens. We have demonstrated the presence of the Hu antigen in neuroblastoma cell lines such as SKN-SH, LAN-1 and IMR-32 by both immunocytochemistry and immunoblots of nuclear extracts. The Yo paraneoplastic antigen has been found to be expressed in HeLa cells. These methods may be used for screening of patients with suspected paraneoplastic disease and/or malignancy. Western blot analysis appears to be superior to immunohistochemistry alone. In our experience only 11 out of 122 SCLC patients were positive for the anti-Hu antibody, whereas 5 out of 5 patients with SSN/SCLC were positive, and all of the neurological controls were negative. The availability of cell lines expressing paraneoplastic antigens offers an easy diagnostic assay which may complement or replace conventional immunohistochemistry.
Paraneoplastic cerebellar degeneration is a well-defined clinical entity typically associated with ovarian or breast carcinoma. Serum or CSF of these patients has been shown to contain autoantibodies against the cytoplasm of Purkinje cells. These antibodies have been referred to as anti-Yo. We report the case of a 67-year old woman with ovarian adenocarcinoma presenting with subacute cerebellar dysfunction. Her serum was found to react with the cytoplasm of Purkinje cells and HeLa cells. On Western blot analysis a 60 to 70 kD reaction was obtained. This suggests the presence of a classical anti-Yo antibody.
Myelin-associated glycoprotein (MAG) is a neural cell adhesion molecule expressing the L2/HNK-1 carbohydrate epitope. MAG is heavily glycosylated containing 30% carbohydrate by weight. In this study, human MAG glycopeptides were isolated and sequenced. Of the 9 MAG sequons 7 were glycosylated and 1 was partially glycosylated at Asn106. Asn332 which was not recovered in the glycopeptide fractions was probably not glycosylated. Furthermore, preliminary data indicate that all MAG glycosylated sequons might bear the L2/HNK-1 epitope.
A patient who had a polyneuropathy compatible with a chronic inflammatory demyelinating polyneuropathy and was initially negative for anti-myelin-associated glycoprotein (MAG) antibodies developed a double monoclonal gammopathy, IgM kappa and IgM lambda, two years after the diagnosis. The IgM kappa, but not the IgM lambda, exhibited strong anti-MAG antibody activity. The late appearance of the anti-MAG immunoreactivity suggests that in patients with an initial diagnosis of chronic inflammatory demyelinating polyneuropathy, the search for anti-MAG antibodies should be repeated during the course of the neuropathy.
Myelin/oligodendrocyte glycoprotein (MOG) is a quantitatively minor component of CNS myelin. In this study, human MOG was found to express the L2/HNK-1 epitope on N-linked oligosaccharide structures. This carbohydrate epitope has been found previously in three other characterized human myelin glycoproteins: the myelin-associated glycoprotein, P0, and the oligodendrocyte-myelin glycoprotein. It seems, therefore, that the L2/HNK-1 epitope is expressed frequently in human myelin glycoproteins. Serial lectin affinity chromatography of 14C-glycopeptides indicated that MOG N-oligosaccharide structures are mainly of the complex type, accounting for 77.8% of total radioactivity. In contrast with myelin-associated glycoprotein and P0, which express the L2/HNK-1 epitope on fucosylated structures, in MOG the epitope was detected on all glycopeptide fractions obtained by serial lectin affinity chromatography, although a preferential expression of the L2/HNK-1 epitope was observed on fucosylated structures. Finally, the data indicated that, as for other human myelin glycoproteins, only a subpopulation of MOG molecules expresses the L2/HNK-1 epitope.
In addition to myelin basic protein (MBP), other minor components of myelin, such as myelin-associated glycoprotein (MAG), may be important autoantigens in MS. To determine whether MAG might be involved in an autoimmune reaction in MS, we screened peripheral blood lymphocytes from MS patients and normal subjects for their sensitization to human MBP and MAG antigen using a [3H]thymidine incorporation assay. We recorded three patterns in MS patients: (1) patients who responded neither to MAG nor to MBP (4/11); (2) patients who responded to both MBP and MAG (5/11); and (3) patients who gave an exclusive response to MAG (2/11). The 10 healthy controls did not respond to either MAG or MBP. That some individuals with MS expressed a specific response against MAG and that more than 50% of MS patients expressed a sensitization to MAG suggest that MAG may have a role in the pathogenesis of MS.
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Paraneoplastic neurological syndromes are well established causes of neurological dysfunction in cancer patients. Recently an auto-immune origin has been found for the Lambert-Eaton myasthenic syndrome, the subacute sensory neuronopathy of Denny-Brown, the paraneoplastic cerebellar degeneration, the paraneoplastic opsoclonus-myoclonus as well as a few other minor syndromes. Indeed patients suffering from these diseases have been found to produce antibodies directed against the central nervous system. These antibodies can be detected as much as a year before the cancer is found and they are therefore interesting for neurological and oncologic diagnosis.
The N-linked oligosaccharide structures of human myelin-associated glycoprotein (MAG) and P0 have been characterized by serial lectin affinity chromatography (SLAC) of 14C-glycopeptides. 14C-Glycopeptides were prepared from purified MAG derivative and P0 by extensive proteolytic digestion and N-14C-acetylation. Assuming that all the 14C-glycopeptides were radiolabelled to the same specific radioactivity, the relative occurrence of the oligosaccharide structures was correlated to the amount of incorporated radioactivity. Sixteen and 15 fractions were generated by SLAC of MAG and P0 14C-glycopeptides, respectively. Despite this tremendous structural heterogeneity, the oligosaccharide "fingerprints" of MAG and P0 obtained by SLAC displayed similarities: (a) of the three types of N-linked oligosaccharides, the complex type accounted for 80.4% and 94.9% of MAG and P0 radioactivity, respectively; (b) biantennary complex oligosaccharides were the major structures present on MAG and P0; (c) approximately 60% of MAG and P0 oligosaccharides possessed a bisecting N-acetylglucosamine residue; and (d) large amounts of oligosaccharides with an alpha(1-6)fucose residue were found in both MAG and P0 and, noticeably, approximately 25% of the tri- and/or tetraantennary and approximately 90% of the bisected biantennary oligosaccharides of both glycoproteins contained alpha(1-6)fucose residues in the core. This study demonstrates that MAG and P0, both belonging to the immunoglobulin superfamily, display structural similarities in their N-linked oligosaccharide contents.
The epitope for HNK-1 and patient's monoclonal autoantibodies in demyelinating polyneuropathy associated with immunoglobulin M gammopathy is borne by different types of N-linked oligosaccharide structures in human P0 and myelin-associated glycoprotein (MAG). Fourteen glycopeptide fractions bearing different oligosaccharide structures were obtained from either MAG or P0 glycopeptides by serial lectin affinity chromatography on concanavalin A-Sepharose, Phaseolus vulgaris erythrophytohemagglutinin-agarose, Pisum sativum agglutinin-agarose, and Phaseolus vulgaris leucophytohemagglutinin-agarose. As shown by dot-TLC plate immunostaining, the same MAG and P0 glycopeptide fractions were recognized by HNK-1 and patient's immunoglobulin M, confirming that these antibodies display similar specificities. The antigenic carbohydrate was present in glycopeptide fractions that either interact with Pisum sativum agglutinin-agarose or were bound by Aleuria aurantia agglutinin-digoxigenin, indicating that these structures contained alpha(1-6)fucose residues. This study demonstrates that the L2/HNK-1 epitope is borne mainly or even exclusively by N-linked oligosaccharide structures alpha(1-6)fucosylated in the core.
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