Expression of memory, differentiation, and repression of c-myc and p53 genes in human RD/TE-671 cells induced by a ureido-derivative of pyridine (UDP-4).

Human TE-671 cells have been used to study several aspects of neuroectodermal tumors in culture. Since the human TE-671 cell lines has been re-identified as a rhabdomyosarcoma (RD) rather than a medulloblastoma due to the presence of muscle-type nicotinic acetylcholine receptors, we re-investigated the nature of RD/TE-671 cells and characterized their differentiation induced by 2-(3-ethylureido)-6-methylpyridine (UDP-4), a potent inducer of differentiation of neoplastic cells. RD cells were also used for comparative studies. RD/TE-671 cells exposed to UDP-4 were differentiated irreversibly into postmitotic cells expressing mainly neurofilaments and, to a lesser extent, myoid proteins. In contrast to RD cells that expressed preferentially myoid and not neurofilament proteins (NFPs) upon treatment with UDP-4, differentiated RD/TE-671 cells exhibited characteristic dendritic processes and expressed NFPs (NFP68, NFP160, and NFP200), parvalbumin (calcium-binding protein), and neuron-specific enolase, as well as a small amount of vimentin and desmin. In addition, differentiated RD/TE-671 cells expressed memory for differentiation and underwent an irreversible limitation of proliferation, loss of clonogenic potential, selective repression of c-myc and p53 proto-oncogenes, and changes in cell surface architecture. Treatment of RD/ TE-671 cells with nerve growth factor or epidermal growth factor in the presence of UDP-4 did not alter the phenotype of differentiated cells, whereas co-treatment with 12-O-tetradecanoylphorbol-13-acetate and UDP-4 enhanced morphological differentiation. Therefore, we conclude that: (a) RD/TE-671 cells challenged with UDP-4 express memory to differentiate in the absence of inducer; (b) in contrast to RD cells, RD/TE-671 cells appear to be multipotent cells of neuroectodermal origin capable of differentiation into cells expressing neuronal rather than myoid proteins upon treatment with UDP-4; and (c) differentiation of RD/TE-671 cells leads to selective cessation of cell proliferation and repression of c-myc and p53 proto-oncogenes.

The main immunogenic region of the acetylcholine receptor. Structure and role in myasthenia gravis.

Auto-antibodies to the nicotine acetylcholine receptor (AChR) cause the disease myasthenia gravis (MG). Animals immunized with AChR or receiving anti-AChR antibodies acquire MG symptoms. The majority of the monoclonal antibodies (mAbs) raised in rats against intact AChR bind to a region on the extracellular side of the AChR's alpha-subunit, the main immunogenic region (MIR). The major loop of the overlapping epitopes for several anti-MIR mAbs has been localised between residues 67-76 of the alpha-subunit. Anti-MIR mAbs are very potent in accelerating AChR degradation (antigenic modulation) in muscle cell cultures and transferring experimental MG in animals. Fab fragments of single anti-MIR mAbs when bound to the AChR inhibit two-thirds of the MG patients' antibodies from binding and from inducing antigenic modulation of the AChR. This suggest that the majority of the human MG antibodies are also directed against the MIR. It has however to be verified by direct experiments.

Seroepidemiology of Helicobacter pylori infection in Greece.

Helicobacter pylori infection is strongly associated with chronic gastritis and peptic ulceration. As the prevalence of H. pylori infection in southern European populations is not known, a serological survey of 1069 samples from three different age groups in the Greek population was carried out with an enzyme-linked immunosorbent assay (ELISA) for antibodies to these bacteria. The antigen was an ultracentrifuged supernate of whole cell sonicates of 5 isolates of H. pylori assessed by electrophoresis and by immunoblotting with negative and positive sera. The sensitivity of the test was 97.43% and the specificity 100% for IgG antibodies; IgA and IgM antibodies to the antigen preparation were not found. Antibodies to H. pylori were detected among 39.4% of children aged 1-10 years, 67.1% of recruits (20-27 years) and 70% of blood donors (20-50 years). The prevalence of antibodies did not differ with sex in each of the age groups. The proportion of individuals with antibodies to H. pylori was higher in the younger age groups than those reported for similar age groups in western Europe.

Fab fragments of monoclonal antibodies protect the human acetylcholine receptor against antigenic modulation caused by myasthenic sera.

The human cell line TE671 produces large amounts of muscle nicotinic acetylcholine receptor (AChR). TE671 cells were used to determine the specificity of antibodies which can increase the internalization rate of AChR (antigenic modulation) and to test procedures for protecting AChR against this mechanism. The half-life of AChR both in the absence and the presence of anti-AChR antibodies was very similar to that of AChR on human muscle cell cultures. The relative contribution of different anti-AChR antibody fractions to the total antigenic modulation capacity of human myasthenic sera was investigated by competition experiments between Fab fragments of anti-AChR monoclonal antibodies (MoAbs) and intact antibodies (MoAb or myasthenic sera). Fab fragments, which do not induce antigenic modulation, were allowed to shield the corresponding regions of the AChR. Intact antibodies were subsequently added. It was found that protection of the main immunogenic region (MIR), but not of a region on the beta-subunit, essentially blocked the modulatory effect of the intact anti-MIR MoAbs, and approximately 80% of that of myasthenic sera. These data suggest that anti-MIR antibodies are mainly responsible for the loss of human AChR via antigenic modulation. Furthermore the observation that Fab fragments of anti-MIR MoAbs can efficiently protect AChR against antigenic modulation may have therapeutic implications.

Antigenic modulation of human myotube acetylcholine receptor by myasthenic sera. Serum titer determines receptor internalization rate.

Antibodies to the acetylcholine receptor (AChR) added to AChR-bearing muscle cells cross-link the receptors, thus increasing their internalization and degradation rate (antigenic modulation). This mechanism contributes to AChR loss in myasthenia gravis. Until recently, antigenic modulation has been studied in animal tissues, where only a small fraction of human anti-AChR antibodies bind. In the present study, we examined the antigenic modulation of AChR by using patients' sera and cultures of human muscle cells. We aimed to see whether antigenic modulation correlates better with disease severity or with antibody titer. Antibody-containing sera from 29 myasthenic patients in various states of the disease and with different antibody titers against AChR were tested. Control sera from six healthy individuals were also tested. Our experiments showed that all myasthenic sera affected the overall AChR content on the human myotube surface, causing a 49 to 82% loss, whereas control sera had no effect. Although at fixed serum volumes there was some correlation between disease severity and AChR loss, this effect was clearly due to differences in antibody titers. In fact, the antigenic modulation depended mainly on the final concentration of the antibody present. Thus, intrinsic factors other than antibodies to AChR may determine or influence the patients' susceptibility to the disease.

Role of the main immunogenic region of acetylcholine receptor in myasthenia gravis. An Fab monoclonal antibody protects against antigenic modulation by human sera.

Antigenic modulation of acetylcholine receptor (AChR), i.e., acceleration of its internalization and degradation rate by antibody-cross-linking, is considered to be one of the two main causes of AChR loss in myasthenia gravis (MG). The majority of the antibodies to AChR are directed to the main immunogenic region (MIR) on the alpha-subunit of the receptor. We here examine the relative contribution of the anti-MIR antibody fraction (as well as of another fraction) to the antigenic modulation caused by MG patients' sera. Fab fragments of an anti-MIR monoclonal antibody (mAb) or a mAb to the beta-subunit (neither of which causes antigenic modulation) were allowed to shield their corresponding regions on the AChR on the mouse muscle cell line BC3H1. The 27 MG sera subsequently added thus bound to all other regions except to the protected one, and the resulting antigenic modulation was measured. The anti-MIR mAb protected the AChR by 68 +/- 16%. This is interpreted as the contribution to antigenic modulation of the anti-MIR antibody fraction in the human sera. This percentage correlated very well with the occurrence of the anti-MIR antibodies in the same sera. The anti-beta mAb gave only small protection of the AChR. No significant pattern differences were observed between sexes, early and recent onset of the disease, or high and low antibody titers. It is concluded that as far as it concerns the one of the pathogenic mechanisms in MG, i.e., the antigenic modulation, the MIR seems to be the main pathogenic region. The observation that a single mAb can efficiently protect the AChR in this system may prove to be of therapeutic interest.