Josef Köhrle

We describe the validation of a novel assay for the measurement of peroxiredoxin 4 (Prx4), a pote... more We describe the validation of a novel assay for the measurement of peroxiredoxin 4 (Prx4), a potentially secreted antioxidant peroxidase, in human serum. A sandwich immunoluminometric assay (ILMA) was set up applying monoclonal antibodies against the amino-terminus of human Prx4. Prx4 levels have been determined in serum of healthy individuals and patients with sepsis and have been correlated to the clinically established sepsis marker procalcitonin (PCT). The sandwich ILMA detected Prx4 in a range between 0.5 and 128 arbitrary (arb.) U/L and had a functional assay sensitivity of 0.51 arb.U/L. Serum Prx4 was stable for at least 72 h at 4 degrees C and 21 degrees C and circulated in a complex of about 330 kDa as characterized by size-exclusion chromatography. Patients with sepsis (n=45; median, 7.7 arb.U/L) showed significantly higher Prx4 serum levels (P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001) than healthy controls (n=274; median, 0.71 arb.U/L). Serum Prx4 was positively correlated to PCT (r=0.63, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.0001). The newly developed assay reliably detected Prx4 in human serum of healthy and critically ill subjects. Elevated Prx4 in serum of patients with various diseases might serve as a biomarker reflecting increased oxidative stress.

Supplementary Methods, Table 1 from Serum 25-Hydroxyvitamin D and Cancer Risk in Older Adults: Results from a Large German Prospective Cohort Study

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Tentative Application of a Streamlined Protocol to Determine Organ-Specific Regulations of Deiodinase 1 and Dehalogenase Activities as Readouts of the Hypothalamus-Pituitary-Thyroid-Periphery-Axis

Frontiers in toxicology, Mar 21, 2022

In animal studies, both in basic science and in toxicological assessment of potential endocrine d... more In animal studies, both in basic science and in toxicological assessment of potential endocrine disruptors, the state of the thyroid hormone (TH) axis is often described and defined exclusively by the concentrations of circulating THs and TSH. Although it is known that the local, organspecific effects of THs are also substantially regulated by local mechanisms such as TH transmembrane transport and metabolism of TH by deiodinases, such endpoint parameters of the axis are rarely assessed in these experiments. Currently developed in vitro assays utilize the Sandell-Kolthoff reaction, a photometric method of iodide determination, to test the effect of chemicals on iodotyrosine and iodothyronine deiodinases. Furthermore, this technology offers the possibility to determine the iodine content of various sample types (e.g., urine, ex vivo tissue) in a simple way. Here, we measured deiodinase type 1 and iodotyrosine dehalogenase activity by means of the Sandell-Kolthoff reaction in ex vivo samples of hypo-and hyperthyroid mice of two age groups (young; 3 months and old; 20 months). In thyroid, liver and kidney, organspecific regulation patterns emerged across both age groups, which, based on this pilot study, may serve as a starting point for a deeper characterization of the TH system in relevant studies in the future and support the development of Integrated Approach for Testing and Assessment (IATA).

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The flavonoid Xanthohumol interferes with the thyroid hormone axis

Experimental and Clinical Endocrinology & Diabetes, Mar 21, 2005

Strong associations of 25-hydroxyvitamin D levels with all-cause, cardiovascular, cancer and respiratory disease mortality in a large cohort study

Experimental and Clinical Endocrinology & Diabetes, Mar 7, 2013

ABSTRACT BACKGROUND: Serum 25-hydroxyvitamin D [25(OH)D] concentration has been linked to mortali... more ABSTRACT BACKGROUND: Serum 25-hydroxyvitamin D [25(OH)D] concentration has been linked to mortality in several studies, but appropriate cutoffs to define risk categories are under debate. OBJECTIVE: We aimed to conduct a repeated-measurements analysis on the association of serum 25(OH)D concentrations with all-cause and cause-specific mortality, with particular attention given to the shape of dose-response relations. DESIGN: Concentrations of 25(OH)D were measured in n = 9578 baseline and n = 5469 5-y follow-up participants of the ESTHER study, which is a German population-based cohort aged 50-74 y at baseline. Deaths were recorded during 9.5 y of follow-up (median). Restricted cubic splines were used to assess dose-response relations and Cox regression with time-dependent variables to estimate hazard ratios. RESULTS: During follow-up, 1083 study participants died, of whom 350 individuals died of cardiovascular diseases, 433 individuals died of cancer, and 55 individuals died of respiratory diseases. The overall mortality [HR (95% CI)] of subjects with vitamin D deficiency [25(OH)D concentrations &lt;30 nmol/L] or vitamin D insufficiency [25(OH)D concentrations from 30 to 50 nmol/L) was significantly increased [1.71 (1.43, 2.03) and 1.17 (1.02, 1.35), respectively] compared with that of subjects with sufficient 25(OH)D concentrations (&gt;50 nmol/L)]. Vitamin D deficiency was also associated with increased cardiovascular mortality [1.39 (95% CI: 1.02, 1.89)], cancer mortality [1.42 (95% CI: 1.08, 1.88)] and respiratory disease mortality [2.50 (95% CI: 1.12, 5.56)]. The association of 25(OH)D concentrations with all-cause mortality proved to be a nonlinear inverse association with risk that started to increase at 25(OH)D concentrations &lt;75 nmol/L. CONCLUSIONS: In this large cohort study, serum 25(OH)D concentrations were inversely associated with all-cause and cause-specific mortality. In particular, vitamin D deficiency [25(OH)D concentration &lt;30 nmol/L] was strongly associated with mortality from all causes, cardiovascular diseases, cancer, and respiratory diseases.

Chronic Kidney Disease Distinctly Affects Relationship Between Selenoprotein P Status and Serum Thyroid Hormone Parameters

Thyroid, Oct 1, 2015

Chronic kidney disease (CKD) impairs thyroid hormone (TH) metabolism and is associated with low s... more Chronic kidney disease (CKD) impairs thyroid hormone (TH) metabolism and is associated with low serum triiodothyronine (T3) concentrations in patients with a low glomerular filtration rate (GFR). Whether this results from decreased T3 formation from thyroxine (T4) by impaired 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-deiodinase (DIO) activity and/or enhanced degradation of T3 and increased reverse triiodothyronine (rT3) formation from T4 by elevated 5-DIO activity remains unclear. Both activating 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;- and the inactivating 5-deiodination of TH are catalyzed by three selenium (Se)-dependent DIO isoenzymes. Selenoprotein P (SePP) is the major constituent of serum selenium, and functions as Se transport protein from liver to kidney and several other organs. This study tested the hypothesis that serum SePP and TH status are associated with the degree of renal impairment in patients with CKD. A total of 180 CKD patients (stages 1-5) and 70 chronic hemodialysis (CHD) patients undergoing hemodialysis three times per week for at least two years were prospectively investigated for clinical data, parameters of renal function, serum TH profile (thyrotropin, T4, free thyroxine [fT4], T3, free triiodothyronine (fT3), rT3, thyroxine-binding globulin [TBG]), C-reactive protein (CRP), and serum SePP. In CKD patients, renal function was negatively associated with SePP concentration (standardized β = -0.17, p = 0.029); that is, SePP concentrations increased in more advanced CKD stages. In contrast, significantly lower SePP concentrations were found in patients on hemodialysis compared with CKD patients (M ± SD = 2.7 ± 0.8 mg/L vs. 3.3 ± .9 mg/L; p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.001). Notably, in CKD patients, the SePP concentration was negatively associated with T4 (standardized β = -0.16, p = 0.039) and fT4 (standardized β = -0.16, p = 0.039) concentrations, but no association was found with T3, fT3, rT3, T3/T4, rT3/T3, rT3/T4, or TBG concentrations. The SePP concentration was also negatively associated with CRP levels (standardized β = -0.17, p = 0.029). In the CHD group, no association was detected between SePP and the investigated TH parameters. Impaired renal function is positively correlated with serum concentrations of SePP. In patients undergoing CHD treatment, SePP concentrations were significantly reduced, but the TH profile remained unaffected. These findings indicate an important contribution of kidney function on serum SePP homeostasis, and consequently on Se status.

Binding of Thyroxine to Pig Transthyretin, its cDNA Structure, and Other Properties

European journal of biochemistry, Jun 1, 1995

Thyroxine binding to proteins in pig plasma during electrophoresis was observed in the albumin, b... more Thyroxine binding to proteins in pig plasma during electrophoresis was observed in the albumin, but not in the prealbumin and post-albumin regions. Transthyretin could be identified in medium from in vitro pig choroid plexus incubations by size and number of subunits and a very high rate of synthesis and secretion. Its electrophoretic mobility was intermediate between that of thyroxine-binding globulin and albumin. It bound thyroxine, retinol-binding protein, anti-(rat transthyretin) antibodies and behaved similarly to transthyretins from other vertebrate species when plasma was extracted with phenol. Inhibition experiments with the synthetic flavonoid F 21388, analysing the binding of thyroxine, suggested that transthyretin is not a major thyroxine carrier in the bloodstream of pigs. Cloning and sequencing of transthyretin cDNA from both choroid plexus and liver showed that the same transthyretin mRNA is expressed in pig choroid plexus and liver. The amino acid sequence derived from the nucleotide sequence revealed that pig transthyretin differs from the transthyretins of all other studied vertebrate species by an unusual C-terminal extension consisting of the amino acids glycine, alanine and leucine. This extension results from the mutation of a stop codon into a codon for glycine. The unusual C-terminal extensions do not seem to interfere with the access of thyroxine to its binding site in the central channel of transthyretin.

Human cerebral organoids as <i>in vitro</i> platform to assess thyroid hormone system disrupting chemicals

Endocrine Abstracts, Sep 2, 2022

High T3, Low T4 Serum Levels in Mct8 Deficiency Are Not Caused by Increased Hepatic Conversion through Type I Deiodinase

European thyroid journal, 2015

Background: The Allan-Herndon-Dudley syndrome is a severe psychomotor retardation accompanied by ... more Background: The Allan-Herndon-Dudley syndrome is a severe psychomotor retardation accompanied by specific changes in circulating thyroid hormone levels (high T 3 , low T 4). These are caused by mutations in the thyroid hormone transmembrane transport protein monocarboxylate transporter 8 (MCT8). Objective: To test the hypothesis that circulating low T 4 and high T 3 levels are caused by enhanced conversion of T 4 via increased activity of hepatic type I deiodinase (Dio1). Methods: We crossed mice deficient in Mct8 with mice lacking Dio1 activity in hepatocytes. Translation of the selenoenzyme Dio1 was abrogated by hepatocyte-specific inactivation of selenoprotein biosynthesis. Results: Inactivation of Dio1 activity in the livers of global Mct8-deficient mice does not restore normal circulating thyroid hormone levels. Conclusions: Our data suggest that although hepatic Dio1 activity is increased in Mct8-deficient mice, it does not cause the observed abnormal circulating thyroid hormone levels. Since global inactivation of Dio1 in Mct8-deficient mice does normalize circulating thyroid hormone levels, the underlying mechanism and relevant tissues involved remain to be elucidated.

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Hyperthyroidism and Hypothyroidism in Male Mice and Their Effects on Bone Mass, Bone Turnover, and the Wnt Inhibitors Sclerostin and Dickkopf-1

Endocrinology, Jul 28, 2015

Thyroid hormones are key regulators of bone homeostasis, and Wnt signaling has been implicated in... more Thyroid hormones are key regulators of bone homeostasis, and Wnt signaling has been implicated in thyroid hormone-associated bone loss. Here we tested whether hyperthyroidism and hypothyroidism interfere with dickkopf-1 (DKK1) and sclerostin, two inhibitors of Wnt signaling. Twelveweek-old male C57BL/6 mice were rendered either hyperthyroid or hypothyroid. Hyperthyroid mice displayed decreased trabecular (Ϫ54%, P Ͻ .001) and cortical bone density (Ϫ5%, P Ͻ .05) and reduced cortical thickness (Ϫ15%, P Ͻ .001), whereas hypothyroid mice showed a higher trabecular bone density (ϩ26%, P Ͻ .001) with unchanged cortical bone parameters. Histomorphometry and biochemical markers of bone remodeling indicated high bone turnover in hyperthyroid mice and low bone turnover in hypothyroid mice. In vivo, serum DKK1 concentrations were decreased in hyperthyroid mice (Ϫ24%, P Ͻ .001) and increased in hypothyroid mice (ϩ18%, P Ͻ .01). The increase of the number of DKK1-positive cells in hypothyroid mice was confirmed at the tissue level. Interestingly, sclerostin was increased in both disease models, although to a higher extent in hyperthyroid mice (ϩ50%, P Ͻ .001, and ϩ24%, P Ͻ .05). Serum sclerostin concentrations adjusted for bone mass were increased by 3.3-fold in hyperthyroid (P Ͻ .001) but not in hypothyroid mice. Consistently, sclerostin mRNA expression and the number of sclerostin-positive cells were increased in hyperthyroid but not in hypothyroid mice. Our data show that thyroid hormone-induced changes in bone remodeling are associated with a divergent regulation of DKK1 and sclerostin. Thus, the modulation of Wnt signaling by thyroid hormones may contribute to thyroid hormoneassociated bone disease and altered expression of Wnt inhibitors may emerge as potential therapeutic targets.

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An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases

Thyroid, Aug 1, 2015

Deiodinases (DIO1, 2 and 3) are key enzymes in thyroid hormone (TH) activation and inactivation w... more Deiodinases (DIO1, 2 and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH-status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation, e.g. the low T3 syndrome in critical illness. Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil (PTU) is in use for treatment of severe hyperthyroidism. Other well-known inhibitors, e.g. iopanoic acid (IA) or aurothioglucose (ATG), are non-selective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening (HTS) is needed. We recently described a non-radioactive screening assay, utilizing the classical Sandell-Kolthoff-reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine Dio1 activity testing. Now, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2 and DIO3. As prove-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various non-radioactive substrates and confirmed by LC-MS/MS. The contrast agent and known DIO-inhibitor IA was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor while xanthohumol turned out to potently block the activity of all three isoenzymes.

Establishment of an Effective Radioiodide Thyroid Ablation Protocol in Mice

European thyroid journal, 2015

followed by injection of bovine TSH before the application of 150 μCi (5.5 MBq) 131 I decreased s... more followed by injection of bovine TSH before the application of 150 μCi (5.5 MBq) 131 I decreased serum T 4 concentrations below the detection limit and significantly increased pituitary TSHβ concentrations. The systemic effects of induced hypothyroidism were shown by growth arrest and a decrease in liver DIO1 expression below the detection limit. 99m Tc-pertechnetate scintigraphy revealed absence of thyroidal 99m Tc-pertechnetate uptake in ablated mice. In summary, we report a revised protocol for radioiodide ablation of the thyroid gland in the mouse to generate an in vivo model that allows the study of thyroid hormone action using NIS as a reporter gene.

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Data from Serum 25-Hydroxyvitamin D and Cancer Risk in Older Adults: Results from a Large German Prospective Cohort Study

Background: Several observational studies assessed the relationship between serum 25-hydroxyvitam... more Background: Several observational studies assessed the relationship between serum 25-hydroxyvitamin D [25(OH)D] concentrations and the risk of cancer but results were inconclusive. Methods: We measured 25(OH)D concentrations in a population-based cohort study of 9,949 men and women ages 50 to 74 years in Saarland, Germany. Comprehensively adjusted Cox regression models were applied to estimate HRs and 95% confidence intervals (CI) for the association between season-standardized 25 (OH)D concentrations and total and site-specific cancer incidence. Results: Overall, during a median of 8 years of follow-up, 873 subjects developed cancer; the most common being prostate (171), breast (137), lung (136), and colorectal (136) cancer. Low season-standardized 25(OH)D (<30, 35, 40, or 36 nmol/L in winter, spring, summer, and autumn, respectively) was neither significantly associated with total cancer incidence (HR, 1.10; 95% CI, 0.93-1.30) nor with site-specific cancer incidence. However, a significantly increased overall cancer risk was observed for low 25(OH)D among men, nonobese subjects and subjects reporting low fish consumption and for high 25(OH)D in nonsmokers and nonobese subjects. Accordingly, restricted cubic splines to investigate dose-response relationships curves showed an inverse association of 25(OH)D levels and total cancer risk in men but not in women. Conclusions: 25(OH)D concentrations were significantly associated with overall cancer incidence in subgroups of this large cohort from Germany. No significant association was observed with site-specific cancers but this could be due to a limited statistical power for these endpoints. Impact: Further research should clarify whether and to what extent specific risk groups might profit from vitamin D supplementation. Cancer Epidemiol Biomarkers Prev; 22(5); 905-16. Ó2013 AACR.

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The Role of Dickkopf-1 in Thyroid Hormone–Induced Changes of Bone Remodeling in Male Mice

Endocrinology, Jan 25, 2019

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Canonical TSH Regulation of Cathepsin-Mediated Thyroglobulin Processing in the Thyroid Gland of Male Mice Requires Taar1 Expression

Frontiers in Pharmacology, Mar 20, 2018

Trace amine-associated receptor 1 (Taar1) has been suggested as putative receptor of thyronamines... more Trace amine-associated receptor 1 (Taar1) has been suggested as putative receptor of thyronamines. These are aminergic messengers with potential metabolic and neurological effects countering their contingent precursors, the thyroid hormones (THs). Recently, we found Taar1 to be localized at the primary cilia of rodent thyroid epithelial cells in vitro and in situ. Thus, Taar1 is present in a location of thyroid follicles where it might be involved in regulation of cathepsin-mediated proteolytic processing of thyroglobulin, and consequently TH synthesis. In this study, taar1 knockout male mice (taar1 −/−) were used to determine whether Taar1 function would entail differential alterations in thyroid states of young and adult animals. Analyses of blood serum revealed unaltered T 4 and T 3 concentrations and unaltered T 3-overT 4 ratios upon Taar1 deficiency accompanied, however, by elevated TSH concentrations. Interestingly, TSH receptors, typically localized at the basolateral plasma membrane domain of wild type controls, were located at vesicular membranes in thyrocytes of taar1 −/− mice. In addition, determination of epithelial extensions in taar1 −/− thyroids showed prismatic cells, which might indicate activation states higher than in the wild type. While gross degradation of thyroglobulin was comparable to controls, deregulated thyroglobulin turnover in taar1 −/− mice was indicated by luminal accumulation of covalently cross-linked thyroglobulin storage forms. These findings were in line with decreased proteolytic activities of thyroglobulin-solubilizing and-processing proteases, due to upregulated cystatins acting as their endogenous inhibitors in situ. In conclusion, Taar1-deficient mice are hyperthyrotropinemic in the absence of respective signs of primary hypothyroidism such as changes in body weight or TH concentrations in blood serum. Thyrocytes of taar1 −/− mice are characterized by non-canonical TSH receptor localization in intracellular compartments, which is accompanied by altered thyroglobulin turnover due to a disbalanced proteolytic network. These finding are of significance considering the rising popularity of using TAAR1 agonists or antagonists as neuromodulating pharmacological drugs. Our study highlights the importance of further

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Circulating Levels of Peroxiredoxin 4 as a Novel Biomarker of Oxidative Stress in Patients with Sepsis

Shock, May 1, 2011

Oxidative stress, a situation with increased reactive oxygen species production and/or decreased ... more Oxidative stress, a situation with increased reactive oxygen species production and/or decreased antioxidant defense mechanisms, is evident in the pathogenesis of sepsis. Peroxiredoxin 4 (Prx4) is a hydrogen peroxide degrading peroxidase recently found circulating in blood of septic patients and potentially reflecting an antioxidant system in imbalance. We studied Prx4 serum levels of 79 consecutively enrolled medical intensive care unit patients. The diagnostic and prognostic performance of Prx4 was compared with other biomarkers, the APACHE II score and the SOFA score. Median Prx4 serum levels gradually increased with disease severity in patients classified on admission as having systemic immune response syndrome (2.

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Identification of Iopanoic Acid as Substrate of Type 1 Deiodinase by a Novel Nonradioactive Iodide-Release Assay

Endocrinology, Mar 20, 2012

Enzymatic 5'-and 5-deiodination are key reactions for local and systemic activation and inactivat... more Enzymatic 5'-and 5-deiodination are key reactions for local and systemic activation and inactivation of iodothyronines and thyronamines. Expression of the three deiodinase (DIO) isoenzymes is regulated by a number of parameters, including thyroid status, genotype, micronutrient availability, and disease-related signaling. In addition, DIO are potential targets of pharmacological as well as environmentally derived substances, which might affect their enzymatic activity (endocrine disruptors). With the classical DIO activity assay, testing depends on the availability of radioactively labeled substrates (e.g. 125 I-rT 3) to monitor the release of radioactive iodide. Recently, liquid chromatography-tandem mass spectrometry was described as an alternative method apparently resolving this limitation. However, it has a high demand in technical equipment and analytical routine and is limited in sample number by considerable measuring time. We therefore combined the classical deiodination assay with an easily accessible photometric method taking advantage of the Sandell-Kolthoff reaction for measuring iodide release. In brief, iodine works as a catalyst within this redox reaction between Ce 4ϩ and As 3ϩ leading to an acceleration of destaining. Furthermore, the protocol was adapted to minimize handling effort and time consumption. Because this method is not dependent on radioactivity, it expands the substrate spectrum of the classical method. Suitability of this assay was tested with tissue samples from animal experiments (hepatic Dio1 activity in hypo-and hyperthyroid mice) and established DIO inhibitors. As a new but not unexpected finding, the alleged inhibitor iopanoic acid turned out to be a DIO substrate. This finding was confirmed by liquid chromatography-tandem mass spectrometry, and its potential clinical impact requires further studies.

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Autoantibodies to the IGF1 receptor in Graves' orbitopathy

The Journal of Clinical Endocrinology and Metabolism, Feb 1, 2013

Context: Graves' disease (GD) is maintained by stimulating antibodies against the TSH receptor. G... more Context: Graves' disease (GD) is maintained by stimulating antibodies against the TSH receptor. Graves' orbitopathy (GO) is the main extrathyroidal manifestation of GD, potentially involving autoimmunity against the IGF1 receptor (IGF1R). Objective: We tested for autoantibodies against the IGF1R (IGF1R-Abs) in sera of GD patients and controls and elucidated their possible implication in the disease. Design: A diagnostic assay for IGF1R-Ab was established with recombinant human IGF1R as autoantigen. Serum samples or purified Ig preparations were analyzed for IGF1R binding and modulation of IGF1 signaling in vitro. A total of 108 consecutive GO patients represented on average by 5.4 separate serum samples per individual along with 92 healthy controls were analyzed. Results: IGF1R-Ab were detected in 10 serum samples from control subjects (11%) and in 60 samples (10%) from the GO patient serum bank. The positive patient samples were derived from 15 individuals yielding an IGF1R-Ab prevalence of 14% in GO. More than three consecutive samples were available from 11 of the 15 positive GO patients spanning an average disease period of 2 years. IGF1R-Ab concentrations were constantly elevated in these patients demonstrating relatively stable IGF1R-Ab expression over time. IGF1R-Ab failed to stimulate IGF1R autophosphorylation but instead inhibited IGF1-induced signaling in hepatocarcinoma HepG2 cells. Similarly, growth of MCF7 breast cancer cells was inhibited by IGF1R-Ab, supporting their classification as IGF1 antagonists. Conclusions: Our data demonstrate the existence of IGF1R-Abs in humans but do not support the hypothesis that the IGF1R-Abs contribute to GO pathogenesis.

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