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Biomedical subjects

M Zakarija

Publications and source records attributed to M Zakarija.

At least 37 records · Page 2Linked to original sources
PubMed1988

Effects of gamma-interferon and tumor necrosis factor alpha on thyroid cells: induction of class II antigen and inhibition of growth stimulation.

A functioning rat thyroid cell line (FRTL5) was used to study interactions of thyrotropin (TSH) and various cytokines on expression of class I and II major histocompatibility complex (MHC) antigens and on growth stimulation. Only gamma-interferon (gamma-IFN) affected MHC antigen expression, i.e., to enhance class I, that was constitutive, and to induce class II. A concomitant, but probably not directly related, effect of gamma-IFN was to diminish growth stimulation, as effected by TSH and other activators of adenylate cyclase and measured by DNA increase and enhanced incorporation of [3H]thymidine into DNA. Stimulation of growth by tetradecanoylphorbol ester was also decreased by gamma-IFN. These effects of gamma-IFN were mimicked to some degree by tumor necrosis factor but there was major synergism between the two cytokines. Enhanced accumulation of cAMP by TSH and other agents was not diminished in these experiments. Flow cytometry analysis showed that inhibition of growth stimulation involved blocking of the passage of cells from the G0/1 phase to the S phase. The data may have relevance to goiter size in autoimmune thyroid disease.

Animals↗
PubMed1988

The effect of thyroxine on spontaneous thyroiditis in BB/W rats.

The effect of T4 on the incidence of lymphocytic thyroiditis and on titers of antibodies to thyroglobulin and thyroid microsomal antigen was studied in BB/W rats that are prone to develop spontaneously autoimmune thyroiditis and diabetes. Thirty-three animals were separated in two groups. Rats in one group had 1 mg T4 per liter of drinking water, and the other group was given no T4. After 3-4 months of therapy the T4-treated group had a reduced production of antibody to thyroglobulin (p less than 0.05) and to microsomal antigen (p less than 0.005) and a significantly lower frequency of lymphocytic thyroiditis (p less than 0.05). Our data are consistent with previous findings in experimentally induced thyroiditis in rats and suggest that T4 has an immunosuppressive effect.

Animals↗
PubMed1988

Evidence supporting the identity in Graves' disease of thyroid-stimulating antibody and thyroid growth-promoting immunoglobulin G as assayed in FRTL5 cells.

This paper addresses the question: in Graves' disease is there a thyroid-growth stimulating IgG (TGI) separate from thyroid-stimulating antibody (TSAb)? Using the functioning rat thyroid line (FRTL5) cells for TGI (incorporation of [3H]-thymidine into DNA) and TSAb (increase in cAMP concentration) assays, we tested IgG from 30 Graves' patients. Positive TGI assay occurred only if cAMP increased in the cells and responses correlated, i.e., r = 0.95, P less than 0.001. With one very potent TSAb-IgG we showed that Fab was active as TGI and TSAb, IgG with pI of 8.5-9.0 was the most potent fraction in both systems and an inhibitory IgG prevented the action of both TSAb-IgG and TSH in both the TSAb and TGI assays. In the last example, the action was on the cell membrane and not on the TSH or IgG. These data are entirely compatible with the view that in Graves' disease, at least as tested in FRTL5 cells, the same IgG is active in stimulating both growth and adenylate cyclase.

Animals↗
PubMed1987

Hyperthyroxinemic mice have reduced natural killer cell activity. Evidence for a defective trigger mechanism.

Natural killer (NK) activity of peripheral blood lymphocytes from hyperthyroxinemic patients (Graves' disease or thyroxine (T4)-treated) is severely depressed. In order to study the relationship of thyroid hormone to NK activity, a model for hyperthyroxinemia was induced in mice by addition of T4 to the drinking water. Control mice were hypothyroid (fed propylthiouracil) or normal. Serum T4 levels were elevated (within 2 wk) in mice fed thyroid hormone. Six weeks after initiation of the diets, in vitro NK activity was undetectable in the peripheral blood, spleen, or lung mononuclear cell populations harvested from hyperthyroxinemic mice. Control mice had NK activity within the normal range. Spleen cells from mice fed thyroid hormone and control mice were tested for their ability to release lytic factors (natural killer cytotoxic factors). Lymphoid cells were incubated for 20 hr with unlabeled Yac-1 cells. Supernatants were tested for their capacity to lyse 51Cr-labeled Yac-1 cells in a 20-hr chromium release assay. Unlike controls, supernatants from hyperthyroxinemic spleen cells incubated with Yac-1 targets were unable to lyse 51Cr-Yac-1 cells. The NK cells from the mice fed T4 synthesized lytic factors because nonspecific stimuli, such as 12-O-tetradecanoyl phorbol-13-acetate and the calcium ionophore A23187, induced release of lytic factors capable of lysing Yac-1 targets into the media. These data support the hypothesis that excess thyroid hormone interferes with the triggering mechanism used by NK targets to cause release of lytic molecules from NK cells.

Animals↗
PubMed1987

Suppression of peripheral blood natural killer cell activity by excess thyroid hormone.

Natural killer (NK) cells were assessed in patients with hyperthyroxinemia due to Graves' disease or treatment with thyroxine (T4). Cytolytic activity was measured with 51Cr-labeled K562 tumor cells and NK enumeration was by flow cytometry using NKH-1 monoclonal antibody to identify the relevant surface marker. Activity was uniformly decreased in association with hyperthyroxinemia, regardless of the underlying pathology; however, there was no reduction in the number of NKH-1+ cells. NK activity was enhanced by addition of interleukin 2 (IL-2) in both control and patients' cells although the value in the latter instance failed to reach the basal control level. Production of IL-2 by lymphocytes from hyperthyroxinemic subjects, in response to phytohemagglutinin, was also reduced. Since NK cells are thought to act as a defense against viral infections and some malignancies and may play a role in autoregulation of the immune system, this effect of T4 may have significant biological implications.

Cell Line↗
PubMed1987

The spectrum and significance of autoantibodies reacting with the thyrotropin receptor.

Autoantibodies that occur in autoimmune thyroid disease and that interact with the TSH receptor are reviewed. Current limited understanding of the nature of the TSH receptor is detailed, and available assays for the different types of antibodies are described. The incidence, clinical significance, and interactions of the various antibodies are summarized.

Autoantibodies↗
PubMed1986

Characteristics of patients with normal T3 and T4 and a low TSH response to TRH.

The nature of the thyroid disorder presented by patients with normal T4 and T3 but blunted TSH response to TRH has not been clarified. In this study, we compared thyroid function tests in 16 such patients with those of 14 controls and 10 hyperthyroid patients. Basal total T4, free T4, total T3, iodine uptake and cholesterol of the study group were similar to controls but significantly (P less than 0.001) lower than in hyperthyroid patients, except for cholesterol which was higher. In contrast, the basal TSH, increase in TSH after TRH stimulation, and decrease of T4 during T3 suppression tests were similar to data obtained in hyperthyroid patients but significantly (P less than 0.001) lower than in controls. Pulse rate was mid-way between the control and the hyperthyroid group. Thyroid stimulating antibody (TSAb) was measured with human thyroid cells in culture; the assay was positive in four subjects in the 16-patient group and in all hyperthyroid patients tested. TSH stimulation test showed a hyporesponse in iodine uptake in the four patients with positive TSAb (26 +/- 29%), as well as in hyperthyroid patients (6 + 5%). However, there was a hyper-response to TSH (213 +/- 52%) in the remaining 12 patients in the group, none of whom had TSAb. Thus TSAb is not seen as responsible for the thyroid disorder in the majority of patients with normal T3 and T4 and absent or blunted TSH response to TRH; surprisingly, most of these patients have thyroid hypersensitivity to TSH. These two characteristics, absence of TSAb and hypersensitivity to TSH, delineate a thyroid disorder clearly different from Graves' disease.

Adult↗
PubMed1986

Evidence that adenosine 3',5'-monophosphate mediates stimulation of thyroid growth in FRTL5 cells.

Thyroid function, including growth, is TSH dependent, and most metabolic functions of TSH are thought to be mediated by cAMP. Recently, it has been suggested by several groups that growth may be an exception and that it may not be related to cAMP action. In addition, evidence has accrued indicating that the thyroid-stimulating antibody (TSAb) of Graves' disease, the metabolic actions of which are also cAMP mediated, may not be the goitrogenic agent in that syndrome. To evaluate these concepts, we used functioning rat thyroid cells (FRTL5) in monolayer culture and, as indices of growth, the incorporation of [3H]thymidine ([3H]Tdr) into DNA, the concentration of DNA measured directly, and the percentage of cells in S phase, as assessed by flow cytometry, all studied over 72 h of incubation. TSH, forskolin, and cholera toxin enhanced growth by each criterion and increased the concentration of cAMP in parallel; the effect on cAMP occurred rapidly and was maximal well in advance of influences on growth. In all instances, measures of growth promotion were minimal at 24 h and maximal at 48 h, except for [3H]Tdr incorporation, which was greater at 72 h than at 48 h. 3-Isobutyl-1-methylxanthine (IBMX) and (Bu)2 cAMP were also tested. Both enhanced all indices of growth and were as effective as TSH. Maximal responses to TSH were obtained at 100-200 microU/ml, maximal responses to both IBMX and (Bu)2cAMP occurred at 5 X 10(-4) M, and all three stimulators increased the DNA concentration and [3H]Tdr uptake and induced S phase in at least 20% of all cells in culture. The peak effect on DNA and S phase was consistently at 48 h. Epidermal growth factor (EGF) was shown to increase [3H]Tdr incorporation in a nondose-dependent fashion (10(-10) to 5 X 10(-9) M gave approximately 250% of control) over 1, 2, 3, 5, and 7 days, with no increase in DNA and a slight decrement in the concentration of cAMP. A laboratory standard TSAb-immunoglobulin G was shown to parallel TSH in both increasing cAMP (over 2 h of incubation) and growth stimulation (over 72 h). The data are entirely consistent with the view that TSH-stimulated thyroid growth is mediated by cAMP and that the established action of TSAb on adenylate cyclase is sufficient to explain goiter as well as hyperthyroidism in Graves' disease.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Prediction and therapy of intrauterine and late-onset neonatal hyperthyroidism.

These studies in a mother and child describe the effects of multiple antibodies directed against the TSH receptor that influenced thyroid function in the fetus and infant. Blood was taken periodically for 6 months from a child (C3) whose mother (M) was known to have in her serum immunoglobulin G (IgG) that contained thyroid-stimulating antibody (TSAb), an inhibitor of TSAb and TSH binding and action, and an enhancer of TSH binding to its receptor, the last activity presumed to enhance both TSH and TSAb action. We correctly predicted that C3 and an older sibling, C2, would have delayed onset of hyperthyroidism (approximately 45 days of age) due to interaction of these antibodies. In addition, in both C2 and C3, fetal hyperthyroidism in the second trimester was postulated, and therefore, M was given propylthiouracil from then until term (C2) or 8 months (C3), with associated return of the fetal heart rate to normal in the one fetus (C3) in whom this was monitored. IgG was purified from C3's serum samples and tested for TSAb activity using human thyroid cells in monolayer culture and its ability to alter binding of [125I]TSH to human thyroid cell membranes. In the TSAb assays, samples obtained from birth to 4 months of age produced a negative dose response, and those obtained from 4-6 months showed a positive dose response. The effect on the binding of [125I]TSH was enhancement with all IgG obtained for 6 months, except that for the first 52 days a high concentration was inhibitory. The combined clinical and assay data are compatible with the following interpretations. M's IgG contained TSAb and both an inhibitor and an enhancer of that activity, the total effects varying at different times. In the second trimester, the net effect of transplacentally transferred IgG was to induce hyperthyroidism. At birth and perhaps during the third trimester, the inhibitor of TSAb prevented hyperthyroidism; from about 45 days of life, the enhancing effect facilitated TSAb action to produce hyperthyroidism until maternal IgG was completely cleared from the infant's circulation at about 7 months of age.

Antibody Affinity↗
PubMed1985

Studies on multiple thyroid cell membrane-directed antibodies in Graves' disease.

Immunoglobulin G was obtained from the serum of a woman who had given birth to three children with a delayed onset of hyperthyroidism; the clinical events were due to the coexistence of thyroid-stimulating antibody (TSAb) and an inhibitor of TSAb in the maternal serum. The current studies explore the possible existence of additional thyroid membrane-directed antibodies. Human thyroid slices, cells in monolayer culture, and functioning rat thyroid cells (FRTL5), with measurement of cyclic AMP concentration, were used for TSAb assays. Assays of the inhibition of binding of 125I-thyrotropin (TSH) to its receptor used human thyroid and FRTL5 cells, and human thyroid and guinea pig fat cell membranes as receptors. All activities were associated with IgG kappa. Fractions of IgG kappa obtained by adsorption to and the desorption from human thyroid and guinea pig fat cell preparations and F(ab')2 and Fab fragments of the parent IgG were tested. Results indicated that there were three activities in the IgG, namely, TSAb; an inhibitor of TSH-binding that was active in all species and preparations tested, and was effective as Fab and F(ab')2 on both particulate and solubilized thyroid membranes; and an enhancer of TSH-binding (e.g., approximately equal to 220% increase in binding) that was relatively specific for human thyroid membranes only in particulate form, was not adsorbed by fat, and was active as F(ab')2, but minimally as Fab. The concept is developed that dilution of the total IgG, experimentally in vitro or by metabolic clearance in vivo in neonates, determines the effect on either thyroid stimulation or TSH-binding. The incidence of such multiple antibodies and their interaction remains to be determined.

Adipose Tissue↗
PubMed1983

Immunoglobulin G inhibitor of thyroid-stimulating antibody is a cause of delay in the onset of neonatal Graves' disease.

Studies were carried out with the serum IgG from a mother and her two children who developed neonatal Graves' disease several weeks after birth. The maternal IgG: (a) stimulated the human thyroid in vitro, but maximal stimulation was found only with dilution of the IgG; (b) was very potent in the long-acting thyroid stimulator (LATS)-protector assay, but only when an inhibitor of the system was diluted out; (c) inhibited a standard preparation of LATS in the mouse bioassay; (d) was biphasic in the thyrotropin-binding inhibition (TBI) assay, i.e., enhanced binding at low concentrations of IgG and inhibited binding at high levels. Enhancement in the TBI assay was found only with particulate preparations of human thyroid membranes as receptor and not when that material was solubilized, nor with guinea pig fat cell membranes as receptor. Serial blood samples from the second child were obtained at birth and until 3 mo of age. In the thyroid slice (cyclic AMP) assay system there was a negative dose-response relationship in testing IgG until age 45 d when it became positive, coinciding with the clinical recognition that hyperthyroidism had developed. The data are compatible with a concept that this mother's IgG contained thyroid-stimulating antibody (TSAb) and another moiety that inhibited TSAb through an action on the thyroid cell membrane, thus delaying the onset of hyperthyroidism in the neonate until the inhibiting IgG was metabolically cleared to an ineffective concentration.

Animals↗
PubMed1983

Comparisons of different assays for the thyroid-stimulating antibody of Graves' disease.

Various current methods for the assay of the thyroid-stimulating antibody of Graves' disease (TSAb) were assessed for comparative sensitivity and specificity. Five procedures were examined in detail: I) increase in cAMP in human thyroid slices; II) inhibition of [125I]TSH binding to human thyroid membranes, particulate or III) solubilized; IV) inhibition of [125I]TSH binding to guinea pig fat cell membranes, particulate or V) solubilized. The minimum effective concentration of TSH (microunits per ml) in these assays was: I) 5; IV) 12.5; V) 25 and II and III) 100. The guinea pig fat systems (IV and V) were more sensitive than the human thyroid assays (II and III) in terms of the concentration of TSH required for 50% inhibition of binding. Five preparations of TSAb-immunoglobulin G were found in general to have constant relative potency in the five assay systems, but individual immunoglobulins G were variable in their effects when data in the five procedures were compared directly. It is probable that, in addition to TSAb, other antibodies, more or less specific for the human thyroid, influenced the TSH binding inhibition assays to a variable extent. The above TSH binding inhibition assays were carried out with solutions or suspensions of receptor preparations. Solid phase systems, using microtiter plates for TBI assays, were assessed in less detail. Human thyroid, particulate or solubilized, was ineffective (little specific binding of [125I]TSH), but a particulate preparation of guinea pig fat cell membranes was an efficient basis for a solid phase TBI system. Solid phase assessment with [125I]staphylococcal protein A of binding of thyroid autoantibodies to human thyroid membranes was not specific for TSAb.

Adipose Tissue↗
PubMed1983

Pregnancy-associated changes in the thyroid-stimulating antibody of Graves' disease and the relationship to neonatal hyperthyroidism.

Assays for the thyroid-stimulating antibody (TSAb) of Graves' disease were performed with serum obtained from 17 women with 20 pregnancies who either had or had had Graves' disease, or who had delivered a child with neonatal hyperthyroidism. Ten of the children, of eight women, were diagnosed as being hyperthyroid; all eight mothers had high concentrations of TSAb, as measured by adenylate cyclase stimulation. In the infants with neonatal hyperthyroidism, a minimum 500% increase in cAMP was found on assay of maternal immunoglobulin G, and no such high values were associated with a euthyroid infant. Blood samples for TSAb assay were available from 11 mothers on both the day of delivery and at least 1 other time in the pre- or postpartum period. In 7 mothers, the lowest value (negative in 4) was obtained coincident with delivery, and in the remaining 4, there was no pregnancy-associated change. Thus, a pattern, related to pregnancy, of a decline in TSAb concentration or a subsequent postpartum increase was observed in the majority of subjects. Apparently, neonatal hyperthyroidism due to transplacental passage of TSAb occurred only when this decline did not reduce the concentration to a low value.

Antibodies↗
PubMed1983

Immunochemical characterization of the thyroid-stimulating antibody (TSAb) of Graves' disease: evidence for restricted heterogeneity.

The thyroid-stimulating antibody (TSAb) of Graves' disease is an IgG; its immunochemical characteristics were analyzed using an increase in the concentration of cyclic AMP in the human thyroid slice in vitro for its assay. IgG from serum known to contain TSAb in high concentration was purified and H chains from this IgG were combined with Bence Jones k or lambda L chains or original L chains; reconstitution of IgG was confirmed by agar gel and immunoelectrophoresis. In studies with 6 sera there was no thyroid stimulation on testing such recombined molecules except for minimal activity in one instance of recombination of original H and L chains. In 8 other experiments TSAb-IgG containing only k or lambda L chain was isolated by affinity chromatography. In 7 instances thyroid-stimulating potency was entirely with IgG lambda, except for minor activity in IgGk in 3 of these studies; in the eighth preparation TSAb was predominantly in IgGk with minimal activity in IgG lambda that was assayed at a much higher concentration. By chromatography on Protein A Sepharose, IgG3 was obtained and it was found to contain no TSAb; activity was, however, recovered in the Protein A-retained fraction (i.e. IgG 1 + 2 + 4). With 3 preparations of TSAb-IgG lambda, subclasses 1 + 2 + 4, removal by affinity chromatography of subclass 4 led to full recovery of TSAb in subclasses 1 + 2. On subsequent removal of subclass 2, in two instances all activity was retained by subclass 1; in the third preparation some TSAb was not accounted for. These data, together with previous reports from this laboratory showing a relatively constant pI for TSAb-IgG on preparative isoelectric focusing, are compatible with its having restricted heterogeneity, instead of being polyclonal as described by others.

Animals↗
PubMed1982

Solubilization, purification, and partial characterization of thyrotropin receptor from bovine and human thyroid glands.

In an attempt to purify the thyroid receptor for TSH and to study interaction with the thyroid-stimulating antibody (TSAb) of graves' disease, we used both bovine and human thyroid glands. With either tissue, the 10,000 X g pellet of homogenized material was solubilized with 0.5% Triton N-101; excess Triton was removed by Amberlite XAD-2, and purification was effected by TSH affinity chromatography, followed by gel filtration on Sepharose 6B. Greatest purification was achieved with bovine tissue; this receptor preparation was 183-fold concentrated over the starting material, but contained only 6% of the original TSH-binding activity, due in part to spontaneous loss over the 4 days required for processing. On polyacrylamide gel electrophoresis, there were at least three protein bands, one of which was probably a subunit of thyroglobulin. Purified immunoglobulin G with thyroid-stimulating antibody activity inhibited the binding of TSH at all stages of purification of the receptor.

Animals↗
PubMed1981

Absence of thyroid-stimulating antibody and long acting thyroid stimulator in relatives of Graves' disease patients.

None of the 76 euthyroid relatives of patients with Graves' disease had detectable LATS in their serum nor was thyroid-stimulating antibody (TSAb), assessed by an increment of cAMP in human thyroid slices, detected in any of the 60 sera tested. Seven of 41 were slightly positive for TSH binding-inhibiting immunoglobulin (TBII). Nineteen of 73 sera were positive (greater than 1:1600) for antibody to thyroid antimicrosomal antigen, of which 4 were also positive (greater than 1:400) for antibody to thyroglobulin; 3 of the 19 had a slightly elevated basal TSH which rose excessively after TRH. Thus, although these euthyroid relatives had evidence of thyroid immunological defects, a thyroid-stimulating antibody was not found.

Antibodies↗