Ca(2+)-binding protein expression in primary human thyrocytes.

We recently identified several Ca(2+)-binding proteins (CaBP) from the S100 and annexin family to be regulated by TSH in FRTL-5 cells. Here, we study the regulation of S100A4, S100A6 and ANXA2 in primary human thyrocytes (PHT) derived from surrounding tissues (ST), cold benign thyroid nodules (CTN) and autonomously functioning thyroid nodules (AFTN). We investigated the expression and regulation of CaBP and the effect of their expression on Ca(2+) and TSHR signaling. We used an approach that accounts for the potential of an individual PHT culture to proliferate or to express thyroid differentiation features by assessing the expression of FOS and TPO. We found a strong correlation between the regulation of CaBP and the proliferation-associated transcription factor gene FOS. PKA and MEK1/2 were regulators of ANXA2 expression, while PI3-K and triiodothyronine were additionally involved in S100 regulation. The modulated expression of CaBP was reflected by changes in ATP-elicited Ca(2+) signaling in PHT. S100A4 increased the ratio of subsequent Ca(2+) responses and showed a Ca(2+) buffering effect, while ANXA2 affected the first Ca(2+) response to ATP. Overexpression of S100A4 led to a reduced activation of NFAT by TSH. Using S100A4 E33Q, D63N, F72Q and Y75K mutants we found that the effects of S100A4 expression on Ca(2+) signaling are mediated by protein interaction. We present evidence that TSH has the ability to fine-tune Ca(2+) signals through the regulation of CaBP expression. This represents a novel putative cross-regulating mechanism in thyrocytes that could affect thyrocyte signaling and physiology.

Good clinical outcome after accidental intra-arterial injection of flunitrazepam tablets in 16 drug abusers with critical limb ischaemia.

OBJECTIVES: Inadvertent intra-arterial injection of flunitrazepam tablets intended for intravenous use by drug abusers has devastating effects. We report here on the clinical outcome of 16 drug abusers developing critical limb ischaemia after flunitrazepam injection. METHODS: Treatment combined immediate analgesia and anticoagulation, long-lasting local thrombolysis and vasodilatation, antibiotic prophylaxis, and physical mobilization. The immediate bolus injection of 5,000 IU heparin was followed by a continuous heparin infusion up to the target partial thromboplastin time. Under arteriographic control local intra-arterial infusion with alternating 4-h cycles of 5 mg recombinant tissue plasminogen activator followed by 5 µg prostaglandinE1 (PGE1) was performed for 24-48 hours. Subsequently, 60 µg PGE1 was applied once daily. RESULTS: Drug abusers, having been injected with 4-30 mg flunitrazepam, were treated 3-72 hours after the accident, with six of them not being treated until after 24 hours. All showed a high tissue ischaemia score. At the time of being discharged from hospital 13 patients had a normal extremity. In one patient, first receiving treatment 72 hours after injection, minor amputation of fingers was necessary. The life of the patient who injected 30 mg flunitrazepam in the leg was saved after hip disarticulation. One patient developed neurological dysfunction in the affected toes. CONCLUSIONS: Intensive treatment after inadvertent intra-arterial drug injection normalized the affected extremity in most drug abusers, even after the late onset of therapy.

[Ischemic preconditioning in a rat adipocutaneous flap model]. / Der epigastrisch adipokutane Lappen am Tiermodell zur Evaluierung der ischämischen Präkonditionierung.

INTRODUCTION: Flap necrosis in ear, nose, and throat surgery, especially in high-risk groups, is not rare, but not all of the individual pathophysiological processes are known. The objective of this study was to establish an animal model to determine whether acute ischemic preconditioning, which has been reported to be successful in organ transplantation, will result in enhanced flap survival. METHODS AND MATERIALS: Forty-two Wistar rats were divided into three experimental groups. An epigastric adipocutaneous flap, based on both superficial epigastric arteries and veins, was raised. The flap was either raised (control), clamped for 2 h (ischemic), or subjected to ischemia of 30 min, followed by 30 min of reperfusion and another 2 h of induced ischemia (IP). The mean flap necrosis area was assessed in all groups on the 5th postoperative day. RESULTS: All animals were doing well on the final day. The average necrosis in the ischemic group was significantly greater than in the control group. No significant superiority in the IP group was demonstrated. CONCLUSION: The data show that the experimental animal model is practicable and that additional approaches to ischemic preconditioning should be verified.

Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency.

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer. To get mechanistic insights into the time scale, sequence and frequency of intestinal stem cell (ISC) transformation, we quantified MSI and growth characteristics of organoids of Msh2-deficient and control mice from birth until tumor formation and related them to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Consistently, Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency. Transient cyst-like growth but not MSI was suppressed by aspirin. In summary, as highlighted by organoids, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation.

Graves' disease and Hashimoto's thyroiditis in monozygotic twins: case study as well as transcriptomic and immunohistological analysis of thyroid tissues.

OBJECTIVE: To report on the rare simultaneous occurrence of Graves' disease (GD) and Hashimoto's thyroiditis (HT) in monozygotic twins. DESIGN: We compared the pattern of thyroid tissue-derived cDNAs to gain insight into previous and ongoing immune destruction and reconstruction processes using microarrays. The results were confirmed by immunohistology and real-time PCR. RESULTS: Destruction of thyroid tissue in HT reduced levels of thyrocyte-related cDNAs and cDNAs encoding extracellular matrix components, but increased levels of proteases involved in extracellular matrix degradation compared with GD. Lymphocytic infiltrates forming ectopic follicles replaced the thyroid tissue almost completely in HT. Thus, lymphocyte-related cDNA levels were higher in HT than in GD. The same was true for many chemokines and their receptors, which not only enable migration towards the thyroid but also maintain the lymphocytic infiltrate. HT also showed increased levels of cDNAs encoding molecules related to apoptosis than did GD. Surprisingly, the Th1- and Th2-specific cytokine profiles suggested for HT and GD respectively could not be confirmed. cDNAs encoding factors and receptors involved in angiogenesis were increased in GD compared with HT. CONCLUSIONS: Comparison of gene expression reflects the cellular differences between the two types of autoimmune thyroid disease in twins with identical genetic and similar environmental background.

CD97: a dedifferentiation marker in human thyroid carcinomas.

CD97 is a dimeric glycoprotein of Mr 75,000-85,000 and 28,000 belonging to a novel subfamily of seven-span transmembrane region leukocyte cell surface molecules. It is expressed abundantly in cells of hematopoietic origin. This is the first report demonstrating the expression of CD97 outside the hematopoetic system. CD97 was studied in normal human and neoplastic follicular epithelium of the thyroid and anaplastic (n = 3) and papillary (n = 1) thyroid carcinoma cell lines. In normal thyroid tissue (n = 11), no immunoreactivity of CD97 could be found, whereas in differentiated thyroid carcinomas (n = 10), CD97 expression was either lacking or low. Eleven of 12 undifferentiated anaplastic carcinomas revealed high CD97 presentation. CD97 was absent or only weakly present in patients with postoperative T1 tumors but increased greatly with the progression to postoperative T4 tumors. CD97 is clearly present in thyroid carcinoma cell lines but only at a very low level in normal human thyrocytes. Quantitation of CD97 cell surface expression levels revealed that C 643 and SW 1736 cells showed a two to four times higher specific antibody-binding capacity than did 8505 C and HTh 74 cells and a nearly 20 times higher specific antibody-binding capacity than normal thyrocytes. Phorbol 12-myristate 13-acetate treatment progressively caused a decrease of CD97 antigen expression in all cell lines to about 30% of their initial levels after 48 h. Immunohistochemical staining of SW 1736 cells revealed that CD97 is located in most of the cell compartments and suggested a CD97 internalization process after phorbol 12-myristate 13-acetate treatment. Semiquantitative reverse transcription-PCR showed a correlation of CD97 mRNA and cell surface CD97 expression level in the cell lines. SW 1736, HTh 74, and 8505 C cells apparently expressed CD97 with alternative glycosylation compared to peripheral lymphocytes, whereas most of the CD97 antigen presented on thyrocytes and C 643 cells had glycosylation sites resembling those of lymphocytes. The data suggest that CD97 expression may be a sensitive marker of dedifferentiation and of lymph node involvement in human thyroid tumors.

Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells.

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze beta-cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase-polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 +/- 85 and 338 +/- 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 micromol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 +/- 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 micromol/l, 24-48 h) increased levels of IA-2 mRNA (190 +/- 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a time- and dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 +/- 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.

CXCR6 within T-helper (Th) and T-cytotoxic (Tc) type 1 lymphocytes in Graves' disease (GD).

OBJECTIVE: In Graves' disease (GD), stimulating anti-TSH receptor antibodies are responsible for hyperthyroidism. T-helper 2 (Th2) cells were expected to be involved in the underlying immune mechanism, although this is still controversial. The aim of this study was to examine the expression of CXCR6, a chemokine receptor that marks functionally specialized T-cells within the Th1 and T-cytotoxic 1 (Tc1) cell pool, to gain new insights into the running immune processes. METHODS: CXCR6 expression was examined on peripheral blood lymphocytes (PBLs) and thyroid-derived lymphocytes (TLs) of GD patients in flow cytometry. CXCR6 cDNA was quantified in thyroid tissues affected by GD (n = 16), Hashimoto's thyroiditis (HT; n = 2) and thyroid autonomy (TA; n = 11) using real-time reverse transcriptase PCR. RESULTS: The percentages of peripheral CXCR6(+) PBLs did not differ between GD and normal subjects. CXCR6 was expressed by small subsets of circulating T-cells and natural killer (NK) cells. CXCR6(+) cells were enriched in thyroid-derived T-cells compared with peripheral CD4(+) and CD8(+) T-cells in GD. The increase was evident within the Th1 (CD4(+) interferon-gamma(+) (IFN-gamma(+))) and Tc1 (CD8(+)IFN-gamma(+)) subpopulation and CD8(+) granzyme A(+) T-cells (cytotoxic effector type). Thyroid-derived fibro-blasts and thyrocytes were CXCR6(-). There was no significant difference between the CXCR6 mRNA levels in GD compared with HT and normal TA tissues. The lowest CXCR6 mRNA levels were obtained from thyroid nodules from TA patients and GD patients with low thyroid peroxidase autoantibody levels. CONCLUSIONS: CXCR6 was overexpressed in Th1 and Tc1 TLs compared with PBLs in GD. CXCR6 could be a marker for lymphocytes that have migrated into the thyroid and assist in the thyroid, independently of the bias of the underlying disease.

Expression of ADAM15 in lung carcinomas.

ADAM15, a member of the ADAM (a disintegrin and metalloprotease) family, is a membrane protein containing both protease and adhesion domains and may, thus, be involved in tumor invasion and metastasis. The aim of this study was to analyze the expression of ADAM15 and its potential ligand, integrin alpha(v)beta3 (CD51/CD61), in lung carcinoma cell lines and tissues. Most small cell lung carcinomas (SCLCs) and non-SCLC cell lines were ADAM15, alpha(v) and beta3 integrin mRNA positive. Half of the cell lines expressed ADAM15, and three expressed the alpha(v)beta3 heterodimer at the cell surface as shown using flow cytometry. Paraffin sections of pulmonary epithelial tumors, including SCLCs (n=26), squamous cell cancer (SCCs, n=27) and adenocarcinomas (ACs, n=17) were stained with antibodies to the ectosolic and cytosolic domain of ADAM15 and alpha(v)beta3 integrin complex. The results were scored (0-12, according to Remmele's score). Normal epithelial cells of the lung were negative or slightly positive for ADAM15 (score<2). The score was always significantly higher for tumor cells. ACs showed the strongest staining (tumor center; ADAM15ecto; mean+/-SEM; 5.47+/-1.04), whereas SCLCs only showed weak ADAM15 expression (2.67+/-0.42; SCCs: 3.62+/-0.62). Frequently, significantly stronger ADAM15 expression has been shown in tumor cells located at the front of invasion compared with those within solid formations. Overall analysis of all tumor specimens and each tumor type revealed no significant correlation between tumor stage or degree of differentiation and ADAM15 ectosolic or cytosolic domain expression in tumor cells. Both molecules are often co-localized in the same tumor cells in ADAM15- and alpha(v)beta3 integrin-positive carcinomas. In summary, lung carcinoma cell lines and tissues were frequently ADAM15 positive.

Bovine eotaxin (CCL11)--an unusual member of the eotaxin group--attracts eosinophils in vitro but is not responsible for eosinophilia in the ovary.

Under physiological conditions normally characterised by low tissue infiltration of eosinophils, a conspicuous number of these cells are attracted into the human and ruminant ovary. Eosinophils suddenly increase in the thecal layer of the preovulatory follicle and corpus luteum at very early development. Currently, we only have a limited understanding of the mechanism for the recruitment of the ovarian eosinophils. Eotaxin (CCL11) may be one of the chemoattractants involved in stimulating eosinophils to migrate selectively into ovary. As a prerequisite for the analysis of eotaxin expression in the bovine ovary, we determined the complete bovine eotaxin mRNA sequence since it was not available from databases. The bovine eotaxin is the first member of the monocyte chemoattractant protein (MCP)/eotaxin subfamily with two mRNA isoforms varying in length in the untranslated 3'-untranslated region. The unusual amino-acid sequence of bovine eotaxin contains structural features that are so far known to be characteristic for MCP, but not eotaxin. In our microchemotaxis assays, recombinant bovine eotaxin showed a functional pattern orthologous to known eotaxins. Thus, the chimeric structure of bovine eotaxin did not affect the favoured chemotactic activity on eosinophils. Semiquantitative RT-PCR was used to investigate the expression of eotaxin in different regions of the bovine ovary. We only detected faint eotaxin mRNA signals that did not indicate physiological significance even in stimulated granulosa cell cultures, follicle-derived macrophages or fibroblasts. Taken together, bovine eotaxin attracts eosinophils in vitro but is not responsible for eosinophilia in the ovary. Its unusual chimeric structure confirms the unity of the MCP/eotaxin subfamily of CC chemokines and distinguishes it from other CC chemokine subfamilies.

Expression and regulation of regulated on activation, normal T cells expressed and secreted in thyroid tissue of patients with Graves' disease and thyroid autonomy and in thyroid-derived cell populations.

Thyroid glands affected by Graves' disease (GD) show striking lymphocytic infiltration, mainly by CD45RO(+) T cells. The mechanisms by which the various lymphocytic subsets are recruited and maintained in the thyroid are unknown. RANTES (regulated on activation, normal T cells expressed and secreted) in interaction with its receptors (CCR1, CCR3, CCR4 and CCR5) may be one of the favorite chemokines involved in the cell trafficking and maintenance. RANTES messenger RNA (mRNA) was quantified in the thyroid tissue of 16 patients with GD and 7 patients with thyroid autonomy (TA), using competitive RT-PCR. We found a clear correlation between the RANTES mRNA level and 1) the degree of T-cell infiltration (r = 0.68), and 2) the level of serum antibodies to thyroid peroxidase (r = 0.76) in GD but not in TA patients. There was no difference between the autonomous nodules and the quiescent surrounding tissue in TA patients. To define the cellular source of RANTES mRNA and protein, we examined various thyroid-derived cells. Lymphocytes showed a markedly higher basal RANTES mRNA and protein level (mean +/- SEM; pg/mL, n = 3; 140 +/- 30) than thyrocytes (12 +/- 5) and fibroblasts (9 +/- 2). Lymphocyte stimulation with PMA enhanced RANTES secretion significantly (4490 +/- 200). Fibroblasts responded to stimulation with interleukin 1 (530 +/- 220) and tumor necrosis factor alpha (2780 +/- 1790), whereas thyrocytes did not. However, some thyroid carcinoma cell lines showed very high basal and stimulated RANTES expression. Lymphocytes expressed the mRNA of all chemokine receptors that bind RANTES. The number of CCR3(+) and CCR5(+) T cells was significantly higher in thyroid-derived leukocytes than in those in the peripheral blood stream. We conclude that RANTES expression, mainly by lymphocytes, is perhaps involved in the maintenance of lymphocytic infiltration and, therefore, in the autoimmune responses in GD.

Reduced expression of stromal-derived factor 1 in autonomous thyroid adenomas and its regulation in thyroid-derived cells.

Stromal-derived factor 1 (SDF-1) and CXCR4 comprise a unique chemokine/chemokine receptor pair, exhibiting important functions in morphogenesis and growth regulation as well as attractant properties on T lymphocytes. No data are available on SDF-1 and CXCR4 in normal or pathological thyroid tissues. SDF-1, CXCR4, and CD18 messenger ribonucleic acid (mRNA) as a marker of leukocytic infiltration were quantified in tissues affected by thyroid adenoma (n = 11) and Graves' disease (GD; n = 16) using competitive RT-PCR. SDF-1 mRNA levels differed significantly between autonomous adenomas and the corresponding normal tissue, but not in GD between patients with low or high leukocyte infiltration, thyroid peroxidase, and TSH receptor autoantibodies, respectively. We found a strong correlation between CXCR4 and CD18 mRNA, which indicates CXCR4 expression by leukocytes. To define the cellular source of SDF-1 and CXCR4 in thyroid tissue, we examined various thyroid-derived cells. Fibroblasts are the most potent producers of SDF-1, although thyrocytes also secrete SDF-1 in vitro. Leukocytes showed very weak SDF-1 mRNA levels and no secretion of the chemokine. Immunohistology confirmed and extended these results; SDF-1 expression was found in fibroblasts, but not or very weakly in CD45(+) leukocytes and thyrocytes. Only leukocytes were CXCR4(+). As examined by flow cytometry, the number of CD3(+) T cells expressing CXCR4 is significantly higher in the thyroid than in peripheral blood. SDF-1 seems to be involved in thyroid tissue homeostasis in thyroid adenoma, but not in the maintenance of lymphocytic infiltration in GD.

Regulation of CD97 protein in thyroid carcinoma.

CD97 is a dimeric glycoprotein belonging to the secretin receptor superfamily and is abundantly expressed in cells of hematopoietic origin. The aim of this study was to analyze the expression of the CD97 protein in thyroid carcinomas and the role of all-trans-retinoic acid (RA) in the regulation of CD97 protein in monolayer culture of the human follicular thyroid carcinoma cell line FTC-133. In normal thyroid tissue, no immunoreactivity of CD97 could be found, whereas in differentiated thyroid carcinomas, CD97 expression was either lacking or low. Undifferentiated anaplastic thyroid carcinomas revealed high CD97 expression. The expression of CD97 protein seems to be correlated to the postoperative histopathological classification staging. Approximately 50% of FTC-133 cells expressed the CD97 protein under basal culture conditions. No differences were found in the number of CD97-positive cells after TSH, forskolin, and insulin treatment compared to control values. Epidermal growth factor treatment led to an increase in CD97 immunostaining (up to 90%), whereas phorbol 12-myristate 13-acetate slightly decreased the immunoreactivity of CD97 (from 50% to 30%). Under basal conditions, RA treatment for 72 h led to a decrease in total cell number by 33% and in CD97-positive cells from 50% to 30%. TSH, forskolin, phorbol 12-myristate 13-acetate, and insulin showed no effect after 72-h pretreatment with RA, whereas epidermal growth factor treatment led to a slight increase in the number of the CD97-positive cells (from 30% to 40%) compared to the control value. These data suggest that CD97 expression may play an important role in the dedifferentiation of thyroid tumors, and RA might interfere with this process in thyroid carcinoma by suppressing the dedifferentiation marker CD97.

Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period.

RANTES may be one of the chemoattractants involved in stimulating eosinophils and macrophages to migrate selectively into bovine dominant follicles and into developing corpora lutea. We sequenced a 736 bp fragment of the bovine RANTES mRNA encoding the complete protein and defined the ovarian source of RANTES mRNA. As demonstrated by competitive RT-PCR, follicle-derived macrophages showed a 100-1000 times higher RANTES mRNA level compared to unpurified granulosa cells or follicle-derived fibroblasts. By means of in situ hybridization, RANTES mRNA positive macrophages were located in the former thecal layer of the developing corpora lutea.

GRO-alpha in normal and pathological thyroid tissues and its regulation in thyroid-derived cells.

Thyroid glands affected by Graves' disease (GD) show striking leukocytic infiltration, mainly by T-cells. The mechanisms by which the various leukocytes are maintained in the thyroid are unknown. Growth-regulated oncogene-alpha (GRO-alpha) in interaction with its receptor CXCR2 is a chemoattractant for both T-cells and neutrophils and may be one of the chemokines involved in the cell maintenance. GRO-alpha and CD18 mRNA as a marker of leukocytic infiltration were quantified in thyroid tissue using competitive RT-PCR. We found very high GRO-alpha mRNA levels in all thyroid tissues. In GD patients (n=16), the GRO-alpha mRNA did not correlate with the CD18 mRNA level or thyroid peroxidase and TSH-receptor antibodies in patients' sera. In thyroid autonomy (n=10), the GRO-alpha mRNA levels were significantly lower in autonomous single adenomas compared with the corresponding normal tissue. In order to define the cellular source of GRO-alpha mRNA and protein, we examined various thyroid-derived cells. Thyrocytes, thyroid-derived leukocytes and fibroblasts showed basal GRO-alpha mRNA and protein expression, which was remarkably upregulated by different stimuli in vitro. The expression of GRO-alpha by thyroid carcinoma cell lines confirms that thyrocytes may actually produce GRO-alpha. As shown by flow cytometry and immunohistology, CD68+ monocytes/macrophages are the only cell population strongly expressing CXCR2 in the thyroid.

Differential regulation of granulocyte-macrophage colony-stimulating factor mRNA and protein expression in human thyrocytes and thyroid-derived fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha.

In this study, we provide the first report on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by human thyroid epithelial cells. Primary cultures of highly purified thyrocytes and thyroid-derived fibroblasts (n = 3) and three thyroid anaplastic and one largely papillary carcinoma cell lines were exposed to different potent GM-CSF stimulators, employing interleukin 1 alpha (Il-1 alpha) and tumour necrosis factor-alpha (TNF-alpha). Cytokine mRNA levels were monitored by semi-quantitative reverse transcriptase-PCR including an internal heterologous competitor fragment after 3, 6 and 18 h of culture. Culture supernatants were assayed for GM-CSF using a highly sensitive ELISA (detection limit < or = 0.5 pg/ml) after 24 h. Basal GM-CSF mRNA expression was higher in fibroblasts and SW 1736 cells compared with thyrocytes, C 634, 8505 C and HTh 74 cells. GM-CSF was spontaneously secreted by fibroblasts (means +/- S.E.M.; 43 +/- 15 pg/ml), SW 1736 (59 +/- 4 pg/ml), HTh 74 (34 +/- 4 pg/ml) and C 643 cells (12 +/- 1 pg/ml) but not by thyrocytes and 8505 C cells. Treatment with Il-1 alpha (10 U/ml) resulted in a marked increase of GM-CSF mRNA within 3 h and an increase or induction of protein expression in thyrocyte (2350 +/- 214 pg/ml), fibroblast (5242 +/- 1400 pg/ml), SW 1736 (20016 +/- 280 pg/ml) and C 643 cultures (1285 +/- 79 pg/ml). Stimulation with TNF-alpha (10 U/ml) yielded divergent results. No significant increase of GM-CSF mRNA or protein expression was found in thyrocytes although TNF-alpha receptor expression in these cells is well documented. Stimulation with TNF-alpha resulted in an increased GM-CSF production in fibroblasts (361 +/- 14 pg/ml), HTh 74 (148 +/- 51 pg/ml) and SW 1736 cultures (235 +/- 43 pg/ml). TSH (10 mU/ ml) did not stimulate GM-CSF secretion in thyrocytes and HTh 74 cells, both expressing the TSH receptor. Phorbol 12-myristate 13-acetate (10 ng/ml) enhanced GM-CSF mRNA and protein levels in all cell types investigated. Our data suggest that both thyrocytes and fibroblasts synthesize GM-CSF in response to Il-1 alpha, but only fibroblasts respond to TNF-alpha with a significant increase in GM-CSF. Anaplastic thyroid carcinomas are potential GM-CSF producers.

Activated and interferon-gamma producing thyroid-derived T cells are detected in Graves' disease, thyroid autonomy as well as in non-toxic multinodular goiter.

The relative numbers of activated and interferon gamma (IFN-gamma)-producing peripheral blood lymphocytes (PBL) and thyroid-derived lymphocytes (TL) were determined using double surface and intracellular labeling techniques in flow cytometry. Cells were analyzed from 10 patients with Graves' disease (GD), eight patients with thyroid autonomy (TA) and five patients with non-toxic multinodular goiter (NTG). A maximum of 1% IFN-gamma+ cells were detected both in unstimulated PBL and TL. Stimulation caused a two- to threefold higher number of IFN-gamma+ cells in TL (GD, 48 +/- 12%; TA, 48 +/- 11%; NTG, 50 +/- 15%) as compared to PBL (GD, 15 +/- 7%; TA, 16 +/- 8%, NTG, 18 +/- 10%) of the same patients. Nearly all IFN-gamma+ TL in GD were CD3+ T cells, whereas 10-20% of IFN-gamma+ TL in TA and NTG were NK cells. In PBL 80% and in TL almost 100% of IFN-gamma+ cells were antigen-primed CD45RO+ cells. Only 25-35% of IFN-gamma+ thyroid-derived T cells expressed the CD4 antigen. About 42 +/- 10% thyroid-derived T cells in GD, 33 +/- 11% in TA and 34 +/- 13% in NTG expressed the HLA-DR molecule but not the interleukin 2 (CD25) or the transferrin receptor (CD71). Forty per cent of these HLA-DR+ T cells showed an intracellular staining for IFN-gamma and half of them co-expressed the activation antigen CD69. Immunofluorescence double labeling on thyroid cryostat sections demonstrated that HLA-DR+ T cells were also present in situ. The presence of activation antigens on thyroid-derived T cells not only in patients with GD but also in TA and NTG suggests failsafe mechanisms such as anergy, suppression or cytokine regulation in so-called non-immunogenic goiter.

The role of CXCR5 and its ligand CXCL13 in the compartmentalization of lymphocytes in thyroids affected by autoimmune thyroid diseases.

OBJECTIVE: Graves' disease (GD) and Hashimoto's thyroiditis (HT) are characterized by lymphocytic infiltrates partly resembling secondary lymphoid follicles in the thyroid. CXCR5 and its ligand CXCL13 regulate compartmentalization of B- and T-cells in secondary lymphoid organs. The aim of the study was to elucidate the role of this chemokine receptor-ligand pair in thyroid autoimmunity. METHODS: Peripheral blood and thyroid-derived lymphocyte subpopulations were examined by flow cytometry for CXCR5. CXCR5 and CXCL13 cDNA were quantified in thyroid tissues by real-time RT-PCR. RESULTS: We found no differences between the percentages of peripheral blood CXCR5+ T- and B-cells in GD patients (n=10) and healthy controls (n=10). In GD patients, the number of memory CD4+ cells expressing CXCR5 which are functionally characterized as follicular B helper T-cells is higher in thyroid-derived (18+/-3%) compared with peripheral blood T-lymphocytes (8+/-2%). The highest CXCL13 mRNA levels were found in HT (n=2, 86.1+/-1.2 zmol (10(-21) mol) cDNA/PCR) followed by GD tissues (n=16, 9.6+/-3.5). Only low amounts were determined in thyroid autonomy (TA) (n=11) thyroid tissues, irrespective of whether the autonomous nodule (0.5+/-0.1) or the surrounding normal tissue (1.8+/-0.7) had been analyzed. The same differences were found for CXCR5 (HT: 179.1+/-6.8; GD: 17.4+/-10.6; TA(nodule): 0.8+/-0.5; TA(normal): 4.4+/-3.6). In GD, there is a correlation between CXCL13 and CXCR5 mRNA levels and the number of focal lymphocytic infiltrates and germinal centers as well as anti-thyroperoxidase but not anti-TSH receptor autoantibodies. CONCLUSIONS: CXCR5 and CXCL13 play an essential role in maintaining B- and T-cells in lymphocytic infiltrates and ectopic follicles in thyroid tissue from patients affected by autoimmunity.

Characterisation of the antibody response to the extracellular region of recombinant thyrotropin receptor.

Autoantibodies to the human thyrotropin receptor (TSH-R) are pathogenic in a number of autoimmune thyroid diseases including Graves' disease. We have characterised polyclonal antisera to TSH-R for antibodies which may mimic those present in autoimmune thyroid disease. For immunisations, recombinant extracellular region of human TSH-R which does not interact with its ligand TSH was used. The induced antibodies react with the full length membrane receptor in transfected mammalian cells by flow cytometry showing the presence of antibody capable of recognising the native functional receptor. The properties of the generated antibodies have been compared after two injections or following a multiple immunisation protocol with the receptor in adjuvant. High titre antisera were readily generated after the short injection protocol and further immunisations did not lead to any change in antibody titers. Analysis of the epitopes recognised using synthetic peptides confirmed previous observations that the immunodominant determinants localise to the amino and the carboxyl terminal part of the extracellular region of the receptor. Antisera from both rabbits contain TSH blocking antibody as assessed by inhibition of TSH mediated cAMP stimulation. There was an increase in TSH binding inhibitory immunoglobulin (TBII) activity with multiple injections. Furthermore, the increase in TBII activity was not related to spreading of the antibody response to new determinants on TSH-R. Our results support previous observations on the difficulties in reproducing, by adjuvant immunisation with recombinant TSH-R preparations, the fine specificity of antibodies to TSH-R present in autoimmune disorders such as Graves' disease or primary myxoedema.

The expression of substance P and its neurokinin-1 receptor mRNA in the bovine corpus luteum of early developmental stage.

Related to all leukocytes, 90% of eosinophils are recruited into the bovine corpus luteum of early developmental stage. We here describe a simultaneous appearance of substance P (SP)-positive fibre-like structures and the neurokinin-1 (NK-1) receptor mRNA for SP. Substance P was depicted by using indirect immunohistology and immunofluorescence localization. The dot blot analysis confirmed the presence of SP at the protein level. Using nested reverse transcribed-polymerase chain reaction (RT-PCR) analysis, a 358 bp long partial bovine receptor mRNA for SP (NK-1) was sequenced in the spinal cord. The mRNA for SP and for the NK-1 receptor were then detected in the corpus luteum of early developmental stage with RT-PCR and nested RT-PCR. We conclude: The production of SP and the expression of NK-1 receptor mRNA may be involved in the selective recruitment of eosinophils into the bovine corpus luteum of early developmental stage.