Soluble cell-free extracts of pathogenic Entamoeba histolytica, as well as serum-free minimal media in which trophozoites are incubated, contain substances that cause the rapid rounding up and detachment of tissue-cultured monolayers of mammalian cells (cytopathic activity) and induce fluid secretion in ligated intestinal loops of indomethacin-pretreated rats (enterotoxic activity). A semiquantitative assay for the determination of the cytopathic activity based on the rate of detachment of tissue-cultured baby hamster kidney cells was developed. Two peaks containing cytopathic activity were obtained upon gel filtration of the soluble extracts: peak I, with over 60% of the activity, emerged in the 30,000 to 50,000 molecular weight region, and peak II, containing the remaining activity, was in the 15,000 to 25,000 molecular weight region. The activity of peak I was found to be heat labile and inhibited by sialoglycoproteins such as fetuin and mucin (5 mg/ml), as well as by sialic acid. Protease inhibitors such as antitrypsin, pepstatin, phenylmethylsulfonyl fluoride, metaloprotease inhibitors, and bacitracin had no effect on the cytopathic activity. Marked inhibition of cytopathic activity was observed, however, with iodoacetamide and p-chloromercuribenzoate, which affect sulfhydryl groups. The toxic material in peak II was found to have ionophoric activity and was not inhibited by sialic acid-containing compounds. The materials from both peaks had enterotoxic activity in intestinal ligated loops. The active substance from peak I was further purified (200X) on an agarose-fetuin affinity column, yielding one major protein band with an apparent molecular weight of ca. 30,000 on sodium dodecyl sulfate. Amino acid analysis revealed that the protein was very poor in sulfur amino acids. The sialic acid-sensitive toxic activity was higher in known virulent strains such as HM-1:IMSS and could be markedly augmented after preincubation of the trophozoites with certain Escherichia coli strains.
The involvement of an autoimmune response (cellular and humoral) to the nicotinic acetylcholine receptor ( AChR) in myasthenia gravis ( MG) is well established now. The questions at present are more detailed and specific; for instance, what immunological specificities and which antigenic determinants govern myasthenia and how can we utilize the information concerning these questions for designing approaches for specific regulation of the disease.Detailed immunochemical analysis of AChR should enlighten these questions. In the following, we will summarize studies from our laboratory on various molecular modifications and derivations of AChR and on monoclonal antibodies against defined antigenic determinants in the receptor molecule. In addition, we will also report on some recent experiments with anti-idiotypes.MOLECULAR DISSECTION OF AChR : STRUCTURE AND FUNCTION ANALYSIS Modification of AChR followed by pharmacological and immunological analysis may lead to the isolation and characterization of small fragments of the molecule, which are responsible for the specific cholinergic and/or pathologic myasthenic activity of AChR.The various AChR preparations which have been investigated in our laboratory and their biological activities are summarized in TABLE 1.
Denatured AChR ( R C M -A C h R )AChR, which is a complexed multisubunit molecule, was irreversibly denatured by opening all the disulptide bridges in denaturing conditions (reduction in 6 M guanidine) and blocking the sulphydryl groups with an alkylating agent. The resulting reduced carboxymethylated AChR (RCM-AChR) did not bind a-bungarotoxin (u-Bgt) or other cholinergic ligands, and did not induce experimental autoimmune myasthenia gravis (EAMG) in rabbits1, Nevertheless, it has been demonstrated that RCM-AChR is a good immunogen that induces high titers of antibodies which cross-react with AChR. RCM-AChR
S U M M A R Y Experimental autoimmune myasthenia gravis (EAMG) is an appropriate model for studying the molecular origin, immunological mechanism and regulation of myasthenia gravis. Several approaches are being utilised for the regulation of the immune response to AChR and for immunosuppression of EAMG: Corticosteroids and azathioprine can suppress EAMG concomitantly with suppression of immune responses to AChR. High dose cyclophosphamide treatment in mice facilitates the onset of EAMG and results in a selective suppression of the humoral response to AChR whereas the cellular response is enhanced. Specific immunosuppression of EAMG is achieved by using a nonmyasthenic, denatured AChR preparation which cross reacts with the intact receptor. Various degradations and modifications of AChR are being performed in order to identify the smallest molecular entity responsible for the myasthenic activity of AChR. Studies on specific monoclonal antibodies, anti-idiotypes, and on the effect of measles virus on EAMG are being described and their possible significance in regulating myasthenia are being discussed.
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