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Biomedical subjects

L D Kohn

Publications and source records attributed to L D Kohn.

At least 199 records · Page 11Linked to original sources
PubMed1983

Antibodies that promote thyroid growth. A distinct population of thyroid-stimulating autoantibodies.

We used a strain of differentiated rat-thyroid cells in continuous culture (the FRTL-5 strain) to detect the presence of growth-promoting antibodies in serum samples from patients with autoimmune thyroid disease. We found that IgG preparations from 17 of 20 patients (85 per cent) with active Graves' disease and two of five patients (40 per cent) with Hashimoto's thyroiditis could augment thyroid-cell growth. In parallel with IgG-induced elevations in intracellular cyclic AMP levels in the same cell line, all 20 of the patients with active Graves' disease had thyroid-stimulatory antibodies. Patients' IgG preparations fell into three subclasses: those with both potent cyclic AMP stimulation and potent growth-promoting activity; those with potent cyclic AMP stimulation but low-level growth promotion; and those with potent growth promotion and low-level cyclic AMP action. Growth-promoting antibodies were not detected in patients with Graves' disease in remission (seven patients), nodular goiter (seven), subacute thyroiditis (five), or atrophic thyroiditis (one). Simultaneous assays of growth promotion and cyclic AMP stimulation may be useful in the care of patients with autoimmune thyroid disease.

Adult↗
PubMed1983

Specificity of thyroglobulin interactions with thyroid cells and membranes.

Homologous species specificity is demonstrated with bovine and human thyroglobulin in which the two terminal sugars of the B carbohydrate chain, sialic acid and galactose have been removed by enzymatic hydrolysis. The species specificity is demonstrated by measuring the ability of the deglycosylated thyroglobulin derivatives to inhibit thyrotropin-induced increases in cAMP in human, rat and bovine thyroid cells in culture. Thus human-human or bovine-bovine interactions have higher activity coefficients by at least an order of magnitude than their heterologous counterparts. The homologous interactions are confirmed in binding studies and shown to be associated with negligible degradation of the bound ligand over a 24 hour period.

Animals↗
PubMed1983

Ganglioside dependent return of TSH receptor function in a rat thyroid tumor with a TSH receptor defect.

The 1-8 rat thyroid tumor line with a thyrotropin and cholera toxin receptor defect and a deficiency in higher order membrane gangliosides is shown to regain both receptor functions with the in vivo resynthesis or the in vitro reconstitution of higher order gangliosides. Reconstitution was achieved by exposing primary cell cultures of the tumor to preparations of gangliosides from thyroid cells with functional thyrotropin receptor activity.

Adenylyl Cyclases↗
PubMed1983

Structural changes caused by thyrotropin in thyroid cells and in liposomes containing reconstituted thyrotropin receptor.

Thyrotropin causes a rapid and significant increase in the fluorescence polarization of DPH when this hydrophobic probe is incorporated into a strain of functioning rat thyroid cells (FRTL5). This increase is ligand-specific and is not related to cAMP production. The phenomenon seems to reflect the interaction of thyrotropin with the glycoprotein component of its membrane receptor, as suggested by experiments in which thyrotropin causes increases in DPH fluorescence polarization in liposomes embedded with this receptor component but not with gangliosides. A strain of nonfunctioning rat thyroid cells (FRT), exhibiting no reactivity with monoclonal antibodies to the glycoprotein component of the thyrotropin receptor, requires two orders of magnitude higher concentrations of thyrotropin to exhibit a comparable phenomenon.

Animals↗
PubMed1983

Graves' IgG stimulation of iodide uptake in FRTL-5 rat thyroid cells: a clinical assay complementing FRTL-5 assays measuring adenylate cyclase and growth-stimulating antibodies in autoimmune thyroid disease.

With optimal conditions and cells maintained in the absence of thyrotropin (TSH) for 7-10 days, IgG preparations from approximately 90% of patients with active Graves' disease can exhibit statistically significant stimulation of cAMP levels in rat FRTL-5 thyroid cells as compared to normal controls. FRTL-5 cells maintained in the absence of TSH for 7-10 days lose their ability to take up iodide. Iodide uptake returns upon readdition of TSH over a 60-hour period via a cAMP-mediated process; thus TSH can be replaced by dibutyryl cAMP or other agents which increase cAMP levels, for example, thyroid-stimulating autoantibodies (TSAbs) from Graves' sera. TSAb stimulation of iodide uptake requires the continued presence of TSAb over at least the first 24 hours of a 48-hour reversal period; TSH, in contrast, can be withdrawn after 5 hours and will still achieve maximal effects at 36-48 hours. Iodide uptake, measured as a 30-minute pulse at 48 hours, appears, however, to be faster with TSAb than TSH. With optimized conditions (cells depleted of TSH greater than 7-10 days; 3-isobytyl-1-methyl xanthine, 0.005 mM; TSAb addition for the entire 48-hour assay period; and a 30-minute pulse of 10 microM 125I-sodium iodide at 37 C), TSAb stimulation is concentration-dependent with a half-maximal activity at approximately 10-fold lower concentrations than in the cAMP stimulation assay. In a series of 24 patients with Graves' disease, IgGs with positive values in the cAMP assay were positive in the iodide uptake assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1983

Temperature-mediated interaction of tetanus toxin with cerebral neuron cultures: characterization of a neuraminidase-insensitive toxin-receptor complex.

Energy-dependent internalization of 125I-labeled tetanus toxin into cultured neural cells is shown to follow an energy-independent binding process. A three-step model, involving receptor-mediated binding followed by sequestration and internalization is proposed. In the first step, binding of toxin is enhanced in appearance under low ionic strength medium, at 0-4 degrees C; it is suppressed, however, with increasing incubation temperature under physiological salt concentrations. Cell-bound toxin is displaced by approximately 35.5% when high-salt medium (physiological concentrations) is added to cells at 0-4 degrees C; the effect is further amplified at 37 degrees C. Addition of disialoganglioside GD1b (1-5 micrograms/ml) also lowers the amount of cell-associated toxin. The fraction of 125I-labeled toxin retained by the cells after exposure to high-salt medium at 0-4 degrees C or after addition of GD1b is operationally defined as sequestered toxin. This second step, characterized by a stable association of the toxin with the neural cells, is affected by both physiological salt and by 37 degrees C conditions. Lastly, an energy-dependent phenomenon of firm association of tetanus toxin with neural cells, compatible with internalization, is described. The toxin residing in this fraction is bioactive and cannot be removed by salts, gangliosides, or by treatment with protease or neuraminidase. Binding, sequestration, and internalization are mutually dependent, as they are all blocked by pretreatment of cells with neuraminidase and by an enhanced energy-independent sequestration event, which results in enhanced tetanus toxin internalization by an energy-dependent process.

Animals↗
PubMed1983

Characterization of the optimal stimulatory effects of graves' monoclonal and serum immunoglobulin G on adenosine 3',5'-monophosphate production in fRTL-5 thyroid cells: a potential clinical assay.

Immunoglobulin G (IgG) preparations derived from the sera of patients with hyperthyroidism due to Graves' disease (TSAb) as well as a monoclonal IgG derived from heterohybridoma fusions of Graves' lymphocytes augmented cAMP levels in a continuous strain of functioning rat thyroid cells (clone FRTL-5) in culture. Optimal stimulation was the same for both types of IgG preparations when measured after 2 h of incubation with 5 X 10(4) cells/well and using cells maintained in a nongrowth, TSH-deficient medium for 7 days. At low IgG concentrations, the stimulatory activities of both preparations exhibited a linear dependence on concentration and similar Ka values (approximately 4 X 10(-8) M) despite the fact that 65% of the Graves' serum IgG preparations had a significantly better ability to inhibit TSH binding to membrane preparations. The Ka value for TSH in the same assay was about 5 X 10(-12) M. Using this cell assay, 90% of a series of hyperthyroid Graves' IgG preparations exhibited stimulating activity, a value comparable to the frequency of positive results found by ourselves and others using human thyroid cell and slice systems. In contrast, only 10% of patients who were euthyroid 1 yr after antithyroid drug withdrawal (n = 21) exhibited stimulating activity, and no stimulating activity was detected in patients with nontoxic nodular goiter (n = 11), toxic adenoma (n = 5), or thyroid carcinoma (n = 6). The studies suggest that an optimized rat FRTL-5 thyroid cell system is a clinically useful and convenient alternative to human thyroid cell and slice systems for detecting TSAbs.

Animals↗
PubMed1982

Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease.

Human monoclonal antibodies have been generated from heterohybridomas obtained by fusing mouse myeloma cells with peripheral lymphocytes from patients with active Graves disease. This report characterizes four antibodies as presumptive thyrotropin receptor antibodies because they specifically inhibit thyrotropin binding and competitively inhibit thyrotropin-induced cAMP levels in human thyroid cells. Two of these antibodies, 208F7 and 206H3, are representative of autoimmune stimulators in Graves disease sera because they stimulate thyroid function in all assays, including the mouse bioassay; their ability to inhibit thyrotropin-induced cAMP increases in thyroid cells competitively is complemented by more than additive agonism at low (10 pM) thyrotropin concentrations. These stimulating antibodies interact more potently with human thyroid ganglioside preparations than with bovine thyroid or brain gangliosides; in contrast, they are poor inhibitors of 125I-labeled thyrotropin binding to liposomes containing the glycoprotein component of the human thyrotropin receptor. Antibodies 129H8 and 122G3 appear to be representative of inhibiting or "blocking" antibodies in Graves disease sera. Thus they have no intrinsic stimulatory action in assays of thyroid function but rather inhibit thyrotropin activity in the assays tested. These two antibodies do not react with human thyroid gangliosides but are strong inhibitors of thyrotropin binding to liposomes containing the high-affinity glycoprotein component from human, bovine, and rat thyroid membranes. The data unequivocally establish the pluritopic nature of the immunoglobulins in Graves disease and relate individual components or determinants of the thyrotropin receptor structure with specific autoimmune immunoglobulins.

Antibodies, Monoclonal↗
PubMed1982

Thyrotropin stimulation of the ADP-ribosyltransferase activity of bovine thyroid membranes.

Thyrotropin increases the ADP-ribosylation activity of bovine thyroid membranes. Rapid ADP-ribosylation of membrane components is followed by increasing ADP-ribosylation of components in the supernatant of the reaction mixture. One of the major membrane proteins ADP-ribosylated in the thyrotropin-stimulated reaction has an approximate molecular weight of 40,000; this same protein is also a major ADP-ribosylated product of the A promoter of cholera toxin and appears to be related to the G regulatory subunit of the adenylate cyclase complex. The ADP-ribosylated products appearing in the supernatant solution comigrate with thyrotropin and preparations of 125I-labeled alpha subunit of thyrotropin; the alpha subunit, but not the beta subunit, of thyrotropin can be ADP-ribosylated by the membrane ADP-ribosyltransferase activity. NAD can be shown to enhance the ability of thyrotropin to stimulate the adenylate cyclase activity of bovine thyroid membrane preparations and of membrane preparations of a rat thyroid tumor whose adenylate cyclase activity is otherwise unresponsive to thyrotropin. The beta subunit of thyrotropin inhibits thyrotropin stimulation of both the ADP-ribosylation and adenylate cyclase activities of the thyroid membrane.

Adenylyl Cyclases↗
PubMed1982

Interferon interactions with thyroid cells.

Iodide uptake by functioning rat thyroid (FRTL) cells is increased by mouse interferon. The effect is detectable using purified interferon; it is not accompanied by an increase in intracellular cyclic AMP levels, is measurable within 20 min, and is prevented by cholera toxin, an agent which inhibits interferon's antiviral activity. The effect of interferon is biphasic with maximally increased iodide uptake (approximately 2-fold) evident at about 300 international mouse units per ml (U/ml) and lesser effects evident at higher concentrations (greater than 1000 U/ml). The effect of mouse interferon on iodide uptake is accompanied by an extremely sensitive antiviral response. Thus, significant antiviral protection is evident at 1 U/ml with FRTL cells, as opposed to 1000 U/ml for nonfunctioning rat thyroid (FRT) cells. Functioning FRTL thyroid cells are also more sensitive to mouse interferon (10-fold) with respect to 2',5'-polyadenylate (An) synthetase activity or to 125I-thyrotropin binding to membrane preparations than are nonfunctioning FRT cells. Antiviral protection in FRTL cells is evident as early as 1 h after exposure to mouse interferon, and is accompanied by a nearly 100-fold increase in the measurable titer of 2'5'-An synthetase activity. Actinomycin D blocks the antiviral effect of interferon, but not its effect on iodide uptake. The results are discussed with respect to the unusually sensitive response of heterologous (rat) cells to mouse interferon: a possible relationship between thyrotropin and interferon receptors; and, the difference in interferon sensitivity exhibited by differentiated (functioning) as opposed to undifferentiated (nonfunctioning) thyroid cells.

2',5'-Oligoadenylate Synthetase↗
PubMed1982

[Thyroid-stimulating immunoglobulins. The related antigens].

The presence of thyroid stimulating antibodies (TSAb) in patients with Graves' disease is well established. Considerable evidence has accumulated that these are antibodies to thyroid plasma membrane components related to the TSH receptor. The question of whether thyroid stimulation is mediated by a direct interaction with the TSH binding site is still debated. Recent data obtained by the use of monoclonal antibodies to the TSH receptor are consistent with the view that Graves' immunoglobulins comprise antibodies to at least two different components of the TSH receptor complex, one of which is more strictly related to the binding of TSH and the other to the transmission of the hormonal effect. The causative role of TSAb in the hyperthyroidism of Graves' disease is widely recognized. The use of human specific stimulation assays has circumvented the objection of the relatively low frequency of LATS-positive patients. Individual variations in the thyroid response may account for the lack of correlation between levels of thyroid stimulating immunoglobulins and most parameters of thyroid function. In this respect, the interference of non-stimulatory thyroid antibody and of other autoimmune mechanisms may be of importance. An important clinical implication of TSAb and TSH-binding inhibiting antibody determinations is their prognostic value in predicting the relapse of hyperthyroidism in treated patients. This clinical application has been so far limited by the technical difficulty of the assays. This emphasizes the need for a simple and reliable test, which can be used for routine measurements of TSAb.

Antibodies, Monoclonal↗
PubMed1982

Molecular interactions at the cell surface: role of glycoconjugates and membrane lipids in receptor recognition processes.

The present report has described studies using a particular receptor--the TSH receptor--to raise questions concerned with the role of glycolipids and glycoproteins in receptor recognition events and the relevance of lipid modulation of these components in regard to their functional expression. The importance of carbohydrates in other recognition systems will be even more eloquently detailed in subsequent presentations. It is clear, however, that we have gone beyond their recognition as important and are entered into a research stage where questions of how and why are center stage. It is equally clear, as evidenced by the monoclonal antibody data presented herein, that studies of these questions will provide new insight into the mechanism of disease states and will offer new and better diagnostic and therapeutic approaches.

Gangliosides↗
PubMed1982

Receptor structure and function: an exploratory approach using the thyrotropin receptor as a vehicle.

The purpose of this chapter is not to present the final or even correct model of TSH receptor structure and function. Rather, the current speculative model presented is used to open the door to a more broad view of the receptor problem and controversy as it has evolved today. Questions of how we define a receptor are clearly very much in flux and much more difficult than initially considered when a chemical approach is taken. Numerous binding components will be described and their relevance to the physiologic state will be debated. Some will be clearly erroneous in concept--yet the very debate and data will open new ideas and approaches other than repetitive membrane binding or response measurements. The remainder of this book will explore numerous other aspects of receptor structure, regulation and function. The reader may be disturbed by the complexity and extrapolations of data and the weakness of the models. The reader should, however, remember that the receptor is the key link of the cell to its environment. The complexities of this linkage are evident in our continued concern with knowledge of the mechanisms our bodily senses utilize. The controversy that will exist is evident in the arguments we have today over the agents in our environment which affect us and the mechanisms of these effects. It is hoped that this chapter and book will provide both the desire and some reference to follow and review the data in all receptor fields as they emerge in the next several years.

Adenylyl Cyclases↗