Sven Schinner

IFN-alpha skews monocytes into CD56+-expressing dendritic cells with potent functional activities in vitro and in vivo.

The antitumor effect of IFN-α is mediated by the activation of CTLs, NK cells, and the generation of highly potent Ag-presenting dendritic cells (IFN-DCs). In this study, we show that IFN-DCs generated in vitro from monocytes express CD56 on their surface, a marker which has been thought to be specific for NK cells. FACS analyses of CD56+ and CD56− IFN-DCs showed a nearly identical pattern for most of the classical DC markers. Importantly, however, only CD56+ IFN-DCs exhibited cytolytic activity up to 24% that could almost completely be blocked (−81%) after coincubation with anti-TRAIL. Intracytoplasmatic cytokine staining revealed that the majority of IFN-DCs independently of their CD56 expression were IFN-γ positive as well. In contrast, CD56+ IFN-DCs showed stronger capacity in stimulating allogenic T cells compared with CD56− IFN-DC. Based on these results, five patients with metastasized medullary thyroid carcinoma were treated for the first time with monocyte-derived tumor Ag-pulsed IFN-DCs. After a long term follow-up (in mean 37 mo) all patients are alive. Immunohistochemical analyses of delayed-type hypersensitivity skin reaction showed a strong infiltration with CD8+ cells. In two patients no substantial change in tumor morphology was detected. Importantly, by analyzing PBMCs, these patients also showed an increase of Ag-specific IFN-γ-secreting T cells. In summary, we here describe for the first time that cytotoxic activity of IFN-DCs is mainly mediated by an IFN-DC subset showing partial phenotypic and functional characteristics of NK cells. These cells represent another mechanism of the antitumor effect induced by IFN-α.

Cortical-chromaffin cell interactions in the adrenal gland.

Adrenal catecholamines and steroids are important regulators of the stress response, immune function, blood pressure, and energy homeostasis. Historically, the two cell populations within the adrenal gland—the steroid-producing adrenocortical cells and the catecholamine-producing chromaffin cells—have been regarded as two independent endocrine systems. Research on adrenal physiology and pathophysiology has therefore largely focused on the individual understanding of each cell type. However, adrenal cortex and medulla appear to be interwoven and show multiple contact zones without separation by connective tissue or interstitial membranes. In vitro studies, animal models, and the analysis of human adrenal pathophysiology have demonstrated critical importance of cortical-chromaffin crosstalk for adrenal function and disease. Thus, chromaffin cells regulate steroid-hormone release by the adrenal cortex and steroids induce catecholamine production in the medulla. Consequently, disorders of the adrenal cortex have been shown to affect chromaffin cell function and vice versa. Mouse models of adrenal cortical dysfunction, such as the targeted disruption of the 21-hydroxylase- or the CRHR1 genes, show alterations in chromaffin cell function, while disruption of tyrosine hydroxylase, a key enzyme in catecholamine synthesis, impairs adrenal cortical function. Accordingly, patients with congenital adrenal hyperplasia (CAH) and Addison’s disease show reduced catecholamine biosynthesis. Immense progress in characterizing the mechanisms of chromaffin-cortical interactions has been achieved in recent years. Here, we summarize the current view on intraadrenal communication with respect to adrenal pathophysiology.

Adipocyte-secreted factors increase osteoblast proliferation and the OPG/RANKL ratio to influence osteoclast formation.

Several studies have reported a positive relationship of the body fat mass and bone density. However, it is not clear whether adipocyte-derived signaling molecules directly act on osteoblasts or osteoclasts. Therefore, we investigated the effect of fat cell-secreted factors on the proliferation and differentiation of preosteoblasts and the molecular mechanisms involved. This stimulation led to an increased proliferation of MC3T3-E1 and primary preosteoblastic cells (2.8-fold and 1.5-fold, respectively; p<0.0001), which could be reduced with inhibitors of protein tyrosine kinases, FGFR1 and PI3K. Concordantly, we found human adipocytes to secrete bFGF and bFGF to mimic the effect of adipocyte-secreted factors. The ratio of OPG/RANKL secretion in primary human preosteoblasts increased 9-fold (mRNA and protein) when stimulated with adipocyte-secreted factors. Moreover, osteoblasts which were prestimulated with adipocyte-secreted factors inhibited the formation of osteoclasts. In conclusion, human adipocytes secrete factors that directly act on preosteoblasts and alter their crosstalk with osteoclasts. These in vitro findings reflect the higher bone mass in obese people and attribute it to effects of adipocyte-secreted factors on bone formation.

Association of serum levels of retinol-binding protein 4 with male sex but not with insulin resistance in obese patients

Background: Retinol-binding protein 4 (RBP4) is secreted by adipocytes and by the liver and modulates insulin sensitivity in animal models. However, controversial data exist regarding the association of serum levels of RBP4 with insulin resistance in humans. Obesity confers a major risk to develop insulin resistance. Aim: Therefore, we investigated RBP4 levels in non-diabetic obese patients and analysed the association with insulin sensitivity and other metabolic markers. Subjects and methods: Glucose tolerance was assessed by oral glucose tolerance tests and 70 normal glucose tolerant patients (36 women, 34 men; body mass index >30 kg/m2) were included in our study. We compared the serum level of RBP4 (measured by ELISA) with clinical features (age, sex, BMI, waist-to-hip-ratio, blood pressure) and laboratory findings (total cholesterol, triglycerides, fasting glucose, 2-hour glucose, fasting insulin, HOMA-IR and HOMA-B). The associations between RBP4 and the above mentioned variables were assessed using multiple linear regression models. Results: The mean age (±SD) of the subjects included was 48.1 (± 12.3) years and the mean BMI 41.6 (± 7.4) kg/m2. We found significantly higher RBP4 levels in men (53.0 ± 20.8 µg/ml) than in women (39.7 ± 12.3 µg/ml) (p = 0.0013). However, age and sex-adjusted multiple linear regression models showed no significant association of serum RBP4 levels with BMI, waist-to-hip-ratio, blood pressure, cholesterol, triglycerides, fasting glucose, 2-hour glucose, insulin resistance (as assessed by HOMA-IR), or insulin secretion (as assessed by HOMA-B). Conclusion: Our data show higher RBP4 levels in obese men than in obese women. However, there was no association of RBP4 levels with insulin resistance or other components of the metabolic syndrome. We conclude that obesity might already be associated with elevated RBP4 levels which then show no additional correlation with metabolic markers.

Pathophysiological aspects of Wnt-signaling in endocrine disease.

Wnt-signaling has recently been identified as a regulator of a number of endocrine functions in health and disease in addition to its original attribution to developmental biology. Wnts are extracellular ligands on frizzled receptors and on lipoprotein receptor-related protein co-receptors. Ligand binding leads eventually to the activation of intracellular signaling cascades; based on the involvement of the transcriptional co-activator beta-catenin it can be distinguished between canonical (i.e. beta-catenin) and non-canonical Wnt-signaling. Recent studies revealed that canonical Wnt-signaling regulates the function of endocrine organs and contributes to a number of endocrine disorders. In this review, we would like to focus on a) recent mechanistic data on Wnts in pancreatic beta-cell function; b) human genetic studies on Wnt signaling in type 2 diabetes mellitus; c) crosstalk between adipocytes and endocrine cells through Wnt-signaling molecules (with a focus on the role of Wnt-signaling in adrenocortical cells).

Evidence of a Combined Cytotoxic Thyroglobulin and Thyroperoxidase Epitope-Specific Cellular Immunity in Hashimoto's Thyroiditis

CONTEXT Hashimotos thyroiditis (HT) is a common autoimmune disease leading to thyroid destruction due to lymphocytic infiltration. Only rare data are available regarding the recognition of specific cellular antigens, e.g. of thyroperoxidase (TPO) and thyroglobulin (Tg). OBJECTIVE The aim of this study was to quantify and characterize TPO- and Tg-epitope-specific CD8-positive T cells of HT patients. DESIGN Six different human leukocyte antigen (HLA)-A2 restricted, TPO- or Tg-specific tetramers were synthesized and used for measuring CD8-positive T cells in HT patients and controls. RESULTS The frequency of peripheral TPO- and Tg-specific CD8-positive T cells was significantly higher in HLA-A2-positive HT patients (2.8 ± 9.5%) compared with HLA-A2-negative HT patients (0.5 ± 0.7%), HLA-A2-positive nonautoimmune goiter patients (0.2 ± 0.4%), and HLA-A2-positive healthy controls (0.1 ± 0.2%). The frequency of Tg-specific T cells (3.0%) was very similar to those of TPO-specific CD8-positive T cells (2.9%). Subgroup analyses revealed a steady increase of the number of epitope-specific CD8-positive T cells from 0.6 ± 1.0% at initial diagnosis up to 9.4 ± 18.3% in patients with long-lasting disease. Analyses of the number of thyroid-infiltrating cells as well as the cytotoxic capacity revealed a similar picture for TPO- and Tg-specific T cells. CONCLUSION We here report for the first time that both antigens, TPO and Tg, are recognized by CD8-positive T cells and are involved in the thyroid destruction process leading to clinical disease manifestation.

MicroRNAs miR-146a1, miR-155_2, and miR-200a1 Are Regulated in Autoimmune Thyroid Diseases

Graves’ disease (GD) and Hashimoto’s thyroiditis (HT) are the most common autoimmune thyroid diseases (AITDs). The exact etiology of the immune response in both is still unknown. MicroRNAs (miRNAs) constitute a recently discovered family of small RNAs that control gene expression. Accumulating data suggest that miRNAs crucially control innate and adaptive immune responses, and implicate some miRNAs as having an important role in the pathophysiology of immunity and autoimmunity (1). To the best of our knowledge, there are currently no data on the involvement of miRNA in AITDs. We have determined the levels of some key immunoregulatory miRNAs in the thyroid glands of AITD patients and healthy controls. We investigated the thyroid gland fine-needle aspiration biopsies of patients with HT and GD. There were 28 unrelated Caucasians who were included in this study. The control groups comprised 9 Caucasians without AITDs; there were 9 patients with GD in the GD, and there were 10 patients with HT in the HT group. Ultrasound-guided fine-needle aspiration biopsies were performed using a 20-gauge needle. Total RNA, including miRNA, was extracted from thyroid tissue samples and reverse transcribed with an miRNeasy Mini Kit (Qiagen GmbH). Semiquantitative TaqMan PCR was performed in an ABI PRISM 7700 Sequence Detector (PE Applied Biosystems) using the miScript SYBR Green PCR Kit (Qiagen GmbH). The specific miScript Primer Assays (Qiagen GmbH) were used for the amplification of 13 miRNAs: miR-34a, miR-143_1, miR-143_1*, miR-146a_1, miR-155*_1, miR-155_2, miR-181a*_1, miR-181a2*, miR-181b1, miR-200a1, miR-200a*2, miR-200a1, and miR-1238. All data were analyzed using the nonparametric Kruskal–Wallis test. Results with p < 0.05 were considered statistically significant. Bonferroni-corrected Mann– Whitney U-tests were used as post hoc tests (see Supplementary Data, available online at www.liebertpub.com/thy). In the thyroid tissue of the GD group, miR-146a1 was significantly decreased in comparison to the control group (mean relative expression 5.17 vs. 8.37, respectively, p = 0.019). In the HT group, miR-155_2 was significantly decreased in comparison to the control group (8.30 vs. 11.20, respectively, p = 0.001), and miR-200a1 was significantly increased (12.02 vs. 8.01, p = 0.016). There were no significant differences between the HT and GD groups (see Supplementary Data) or for the other miRNAs tested. Our pilot study was undertaken to evaluate the influence of candidate miRNAs in AITDs that have been suspected of being involved in immunoregulation based on previously published observations (1–4). We found a significant decrease of miR-146a1 in the thyroid tissue of a patient with GD. miR-146a has been reported to be differentially regulated in other autoimmune diseases. Nakasa et al. demonstrated that miR-146a was highly expressed in rheumatoid arthritis (RA) synovial tissue compared to normal synovial tissue (2). This is in contrast to the decrease of miR-146a in our study, and is consistent with the perception that RA is characterized by a predominance of Th1-cytokines, while GD is characterized by the production of Th2-cytokines in thyroid tissue-emigrating mononuclear cells. Furthermore, we report here that miR200a1 is increased in the thyroid tissue of HT patients, but not in that of patients with GD. This further substantiates the findings by Glinsky, who found in an SNP-guided miRNA map that miR-200, miR-34a, miR-143, and miR-1238 were associated with AITDs in general and GD in particular (4). To date, the potential role of miR-200a1 is not understood and warrants further research. This is the first report that there is a significant decrease of miR-155_2 in the thyroid tissue of patients with HT. This miRNA was previously shown to possess important functions in the mammalian immune system by Thai et al. (3). The other miRNA candidates tested did not significantly differ between AITDs, even though evidence exists for their involvement in immunoregulation. We assume that the reported miRNA variations in thyroid tissue are caused by infiltrating activated lymphocytes, because—according to additionally performed microscopy of fine-needle aspirations—these cells are dominant.

Localization and regulation of pancreatic selenoprotein P

Progressive loss of pancreatic β-cell mass is a crucial feature of type 2 diabetes mellitus. As β-cells express very low amounts of the antioxidant enzymes catalase and glutathione peroxidase (GPx), they appear to be particularly vulnerable to oxidative damage in the pathogenesis of diabetes. Here, we investigated the pancreatic expression pattern and regulation of selenoprotein P (Sepp1), which may serve as an additional antioxidant enzyme inside and outside of cells. Sepp1 was detected in rodent pancreas by immunofluorescence and real-time RT-PCR. Regulation of Sepp1 biosynthesis in INS-1 rat insulinoma cells was investigated by real-time RT-PCR, luciferase gene reporter assay, and immunoblotting. Sepp1 and Gpx1 gene expressions in rat pancreas were 58 and 22% respectively of the liver values. Pancreatic Sepp1 expression was restricted to the endocrine tissue, with Sepp1 being present in the α- and β-cells of mouse islets. In INS-1 insulinoma cells, Sepp1 expression was stimulated by the selenium compound sodium selenate and diminished in the presence of high glucose (16.7 vs 5  mM) concentrations. Sepp1 mRNA stability was also lowered at 16.7  mM glucose. Moreover, Sepp1 mRNA levels were decreased in isolated murine islets cultured in high-glucose (22  mM) medium compared with normal glucose (5.5  mM) medium. Pancreatic Sepp1 expression was elevated upon treatment of mice with the β-cell toxin streptozotocin. This study shows that pancreatic islets express relatively high levels of Sepp1 that may fulfill a function in antioxidant protection of β-cells. Downregulation of Sepp1 expression by high glucose might thus contribute to glucotoxicity in β-cells.

Exendin-4 upregulates the expression of Wnt-4, a novel regulator of pancreatic β-cell proliferation

The Wnt-signaling pathway regulates β-cell functions. It is not known how the expression of endogenous Wnt-signaling molecules is regulated in β-cells. Therefore, we investigated the effect of antidiabetic drugs and glucose on the expression of Wnt-signaling molecules in β-cells. Primary islets were isolated and cultured. The expression of Wnt-signaling molecules (Wnt-4, Wnt-10b, Frizzled-4, LRP5, TCF7L2) and TNFα was analyzed by semiquantitative PCR and Western blotting. Transient transfections were carried out and proliferation assays of INS-1 β-cells performed using [(3)H]thymidine uptake and BrdU ELISA. Insulin secretion was quantified. A knockdown (siRNA) of Wnt-4 in β-cells was carried out. Exendin-4 significantly increased the expression of Wnt-4 in β-cells on the mRNA level (2.8-fold) and the protein level (3-fold) (P < 0.001). The effect was dose dependent, with strongest stimulation at 10 nM, and it was maintained after long-term stimulation over 4 wk. Addition of exd-(9-39), a GLP-1 receptor antagonist, abolished the effect of exendin-4. Treatment with glucose, insulin, or other antidiabetic drugs had no effect on the expression of any of the examined Wnt-signaling molecules. Functionally, Wnt-4 antagonized the activation of canonical Wnt-signaling in β-cells. Wnt-4 had no effect on glucose-stimulated insulin secretion or insulin gene expression. Knocking down Wnt-4 decreased β-cell proliferation to 45% of controls (P < 0.05). In addition, Wnt-4 and exendin-4 treatment decreased the expression of TNFaα mRNA in primary β-cells. These data demonstrate that stimulation with exendin-4 increases the expression of Wnt-4 in β-cells. Wnt-4 modulates canonical Wnt signaling and acts as regulator of β-cell proliferation and inflammatory cytokine release. This suggests a novel mechanism through which GLP-1 can regulate β-cell proliferation.

Dendritic Cell Vaccination with Xenogenic Polypeptide Hormone Induces Tumor Rejection in Neuroendocrine Cancer

Purpose: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice). Experimental Design: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid–modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin. Results: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8+ T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-γ-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8+ cells. Immunohistochemistry revealed strong CD8+ tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (−74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group. Conclusion: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.