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C Spitzweg

Publications and source records attributed to C Spitzweg.

At least 19 recordsLinked to original sources
PubMed2000

The immune response to the iodide transporter.

In addition to physiologic, diagnostic, and therapeutic implications, the recently cloned and characterized sodium iodide symporter (NIS) also may play an important role in the pathogenesis of autoimmune thyroid disease. Sodium iodide symporter expression patterns characteristically are changed in autoimmune thyroid disease, including Graves' disease and Hashimoto's thyroiditis, which may be caused, in part, by the regulation of sodium iodide symporter expression of cytokines involved in the pathogenesis of autoimmune thyroid disease. Further, there is increasing evidence that NIS-directed antibodies are present in sera from patients with autoimmune thyroid disease, and these antibodies also may affect NIS functional activity.

Animals↗
PubMed2000

Interleukin-1 receptor antagonist ribonucleic acid and protein expression by cultured Graves' and normal orbital fibroblasts is differentially modulated by dexamethasone and irradiation.

Recent data have indicated that orbital fibroblasts (OF) can be stimulated to produce marked quantities of interleukin-1 receptor antagonist (IL-1RA), a powerful inhibitor of the proinflammatory activities of interleukin-1 in the orbital tissues in Graves' ophthalmopathy (GO). We examined whether the beneficial effects of dexamethasone or irradiation, the two main therapeutic modalities applied in patients with active GO, may be related to their capacity to alter IL-1RA ribonucleic acid (RNA) and protein expression in OF. Early passages of cultured OF were obtained from orbital connective tissue and extraocular muscle of patients with severe active GO and five control subjects. Modulation of the two variants of IL-1RA, intracellular IL-1RA (icIL-1RA) and soluble IL-1RA (sIL-1RA), was studied after exposure of OF to increasing concentrations of dexamethasone (10(-10)-(10(-6) mol/L)), the glucocorticoid receptor antagonist RU 38486 (10(-3) mol/L), or combinations thereof. Alternatively, cell monolayers were exposed to increasing doses of UV irradiation (0.1-1 J/cm2) or ionizing irradiation (0.2-2 Gy). The IL-1RA gene and protein variants were analyzed by RT-PCR, immunocytochemistry, immunoblotting, and enzyme-linked immunosorbent assay. Dexamethasone inhibited IL-1RA RNA steady state levels in GO OF and control OF in a dose-dependent manner. Combined exposure of OF to dexamethasone and RU 38486 completely restored baseline levels of IL-1RA RNA. By contrast, low doses of UV and ionizing irradiation dose dependently up-regulated IL-1RA-specific transcripts in GO OF and control OF, whereas higher doses were less effective. Immunoblotting and enzyme-linked immunosorbent assay revealed suppression of IL-1RA immunoreactivity after treatment with dexamethasone and enhanced expression of IL-1RA by GO OF and normal OF after low doses of UV and ionizing irradiation. Our results indicate that, in contrast to dexamethasone, low doses of irradiation stimulate expression of the IL-1RA gene and protein variants in OF. Induction by irradiation of IL-1RA expression in target cells of the orbital immune process represents an as yet unrecognized mechanism by which orbital radiotherapy may exert some of its beneficial therapeutic effects in patients with active GO.

Cells, Cultured↗
PubMed1999

Prostate-specific antigen (PSA) promoter-driven androgen-inducible expression of sodium iodide symporter in prostate cancer cell lines.

Currently, no curative therapy for metastatic prostate cancer exists. Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) would enable those cells to concentrate iodide from plasma and might offer the ability to treat prostate cancer with radioiodine. Therefore, the aim of our study was to achieve tissue-specific expression of full-length human NIS (hNIS) cDNA in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP and in subcell lines C4, C4-2, and C4-2b in vitro. For this purpose, an expression vector was generated in which full-length hNIS cDNA coupled to the prostate-specific antigen (PSA) promoter has been ligated into the pEGFP-1 vector (NIS/PSA-pEGFP-1). The PSA promoter is responsible for androgen-dependent expression of PSA in benign and malignant prostate cells and was therefore used to mediate androgen-dependent prostate-specific expression of NIS. In addition, two control vectors were designed, which consist of the pEGFP-1 vector containing the PSA promoter without NIS cDNA (PSA-pEGFP-1) and NIS cDNA without the PSA promoter (NIS-pEGFP-1). Prostate cancer cells were transiently transfected with each of the above-described expression vectors, incubated with or without androgen (mibolerone) for 48 h, and monitored for iodide uptake activity. In addition, stably transfected LNCaP cell lines were established for each vector. Prostate cells transfected with NIS/PSA-pEGFP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in a range comparable to that observed in control cell lines transfected with hNIS cDNA. Perchlorate-sensitive iodide uptake was not observed in cells transfected with NIS/PSA-pEGFP-1 and treated without androgen or in cells transfected with the control vectors. In addition, prostate cancer cell lines without PSA expression (PC-3 and DU-145) did not show iodide uptake activity when transfected with NIS/PSA-pEGFP-1. Western blotting of LNCaP and C4-2b cell membranes transfected with NIS/PSA-pEGFP-1 using a monoclonal antibody that recognizes the COOH-terminus of hNIS revealed a band with a molecular weight of 90,000 that was not detected in androgen-deprived cells or in cells transfected with the control vectors, as well as a minor band at Mr 150,000 in transiently transfected LNCaP cell membranes. In conclusion, tissue-specific androgen-dependent iodide uptake activity has been induced in prostate cancer cells by PSA promoter-directed NIS expression. This study represents an initial step toward therapy of prostate cancer with radioiodine.

Adenocarcinoma↗
PubMed1999

Expression of thyroid-related genes in human thymus.

There are several thyroid antigens including human sodium iodide symporter (hNIS), thyrotropin receptor (TSH-R), thyroid peroxidase (TPO), and thyroglobulin (Tg) that have been considered to be thyroid-specific proteins involved in the pathogenesis of autoimmune thyroid diseases. We examined the expression of these thyroid-tolerance related genes in normal human thymus, the lymphoid organ responsible for the induction of central T-cell self. Reverse transcription-polymerase chain reaction (RT-PCR) amplifications were performed with 4 pairs of oligonucleotide primers specific for the hNIS, TSH-R, TPO, and Tg genes, respectively. Gene-specific transcripts were confirmed by Southern hybridization using digoxigenin-labeled internal oligonucleotide probes. To monitor cDNA integrity and quantity, all samples were coamplified with a pair of intron-spanning human beta-actin-specific oligonucleotide primers. Furthermore, using a highly sensitive immunostaining technique and antibodies specific for these 4 antigens, we examined whether NIS-, TSH-R-, TPO-, and Tg-specific immunoreactivity can be detected and localized in normal human thymus. RT-PCR and Southern hybridization revealed expression of each of these 4 thyroid-related genes in normal human thymus. In addition, immunohistochemical analysis of frozen tissue sections derived from normal human thymus showed marked immunoreactivity for NIS, TSH-R, and Tg as well as weaker staining for TPO. Control reactions using isotype matched nonimmune immunoglobulins were consistently negative. Taken together, our results suggest that NIS-, TSH-R-, TPO-, and Tg-RNA are present and actively processed to immunoreactive NIS-, TSH-R-, TPO-, and Tg-like protein in human thymus. These data support the concept that pre-T lymphocytes may be educated to recognize thyroid-related epitopes expressed in thymus, and, thus, to generate self-tolerance against these thyroid-related antigens.

Adult↗
PubMed1999

Analysis of human sodium/iodide symporter, thyroid transcription factor-1, and paired-box-protein-8 gene expression in benign thyroid diseases.

The ability to concentrate iodide, a fundamental property of normally functioning thyroid tissue, is altered in various thyroid diseases. Given the critical role of the Na+/I- symporter (NIS) in controlling iodide access to the thyroid gland, altered expression of NIS may be responsible, at least in part, for an enhanced or diminished capacity to concentrate iodide. In this study, we used Northern blot analysis, a newly established quantitative polymerase chain reaction (PCR) assay and in addition hNIS-directed immunohistochemical analysis to assess the levels of hNIS mRNA and protein expression in various localized and diffuse benign thyroid abnormalities, including Graves' disease (GD), scintigraphically cold solitary benign thyroid nodule (CBTN), nontoxic multinodular goiter (NMNG), solitary autonomously functioning thyroid nodule (AFTN), and mild diffuse iodine deficiency goiter (IDG). In addition, in view of the recent identification of putative binding sites for the transcription factors thyroid transcription factor-1 (TTF-1) and human paired-box-protein-8 (Pax-8) in the human NIS gene promoter, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to assess in these same samples the levels of TTF-1 and Pax-8 gene expression. Northern blot analysis revealed high levels of hNIS gene expression in thyroid specimens derived from patients with GD and AFTN. In contrast, levels of hNIS mRNA expression were moderate in NMNG, low in diffuse IDG, and very low in CBTN. Quantitative RT-PCR analysis of hNIS mRNA transcripts revealed variable but generally low levels of hNIS gene expression in IDG and NMNG, and undetectable or very low levels of hNIS mRNA in all scintigraphically CBTN studied. In contrast, markedly elevated levels of hNIS mRNA transcripts were detected in active GD (up to 17-fold) and AFTN (up to 25-fold). Immunohistochemical analysis revealed abundant hNIS protein expression by thyroid follicular cells in GD, moderate and heterogeneous levels in NMNG, and very low levels in CBTN. hNIS mRNA levels were correlated with TTF-1 and Pax-8 gene expression in GD and, to a lesser degree, in AFTN, NMNG, and IDG, but not in CBTN. In general, hNIS gene expression was more closely correlated with TTF-1 as compared to Pax-8 gene expression. In conclusion, the abundance of hNIS mRNA and protein expression in a broad range of benign thyroid pathologies correlated well with their functional state as assessed by thyroid scintigraphy. In addition to TTF-1 and Pax-8, other transcription factors and enhancer elements may contribute to regulation of NIS gene promoter activity.

Carrier Proteins↗
PubMed1999

Regulation of sodium iodide symporter gene expression in FRTL-5 rat thyroid cells.

The sodium iodide symporter (NIS), first identified in FRTL-5 cells, plays a critical role in iodide transport in the thyroid gland and in the production of the iodine-containing thyroid hormones. The aim of our study was to examine the regulation of NIS RNA steady-state levels and protein expression as well as functional activity in FRTL-5 cells. FRTL-5 cells cycling in media containing thyrotropin (TSH) were incubated for 48 hours with dexamethasone (10(-8)-10(-5) M), triiodothyronine (T3; 10(-9)-10(-6) M), methimazole (100 microM), propylthiouracil (PTU; 100 microM), perchlorate (10 microM) and potassium iodide (40 microM). In other experiments, cells were treated for 48 hours with various cytokines including interleukin-6 (IL-6) (100 U/mL), interferon-gamma (IFN-gamma) (100 U/mL), tumor necrosis factor-alpha (TNF-alpha) (10 ng/ml), IL-1alpha (100 U/mL), and IL-1beta (100 U/mL). Northern blot analysis using a 32P-labeled rat NIS-specific cDNA probe (nucleotides 1397-1937) revealed NIS mRNA as a single species of approximately 3 kb. When normalized for beta-actin mRNA signal intensities, NIS RNA steady-state levels in viable FRTL-5 cells were suppressed by approximately 80% after incubation with dexamethasone and T3 in a concentration-dependent manner. Iodide accumulation was decreased by up to 40% after incubation with dexamethasone and T3, respectively, in a concentration-dependent manner. Using a rabbit polyclonal rNIS-specific antibody, Western blot analysis of FRTL-5 cell membranes revealed a 60% and 70% suppression of NIS protein expression after treatment with T3 (0.1 microM) and dexamethasone (1 microM), respectively. In additon, NIS RNA steady-state levels were decreased by approximately 50% after treatment of monolayers with methimazole, PTU, and potassium iodide, respectively. Incubation with methimazole and PTU resulted in a 20% and 25% decrease of iodide accumulation, respectively, whereas potassium iodide suppressed iodide accumulation by approximately 50%. Treatment of FRTL-5 cells with IL-6 and IL-1beta resulted in a 30% decrease of NIS RNA steady-state levels. IL-6 did not alter NIS functional activity, but IL-1beta suppressed iodide accumulation by approximately 25%. IFN-gamma and perchlorate failed to alter NIS RNA steady-state levels. In contrast to IFN-gamma that had no effect on iodide accumulation, perchlorate almost completely suppressed iodide accumulation. TNF-alpha and IL-1alpha failed to alter NIS RNA steady-state levels in higher passage numbers of FRTL-5 cells, whereas treatment with TNF-alpha and IL-1alpha of early passages of FRTL-5 cells (<20 cell passages) resulted in a 70% and 40% decrease of NIS RNA steady-state levels, respectively, and in a 20% suppression of NIS functional activity. In conclusion, our data suggest that various agents known to affect iodide transport are capable of differentially altering NIS gene expression and function in cultured thyroid cells. Suppression of NIS gene expression and function by certain cytokines may be responsible, at least in part, for the impaired radioiodine uptake by thyroid tissue in certain forms of thyroiditis.

Animals↗
PubMed1999

Analysis of human sodium iodide symporter immunoreactivity in human exocrine glands.

The human sodium iodide symporter (hNIS) is an intrinsic transmembrane protein that mediates the active transport of iodide across the basolateral membrane of thyroid follicular cells. In addition to normally functioning thyroid tissue, various extrathyroidal tissues, including salivary gland, lacrimal gland, gastric mucosa, choroid plexus, and lactating mammary gland, have been demonstrated to accumulate iodide. After cloning and molecular characterization of the sodium iodide symporter, expression of hNIS messenger ribonucleic acid has been detected in a broad range of extrathyroidal tissues using Northern blot analysis and RT-PCR. In this study we used both monoclonal and polyclonal antibodies directed against different portions of hNIS protein together with a highly sensitive immunostaining technique to assess hNIS protein expression in tissue sections derived from normal human salivary and lacrimal glands, pancreas, as well as gastric and colonic mucosa. Immunohistochemical analysis of normal human salivary and lacrimal glands revealed marked hNIS immunoreactivity in ductal cells and less intense staining of acinar cells. Further, immunostaining of gastric and colonic mucosa showed marked hNIS immunoreactivity confined to chief and parietal cells in gastric mucosa and to epithelial cells lining mucosal crypts in colonic mucosa. In normal human pancreas, hNIS immunoreactivity was located in ductal cells, exocrine parenchymal cells, and Langerhans islet cells. In conclusion, our study demonstrates the expression of hNIS protein by several human exocrine glands, suggesting that iodide transport in these glands is a specific property conferred by the expression of hNIS protein, which may serve important functions by concentrating iodine in glandular secretions.

Antibodies, Monoclonal↗
PubMed1998

[Leptin--new knowledge on the pathogenesis of obesity].

Cloning of the ob-gene and characterization of its gene product leptin has led to the identification of a satiety factor, which signals the amount of peripheral fat stores to the central nervous system and regulates further feeding behaviour, thus playing a central role in the regulation of body weight. Soon after cloning of the ob-gene, a leptin-binding receptor has been identified in the central nervous system as well as in various peripheral organs. A feedback loop between peripheral fat stores and leptin receptors in the central nervous system appears to play an important role in normal body weight regulation. In contrast to human obesity, which associated with leptin resistance of uncertain etiology, the obesity syndromes associated with several animal models are now known to result from the interruption of the feedback loop at different points. Moreover, leptin may play a role in manifestation of insulin resistance and type II diabetes. Since the identification of leptin, a vast number of studies have been conducted to assess the molecular mechanisms and signal transduction pathways that are involved in the development and manifestation of obesity. From the large body of data generated to date, novel concepts of the regulation of energy balance and target strategies to control human obesity should soon be forthcoming.

Animals↗
PubMed1998

Thyrotropin receptor expression in Graves' orbital adipose/connective tissues: potential autoantigen in Graves' ophthalmopathy.

It is acknowledged that the TSH receptor (TSHr) on thyroid follicular cells is the autoantigen involved in the hyperthyroidism of Graves' disease. However, whether this receptor is expressed in extrathyroidal tissues, and whether it participates directly in the pathogenesis of Graves' ophthalmopathy (GO) are unclear. We sought to detect the expression of TSHr messenger ribonucleic acid (mRNA) and protein in orbital adipose/connective tissue specimens and in human orbital preadipocyte fibroblast cultures using liquid hybridization analysis and immunohistochemical methods. We demonstrated intact and variant TSHr mRNA transcripts and TSHr-like immunoreactivity in orbital adipose/connective tissue specimens from patients with GO. In addition, TSHr-like immunoreactivity was detected in early passage GO preadipocyte fibroblast cultures that were shown to include some adipose cells. In contrast, neither TSHr mRNA nor protein was detected in normal orbital adipose/connective tissue specimens or in late passage GO orbital fibroblast cultures containing no lipid-laden adipose cells. In conclusion, we showed that TSHr is expressed in the adipose/connective tissue of the diseased orbit in GO. In addition, TSHr is demonstrable in early passage GO preadipocyte orbital fibroblast cultures that contain a subpopulation of adipocytes. Subsequent passaging of these cells results in the loss of both TSHr expression and adipocyte-specific staining. These results suggest that both the expression of this receptor and the accumulation of adipose tissue in the orbit in GO may be induced in vivo by a humoral factor(s) not present in the cell culture environment.

Adipocytes↗
PubMed1998

Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa.

The ability to concentrate iodide is a fundamental property of normally functioning thyroid tissue and represents the first step in the production of thyroid hormones. Iodide uptake has been demonstrated in various extrathyroidal tissues, including salivary gland, gastric mucosa, and lactating mammary gland. Recently, cloning and molecular characterization of the human sodium iodide symporter (hNIS) have been reported; however, the patterns of hNIS gene expression in human tissues have remained unidentified. To examine the profiles of human hNIS gene expression in various normal human tissues, we performed high-stringency Northern blot analysis using a 32P-labeled hNIS-specific complementary DNA (cDNA) probe (nucleotides 1184-1667). To detect rare hNIS transcripts in small tissue samples, RT-PCR was performed with a pair of hNIS-specific oligonucleotide primers designed to amplify a portion (nucleotides 1184-1667) of the hNIS gene. hNIS-specific transcripts were confirmed by Southern hybridization using a digoxigenin-labeled internal hNIS-specific oligonucleotide probe (nucleotides 1460-1477). To monitor cDNA integrity and quantity, and to rule out DNA contamination and illegitimate transcription, all samples were coamplified with two pairs of intron-spanning primers designed to amplify fragments of the human beta-actin and thyroglobulin genes, respectively. Using Northern blot analysis, hNIS transcripts of approximately 4 kb were detected in thyroid gland and parotid gland but not in a broad range of endocrine and nonendocrine tissues. RT-PCR and Southern hybridization revealed hNIS gene expression in thyroid gland, salivary gland, parotid gland, submandibular gland, pituitary gland, pancreas, testis, mammary gland, gastric mucosa, prostate and ovary, adrenal gland, heart, thymus, and lung. By contrast, hNIS transcripts were not detected in normal orbital fibroblasts, colon, and nasopharyngeal mucosa. To further analyze hNIS gene sequences in parotid gland, mammary gland, and gastric mucosa, the EXPAND High Fidelity PCR System and three sets of overlapping NIS oligonucleotide primers were used for amplification and cloning. The resulting PCR products were subcloned into pBluescript-SKII(-)vector, and at least two independent cDNA clones derived from each tissue were subjected to automated sequencing. The nucleotide sequences of hNIS cDNA derived from parotid gland, mammary gland, and gastric mucosa revealed full identity with the recently published human thyroid-derived NIS cDNA sequence. In conclusion, our results demonstrate markedly variable levels of hNIS gene expression in several extrathyroidal tissues. Although the physiological role of hNIS in these tissues awaits further study, our results suggest that the capacity to actively transport iodine may be a feature common to several secretory and endocrine tissues. The diminished capacity to transport and concentrate iodide in extrathyroidal tissues (such as parotid gland, mammary gland, and gastric mucosa), compared with thyroid gland, does not seem to be caused by an altered primary structure of the hNIS cDNA. Variability of NIS gene expression levels in normal extrathyroidal tissues may rather be caused by differences in NIS gene transcriptional activity. Further studies will address this hypothesis and examine the mechanisms of tissue-specific regulation of NIS gene expression.

Blotting, Northern↗
PubMed1998

Lack of association of Graves' disease with the A2 allele of the interleukin-1 receptor antagonist gene in a white European population.

OBJECTIVE: To assess whether the A2-type IL-1RA polymorphism is associated with Graves' disease and Graves' ophthalmopathy. Several reports have described a genetic association between the A2 allele of the interleukin-1 receptor antagonist (IL-1RA) gene and certain inflammatory and autoimmune diseases, suggesting that certain loci within the IL-1-related genes may modulate the autoimmune inflammatory response. Recently, we demonstrated marked differences in the expression and regulation of IL-1RA gene and protein between orbital fibroblasts derived from patients with active Graves' ophthalmopathy and healthy individuals. DESIGN: A total of 144 white European patients with Graves' disease were genotyped to compare their IL-1RA A2 allele frequency with that of 174 healthy controls. METHODS: The polymerase chain reaction was used to amplify the pentallelic variable-number tandem-repeat locus in intron 2 of the IL-1RA gene. RESULTS: We found no significant differences in IL-1RA A2 allele frequencies (0.20 and 0.26 respectively) and IL-1RA A2 carriage rates (31% and 40% respectively) between patients with Graves' disease and the control group. Moreover, presence or absence of Graves' ophthalmopathy in patients with Graves' disease was not related to significant differences in IL-1RA A2 allele frequencies and IL-1RA A2 carriage rates. CONCLUSIONS: Our data do not support an association between the IL-1RA A2 allele and Graves' disease or Graves' ophthalmopathy in our study population. Thus the A2-type IL-1RA gene polymorphism does not appear to indicate an increased susceptibility to develop Graves' disease and Graves' ophthalmopathy. Mechanisms unrelated to the IL-1RA A2 allele may be responsible for altered IL-1RA production within the orbital tissues in Graves' ophthalmopathy.

Alleles↗
PubMed1997

Graves disease associated with autoimmune thrombocytopenic purpura.

Five patients with active Graves disease had moderate to severe autoimmune thrombocytopenic purpura at the time of diagnosis. Correction of hyperthyroidism and restitution of a euthyroid state by carbimazole therapy resulted in complete normalization of platelet counts.

Adult↗