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L J DeGroot

Publications and source records attributed to L J DeGroot.

At least 37 records · Page 2Linked to original sources
PubMed1997

Comparison of administration of recombinant human thyrotropin with withdrawal of thyroid hormone for radioactive iodine scanning in patients with thyroid carcinoma.

BACKGROUND: To detect recurrent disease in patients who have had differentiated thyroid cancer, periodic withdrawal of thyroid hormone therapy may be required to raise serum thyrotropin concentrations to stimulate thyroid tissue so that radioiodine (iodine-131) scanning can be performed. However, withdrawal of thyroid hormone therapy causes hypothyroidism. Administration of recombinant human thyrotropin stimulates thyroid tissue without requiring the discontinuation of thyroid hormone therapy. METHODS: One hundred twenty-seven patients with thyroid cancer underwent whole-body radioiodine scanning by two techniques: first after receiving two doses of thyrotropin while thyroid hormone therapy was continued, and second after the withdrawal of thyroid hormone therapy. The scans were evaluated by reviewers unaware of the conditions of scanning. The serum thyroglobulin concentrations and the prevalence of symptoms of hypothyroidism and mood disorders were also determined. RESULTS: Sixty-two of the 127 patients had positive whole-body radioiodine scans by one or both techniques. The scans obtained after stimulation with thyrotropin were equivalent to the scans obtained after withdrawal of thyroid hormone in 41 of these patients (66 percent), superior in 3 (5 percent), and inferior in 18 (29 percent). When the 65 patients with concordant negative scans were included, the two scans were equivalent in 106 patients (83 percent). Eight patients (13 percent of those with at least one positive scan) were treated with radioiodine on the basis of superior scans done after withdrawal of thyroid hormone. Serum thyroglobulin concentrations increased in 15 of 35 tested patients: 14 after withdrawal of thyroid hormone and 13 after administration of thyrotropin. Patients had more symptoms of hypothyroidism (P<0.001) and dysphoric mood states (P<0.001) after withdrawal of thyroid hormone than after administration of thyrotropin. CONCLUSIONS: Thyrotropin stimulates radioiodine uptake for scanning in patients with thyroid cancer, but the sensitivity of scanning after the administration of thyrotropin is less than that after the withdrawal of thyroid hormone. Thyrotropin scanning is associated with fewer symptoms and dysphoric mood states.

Adult↗
PubMed1997

Oct-1, silencer sequence, and GC box regulate thyroid hormone receptor beta1 promoter.

Thyroid hormone, acting through thyroid hormone receptors (TRs), plays a crucial role in brain development and its insufficiency results in irreversible brain damage. TR alpha mRNA is expressed continuously from early embryonic stages, but the level of TR beta1 mRNA in brain is more abundant in adult than in fetus. To identify important factors which regulate TR beta1 expression, we compared mouse fetal and adult brain nuclear extracts by DNase I footprinting and electrophoretic gel mobility shift assays (EMSA) of the TR beta1 promoter. We carried out transient transfection studies in COS 1 cells using the TR beta1 promoter fused to Luciferase gene, and used mutated promoter vectors and various expression vectors. In DNase I footprinting using the fragment -950 to -717, fetal brain nuclear extracts protected the areas -910 to -884 and -815 to -800 more than did adult extracts. In EMSA, proteins in fetal nuclear extracts bound to a silencer sequence (-924 to -916), GC box (-901 to -887), and E box (-810 to -805), more strongly than did proteins in adult brain extracts. The bands formed on GC box were not supershifted by Sp-1, Sp-2, Sp-3, Sp-4, EGR-1, or EGR-2 antibodies. Three bands were detected on the octamer binding site probe (-913 to -906) and one protein was supershifted by Oct-1 antibody. Adult brain extracts appear to contain more Oct-1 protein than do fetal extracts. The other two bands were more intense in fetal extracts than in adult extracts, but were not supershifted by either Oct-1 or Oct-2 antibodies. Mutation of the silencer response element, mutation of the GC box, and Oct-1 over expression in COS 1 cells increased TR beta1 promoter function as assayed by Luciferase reporter. Mutation of the octamer binding site, to which only Oct-1 bound in COS 1 cells, decreased Luciferase reporter activity. Thus the TR beta1 promoter was regulated negatively by the proteins bound to the silencer sequence and the GC box, and positively by Oct-1. Silencer and GC box binding proteins are more abundant in fetal brain, and Oct-1 is more abundant in adult brain. The results may be responsible for increased amounts of TR beta1 present in late fetal and adult brain.

Animals↗
PubMed1997

Beta-trace gene expression is regulated by a core promoter and a distal thyroid hormone response element.

We isolated and characterized the human beta-Trace protein (betaTP) gene promoter. betaTP, also known as prostaglandin D2 synthase, is a lipocalin secreted from the choroid plexus and meninges into cerebrospinal fluid. Basal transcription of the betaTP gene is directed from a core promoter found within the first 325 bases of the 5'-flanking sequence. The betaTP gene promoter is responsive to thyroid hormone (3,3',5-triiodothyronine, T3) and efficiently repressed by unliganded human thyroid hormone receptor beta (TRbeta). Functional analysis of the betaTP promoter in TE671 cells revealed that responsiveness to T3 occurs in sequences 2.5 kilobase pairs 5' of the start site. Within the hormone-responsive region we identified a thyroid hormone response element (TRE) located from -2576 to -2562 base pairs relative to the transcription start site. The betaTP TRE is composed of two directly repeated consensus half-sites separated by a 3-base pair space (DR3). The betaTP TRE forms specific complexes with TRbeta. We have shown that a gene active in the choroid plexus and meninges is responsive to T3. T3 may play a role in the regulated transport of substances into the cerebrospinal fluid and ultimately the brain.

Base Sequence↗
PubMed1997

The function of retinoid X receptors on negative thyroid hormone response elements.

Retinoid X receptors (RXRs) form heterodimers with thyroid hormone receptors (TRs). RXRs increase DNA binding affinity of TRs and T3-mediated transactivation on positive T3 response elements (TREs). However, the role of RXRs on negative TREs, and the relation of RXRs to the dominant negative effect of mutant TRs, are not defined. To clarify the function of RXRs on negative TREs, we performed transient cotransfection studies using the rat glycoprotein hormone alpha promoter fused to luciferase gene (alphaLuc), and human TRH promoter fused to luciferase gene (TRH-Luc) as reporters. We found that the JEG-3 cell-alphaLuc system was very sensitive to TR regulation. Using TRbeta1 wild-type (WT) expression vector, 6.2 ng/well (170 ng/10 cm dish), and 0.2 ng/well (11 ng/10 cm dish) caused maximal, and half maximal, inhibition of Luc activities in the presence of 1 nM T3. A T3 dose dependent inhibition study was also performed. From these studies, we determined that the appropriate conditions in which to study alphaLuc transactivation, in a linear portion of the dose response curve, was using 0.8 ng/well TRbeta1 expression vector and 0.1 nM T3. Under these conditions, TRbeta1 mutant R316H (GH), but not G345R (Mf), showed a weak dominant negative effect at a 1:1 ratio in the presence of 0.1 nM T3 although neither mutant had detectable T3 binding affinity. Moreover this dominant negative effect of R316H on the alphaLuc reporter was enhanced in the presence of RXRgamma. Mutant G345R showed a stronger dominant negative effect than did R316H when using a double palindromic TRE fused to herpes simplex thymidine kinase-Luc reporter as a positive TRE. These results conform to the clinical features of R316H which is associated with apparent pituitary resistance of thyroid hormone (PRTH). Mutant R316H also showed a weak dominant negative effect with TRH-Luc at a 1:1 ratio in the absence or presence of RXRgamma. However RXRgamma did not enhance the dominant negative effect as it did using alphaLuc reporter gene. Electrophoretic gel mobility shift assay (EMSA) showed that RXR alpha augmented the DNA binding affinity of wild type and R316H TRs as heterodimers on the previously reported negative TREs of glycoprotein hormone alpha promoter, suggesting that RXR does not produce its response by removing TRs from these TREs. RXR alpha augmented DNA binding affinity of TRbeta1WT, and R316H showed a weaker heterodimer band than did the wild type in EMSA. Using the TRH-Luc reporter, basal activity was increased by wild type TRbeta1. However a TRbeta1 DNA binding domain mutant, (C127S) which can not bind to DNA, did not increase the basal activity. This indicates that DNA binding of the TR is required for increasing basal activity of TRH promoter. These results indicate that (1) RXR-TR heterodimers play a role in basal transactivation and T3 suppression of negatively regulated genes, and (2) RXRs increase the dominant negative effect of some mutant TRs on specific negative TREs. (3) This effect occurs without removing TRs from the TRE. (4) The differential dominant negative effect of mutant R316H (negative TRE > positive TRE) may explain, at least in part, the presentation of R316H as PRTH. (5) Augmentation of basal activity by wild type TRs on a negative TRE requires DNA binding.

Animals↗
PubMed1997

Evaluating the role of Th0 and Th1 clones in autoimmune thyroid disease by use of Hu-SCID chimeras.

To study the role of Th0 and Th1 cells in autoimmune thyroid disease, thyroid tissues from patients with Graves' disease (GD), Hashimoto's thyroiditis (HT), and colloid nodular disease were xenografted into SCID mice, followed by ip injection of peripheral blood mononuclear cells (PBMC), T cell lines, and T cell clones (TCC). The antigen-specific TCC reactive to TSH receptor (TSH-R), thyroid peroxidase (TPO), or thyroglobulin (Tg), and their respective peptides, were classified into Th0 (secreting IL-4 and/or IL-5 and IFN-gamma) and Th1 (secreting IFN-gamma) according to their cytokine profile. Engraftment of autologous or HLA-matched allogeneic CD4+ thyroid-specific clones with Th0 or Th1 phenotypes induced the production of total IgG and thyroid-specific autoantibodies by B cells present in xenografted thyroid tissues. TSH-R-specific clones mainly enhanced thyroid-stimulating antibodies (TSAb) production, while clones reactive to TPO and Tg increased the synthesis of TPO and Tg autoantibodies. Total IgG production, but not TSAb, was also stimulated by PBMC and TSH-R lines. TSAb correlated with the viability and hyperplasia of thyroid follicles, but not with the serum T3 levels, which were normal. Thyroid tissue viability was maintained or increased by antigen-specific Th0 clones, and decreased by Th1 clones reactive to TSH-R or TPO. Thyroid lymphocytic infiltration was variable; however, Th0 and Th1 clones from HT patients caused high degree of lymphocytic infiltration compared to the control groups. These results demonstrate for the first time that T cells clones reactive to specific epitopes of TSH-R, TPO, or Tg can generate antibody-mediated and/or cell-mediated responses in the xenografted thyroid tissue microenvironment. Such effects depend on clonal specificity, HLA class II restriction, and cytokine profile of the clone. Th0 clones reactive to TSH-R stimulate both total IgG production and TSAb in SCID mice engrafted with thyroid tissue from GD patients. Th0 and Th1 clones specific for TPO and Tg also function as helper T cells, stimulating total IgG synthesis and autoantibodies against TPO and Tg. Th1 clones may also cause tissue destruction in GD and HT.

Adolescent↗
PubMed1997

Radioiodine and the immune system.

Treatment of Graves' disease patients with radioactive iodide (RAI) can induce two therapeutically important alterations in immune response to thyroid antigens. These may be characterized as a first response and a second phase response. Initial treatment of patients with Graves' disease by RAI leads to killing of thyroid cells and releases antigen into the circulation. In association with this, there is a dramatic increase in levels of thyroid-stimulating immunoglobulins and in cell-mediated immunoreactivity to TSH receptor (TSH-R) and it peptide epitopes. During this phase, ophthalmopathy often is exacerbated. Although it is logical to believe that the release of antigens and stimulation of immunoreactivity is the cause of the worsened ophthalmopathy, a direct cause and effect only can be inferred. Ophthalmopathy often remains a significant problem or develops during the course of treatment of Graves' disease. My observations are that almost all patients who have progressive ophthalmopathy after many form of thyroid treatment usually have residual thyroid tissue stimulated by thyroid stimulating antibodies, even though they may be hypothyroid and on replacement therapy. In this situation, destruction of residual thyroid tissue is associated with amelioration in ophthalmopathy and is presumed to be effective because of diminution in antigenic stimulation, with a subsequent drop in antibody levels and cell-mediated immunoreactivity to TSH-R extracellular domain (ECD). This constitutes a second phase in the radioiodine response, with effects dramatically different from the initial phase, because this phase is associated with a loss in antigenic stimulation rather than an increase. In a series now comprising > 40 patients treated in an uncontrolled prospective manner, comparison to preablation and postablation ophthalmopathy demonstrates clear benefit in almost all patients over a period of 3-12 months. Radioiodine ablation of residual thyroid tissue is the logical first treatment in management of severe ophthalmopathy and should be used before or with the institution of steroids or radiotherapy.

Autoantigens↗
PubMed1997

Thyroid hormone receptor (alpha) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons.

Thyroid hormones appear to play an important role in the seasonal reproductive transitions of a number of mammalian and avian species. These seasonal transitions as well as the effects of thyroid hormones on the reproductive neuroendocrine axis are mediated by the GnRH system. How thyroid hormones affect the GnRH system is unclear. Double label immunocytochemistry was used to examine GnRH- and other neurotransmitter/neuropeptide-containing neurons for thyroid hormone receptor (alphaTHR) colocalization in two seasonal breeders, the golden hamster and the sheep. AlphaTHR was identified in hamster and sheep brain by Western blot analysis. Furthermore, alphaTHR immunoreactivity was widely distributed in brain and was colocalized in identified populations: GnRH neurons (hamster, 28%; sheep, 46%); dopaminergic neurons of the A14 (hypothalamic) and A16 (olfactory bulb) cell groups, but not in the hypothalamic A13 cell group; and neurophysin-immunoreactive neurons of the supraoptic and paraventricular nuclei. The finding of alphaTHR in GnRH and A14 dopamine neurons provides an anatomical substrate for direct thyroid hormone action on the reproductive neuroendocrine system of these two seasonally breeding species. It remains to be determined whether the GnRH gene itself or the gene of another constituent within the same GnRH neuron is responsive to thyroid hormones.

Animals↗
PubMed1997

Thyroid hormone receptor beta 1 expression in developing mouse limbs and face.

Thyroid hormone, acting through thyroid hormone receptors (TRs), plays an important role in amphibian metamorphosis and vertebrate development. To identify where and when TR beta 1 promoter is activated during fetal life, we carried out an in vivo functional study of a 1.3 kilobase (kb) TR beta 1 gene promoter using transgenic mice that express the beta-galactosidase gene under control of the TR beta 1 promoter. Transactivation of the gene was determined by blue staining of tissues after incubation with X-gal. High expression of transgene was detected in the limbs and face of the 12.5-day-old fetus (12.5 F) and 14.5 F, reminiscent of the changes occurring during amphibian metamorphosis, and this disappeared at 17.5 F. The expression was confined to the tip of finger bones, between fingers in the limb buds, and was detected in the root of whisker follicles, nose, and around the eyes. Signal was detected in the oral cavity, nasal cavity, lung, and urogenital sinus of 14.5 F, and disappeared at 17.5 F. Signal was detected in the midbrain and auditory vesicles of 9.5 F but was reduced between 12.5F and 17.5F, and there was no expression in the cerebral cortex layer of 0 days old neonates (PO). Expression was detected in the cortex after P5. There was signal in the cerebral cortex, cerebellum, kidney, and liver of adult mice. TR beta 1 messenger RNA was detected by RT-PCR in the developing limbs and face. Transgene expression in the interdigital tissues, which regress during development, suggests that TR beta 1 is expressed in mammals in areas undergoing apoptosis as well as in areas undergoing differentiation.

Aging↗
PubMed1997

Monoclonal antibodies to rat calcitonin: their use in antigenic mapping and immunohistochemistry.

A library of monoclonal antibodies (mAbs) to rat calcitonin (rCT) was raised from several fusions. Antibodies were screened by enzyme-linked immunosorbent assay with solid phase rCT. Affinities for rCT ranged from 10(9)-10(11) M(-1). Some mAbs reacted preferentially with solid phase rCT, but not with liquid phase, 125I-labeled rCT. Cross-reactivity with human CT (hCT) was assessed using solid phase hCT. Although there are only two amino acid differences, at least nine of the mAbs do not cross-react with solid phase hCT. Several cross-reactive mAbs were chosen for immunohistochemical studies of human medullary thyroid carcinoma samples and showed strong positive staining. A 33 x 33 matrix solid-liquid mAb inhibition assay was carried out to probe the rCT molecule. Five different clusters of mAb were distinguished and interpreted as reflecting five distinct antigenic regions on the surface of the rCT molecule.

Animals↗
PubMed1997

A monoclonal antibody against rat calcitonin inhibits the growth of a rat medullary thyroid carcinoma cell line in vitro.

Medullary thyroid carcinoma (MTC) cells synthesize large amounts of calcitonin (CT), which serves clinically as a useful tumor marker. To examine the possibility of CT serving as a target in immunotherapy for MTC, we raised and characterized more than 40 monoclonal antibodies (mAbs) against rat CT (rCT). The affinity constants for the mAbs were between 2.8 x 10(9) and 1.8 x 10(11) M(-1). Some mAbs react preferentially with solid phase rat CT, but not with liquid phase 125I-labeled rCT. Thirty-nine mAbs cross-react with human CT. We evaluated the antitumor effect of the mAbs in vitro by analysis of [3H]thymidine incorporation into the rat MTC cell line CRL-1607. Some antibodies show an antiproliferative effect, but most are inactive. One mAb (2E5G5, IgG2b), which preferentially reacts with solid phase rCT, but not with liquid phase 125I-labeled rCT, exerts an antiproliferative activity on CRL-1607. At 6.25 x 10(-7) M, 2E5G5 killed all of the tumor cells independently of complement in a cytotoxicity assay. We explored the cytotoxic mechanisms by assays for cell cycle arrest and DNA fragmentation. The antitumor effect was manifested by apoptosis and cell cycle arrest. Hence, a secreted peptide may serve as a target in tumor immunotherapy. Therapeutically antibodies may exert antitumor activity by a variety of mechanisms. The antitumor effect of this mAb in a rat animal tumor model is being tested.

Animals↗
PubMed1997

Thyrotropin-receptor and thyroid peroxidase-specific T cell clones and their cytokine profile in autoimmune thyroid disease.

We studied the cytokine profile and the immune responses to thyroid antigens of specific T cell clones (TCC) isolated from patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD). Antigen-specific TCC were reactive to thyroid peroxidase (TPO), thyroglobulin (Tg) or human recombinant TSH-receptor extracellular domain (TSH-R), and/or their respective peptides. Of the 43 clones derived from HT patients, 65% were reactive to TPO, and 59% of the 32 clones derived from GD patients were reactive to TSH-R. TPO epitopes 100-119 and 625-644 were recognized by 75% of HT-derived clones, whereas TSH-R epitopes 158-176, 207-222, and 343-362/357-376 were recognized by 85% of GD-derived TCC. The TCC were classified according to their cytokine profile into T helper cell (Th)0 [secreting interleukin (IL)-4, IL-5, interferon (IFN)-gamma], Th1 (secreting IFN-gamma) and Th2 (secreting IL-4 and/or IL-5). Tumor necrosis factor-beta and IL-10 were produced by all subsets. The specific TCC were predominantly Th1-like cells in HT, and were Th0- and Th1-like cells in GD. Fifty three percent of Th0 clones were derived from GD patients and were reactive to TSH-R, whereas 50% of Th1 clones were derived from HT patients and were reactive to TPO or Tg. Most Th2 clones (82%) were reactive to TPO and were established from peripheral blood. All these clones produced IL-5, and 64% produced IL-4 and IL-10. Interestingly, IFN-gamma was highly produced by TPO- or Tg-specific clones established from HT thyroid tissue. These results confirm at the clonal level our previous studies regarding T cell epitopes on TPO and TSH-R molecules and support the concept that immunodominant T cell epitopes are located on amino acid residues 100-119 and 625-644 of TPO in HT and amino acid residues 158-176, 207-222 and 343-362/357-376 of TSH-R in GD. Our studies also demonstrate that thyroid-specific T cells can be classified into Th0, Th1, and Th2 subsets. TPO- or Tg-specific clones with Th1 phenotype appear to be involved in the pathogenesis of HT, mediating thyroid tissue destruction, whereas TSH-R clones with Th0 phenotype may induce thyroid-stimulating autoantibodies in GD.

Autoantigens↗
PubMed1996

An artificial thyroid hormone receptor mutant without DNA binding can have dominant negative effect.

The syndrome of resistance to thyroid hormone (RTH) encompasses a heterogeneous group of conditions which are caused by mutations of thyroid hormone receptor beta 1 (TR beta 1). Mutations usually cluster in two regions of the ligand-binding domain. The mutant receptors can inhibit normal receptor activity in a dominant negative manner, consistent with the dominant mode of inheritance of RTH. Recent evidence suggested that this dominant negative effect (DNE) of the RTH mutants involves competition for DNA binding and emphasized the essential role of intact DNA binding activity for mutants in order to exert DNE. However, we found that a Cys73Ser substitution in the DNA-binding domain (DBD) of wild-type produces a TR which can inhibit the transcriptional activation by TR alpha 1, either in the presence or absence of T3, on three different TRE-containing reporter genes, in transient co-transfection studies. Co-expression of TRv alpha 2, a TR alpha splicing variant, can enhance this DNE. However, DNE was not observed on the negatively-regulated TSH alpha Luc reporter gene when wild-type and DBD mutant were co-transfected at equimolar ratios. The DNE of DBD mutant is not reversed by co-transfection with excess retinoid X receptor alpha. DBD mutant alone can also inhibit the transactivation from a TK-luciferase reporter gene either linked with rat malic enzyme thyroid response element, or not. These observations parallel those we previously observed using TRv alpha 2. Our results indicate that a DBD mutant can have DNE, possibly through a mechanism similar to that of TRv alpha 2, which may involve interference with basal transcription factors. The clinical significance of these DBD mutants is currently unclear, but it is logical to expect such mutants do occur in nature.

Animals↗
PubMed1996

Expression of thyroid peroxidase in EBV-transformed B cell lines using adenovirus.

Thyroid peroxidase (TPO) is thought to be one of the pathological antigens in Hashimoto's thyroiditis. Epstein-Barr virus-transformed B cell lines (EBVL) can be used for antigen-presenting cells and target cells of cytotoxic T cells. To develop a model for endogenous TPO presentation in EBVL, we constructed a recombinant adenovirus carrying the TPO gene driven by the cytomegalovirus promoter (Ad5-TPO). Enzymatically active human TPO could be expressed in COS cells using Ad5-TPO. the peroxidase activity of the membrane extract from Ad5-TPO-infected COS cells was approximately 6 times higher than that from stably transfected TPO expressing CHO cells. TPO protein expression in the EBVL was analyzed by Western blotting technique. A band at approximately 110 kDa characteristic of hTPO was detected in EBVL infected with Ad5-TPO. hTPO expression in EBVL induced with adenovirus should facilitate understanding of T cell immunity to TPO in patients with autoimmune thyroid diseases.

Adenoviridae↗
PubMed1996

HLA class II associations in African-American female patients with Graves' disease.

Graves' disease (GD) is an autoimmune thyroid disease. Development of the disease phenotype is believed to be dependent on environmental factors and the action of several genes either in concert or in independent groups. Genes of, or closely associated to, the human leukocyte antigen (HLA) complex are important in determining genetic predisposition to GD in various ethnic groups. We previously reported that no significant HLA class I or -DR associations were detected in the total group of African-American patients with GD. To further investigate the associations of GD with HLA class II alleles, 52 unrelated African-American female patients with GD and 55 controls were typed for HLA-DRB1, -DQA1, and -DQB1, using sequence-specific oligonucleotide probes to analyze polymerase chain reaction amplified DNA. There were no significant differences in HLA distribution between patients and controls. These findings indicate that the contribution of HLA-DR and DQ regions to susceptibility to GD is small in African-American women.

Adult↗