[Novel anti-osteoporotic drugs on the horizon]. / Neue Antiosteoporotika am Horizont.

In recent years the great progress in knowledge on bone cell biology has allowed identification of molecular structures that can be targeted with pinpoint precision (druggable targets). Osteoclasts are regulated via the RANK-RANK-ligand (RANKL) signaling pathway and osteoblasts via the Wnt signaling pathway, both of which can be influenced for therapeutic measures. As a result the number of (functional) osteoclasts can be decreased or the genesis of osteoblasts can be increased and bone resorption is inhibited or bone formation is enhanced, respectively. Osteoclasts degrade collagen through cathepsin K and inactivation of this enzyme stabilizes the bone matrix; however, as osteoclasts are still able to maintain a stimulatory cross-talk with osteoblasts, formation of new bone will not be reduced. Parathyroid hormone-related protein plays a role in endochondral ossification and a synthetic analogue of this protein may have potent bone anabolic activity; however, the use of such new and highly efficient therapeutic principles comes with new questions and uncertainties on the sequence of therapies, duration of therapy, long-term side effects, undesired activation of metabolic pathways and effectiveness in comparison to other strategies of fracture prevention.

Control of CYP11B2/CYP11B1 expression ratio and consequences for the zonation of the adrenal cortex.

Access of corticotropin to glucocorticoid synthesis in adrenocortical cells is provided by the expression of the ACTH receptor (MC2R). Activation of the MC2R increases stimulatory G-protein, adenylyl cyclase, and protein kinase A (PKA) activities. Furthermore, PKA phosphorylates transcription factors that have a stimulating effect on glucocorticoid synthesis. Sensitivity of adrenocortical cells to renin/angiotensin-2 is conferred by the expression of the inhibitory G-protein-linked angiotensin-2 type 1 receptor (AT1R) that additionally associates to the phospholipase C-activating G-protein q. The AT1R is connected to the adrenal potassium sensory system and regulates calcium influx as well as phospholipase C-ß (PLC-ß) and thus calmodulin kinase-dependent transcription of steroidogenic enzymes. While AT1R signaling suppresses the influence of corticotropin on the generation of cyclic adenosine monophosphate, the expression of the AT1R and its associated enzyme activities are under the control of glucocorticoids. Thus, dominance of one of the two signaling pathways is dependent on two factors: the extracellular concentration of their ligands and the products of their signaling pathways. These findings are in favor of the hypothesis that the centripetal blood flow through the adrenal gland builds up a glucocorticoid gradient creating a morphogenetic field along which adrenal cortical cells adopt different functional states, leading to the typical zonation of the adrenal cortex.

MicroRNA expressions in PMBCs, CD4+, and CD8+ T-cells from patients suffering from autoimmune Addison's disease.

Autoimmune Addison's disease (AD) is a rare but potentially life threatening disease. The exact etiology of the immune response to the adrenal gland is still unknown. MicroRNAs (miRNAs) critically control gene-expression and play an important role in regulating the immune response. The aim of this study was to determine key immunoregulatory miRNAs influencing autoimmune adrenal insufficiency. For this purpose selected miRNAs were amplified by a semiquantitative SYBR Green PCR from blood mononuclear cells and after purification from CD4+ and CD 8+ cells of 6 patients with autoimmune adrenal insufficiency and 10 healthy controls. In CD4+ T-cells miRNA 181a*_1 (18.02 in AD vs. 11.99 in CG, p=0.0047) is significantly increased whereas miRNA 200a_1 (12.48 in AD vs. 19.40 in CG, p=0.0003) and miRNA 200a_2* (8.59 in AD vs. 17.94 in CG, p=0.0160) are significantly decreased. miRNA 200a_1 (12.37 in AD group vs. 18.12 in control group, p=0.001) and miRNA 200a_2* (10.72 in AD group vs. 17.84 in control group, p=0.022) are also significantly decreased in CD8+ T-cells. This study could show for the first time a significant change of three defined miRNAs in PBMCs, CD4+, and CD8+ T-cells of autoimmune AD patients in vivo. These data may help to better understand the cause of the autoimmune processes leading to autoimmune AD. They extend our very limited knowledge concerning miRNAs in autoimmune Addison's disease.

HLA-A2 phenotype may be protective against Graves' disease but not against Hashimoto's thyroiditis in Caucasians.

Graves' disease (GD) and Hashimoto's thyroiditis (HT) are the most common autoimmune thyroid diseases (AITDs) affecting up to 5% of the general population. In Caucasians HT has a prevalence of up to 4.60% and GD a prevalence of 1-2%. The aim of this study was to investigate the association between HLA-A2 and the AITDs GD and HT among Caucasians. HLA alleles of 33 patients with GD and 75 patients with HT were determined by serological typing. The frequency of HLA A2 was significantly reduced in GD (p=0.033) but not in HT (p=n.s.) as compared to control samples. In individuals positive for HLA-A2 odds ratio for protection from GD was found to be 2.8. This study supports the hypothesis that genetic predisposition to GD is not restricted to MHC class II molecules. The significant negative association between HLA A2 and GD supports the hypothesis that MHC class I genes may be relevant for the protection from GD. In contrast the nonsignificant results for HT indicate that this association may not apply to AITDs in general.

Griseofulvin inhibits the growth of adrenocortical cancer cells in vitro.

Supernumerary centrosomes and aneuploidy are associated with a malignant phenotype of tumor cells. Centrosomal clustering is a mechanism used by cancer cells with supernumerary centrosomes to solve the threatening problem of multipolar spindles. Griseofulvin is an antifungal substance that interferes with the microtubule apparatus and inhibits centrosomal clustering. It has also been demonstrated that griseofulvin inhibits the growth of tumor cells in vitro and in vivo. However, it is not yet known whether treatment with griseofulvin inhibits growth of adrenocortical tumor cells. We studied the viability and antiproliferative effects of griseofulvin on cultured NCI-H295R adrenocortical carcinoma cells using Wst-1-, BrdUrd-, and [³H]-thymidine assays. For the detection of apoptosis we used a caspase 3/7 cleavage assay and light microscopy techniques. We observed that incubation with griseofulvin for 24-48 h leads to a decrease in the viability and proliferation of NCI-H295R cells in a dose-dependent manner. Significant effects could be observed after incubation with griseofulvin as measured by Wst-1-, BrdUrd-, and [³H]dT- uptake assays. Apoptosis of NCI-H295R cells was increased in a dose-dependent manner up to 4.5-fold after incubation with griseofulvin 40 µM for 24 h as shown by caspase 3/7 cleavage assay and light microscopy. With regard to new treatment strategies for adrenocortical cancer, griseofulvin, and possibly other agents, which interfere with the microtubule apparatus and inhibit centrosomal clustering, may turn out to be interesting targets for further research.

Diabetic lipoproteins and adrenal aldosterone synthesis--a possible pathophysiological link?

An increased prevalence of diabetes mellitus (DM) has been reported in patients with primary aldosteronism (PA). DM is associated with abnormal structure and metabolism of circulating lipoproteins, which normally serve as a major source of cholesterol for adrenocortical steroidogenesis. The present study has been designed to investigate the effect of diabetically modified lipoproteins on adrenocortical aldosterone synthesis. Lipoproteins (VLDL, LDL, HDL) isolated from healthy volunteers, were subjected to oxidation or glycoxidation in the presence of sodium hypochlorite (3 mmol/l) or glucose (200 mmol/l), and aldosterone synthesis in human adrenocortical cells (H295R) was examined. Native and glycoxidized VLDL had greatest stimulatory effect on aldosterone production by 15-fold and 14-fold, respectively. At the molecular level, these VLDL produced maximum increases in Cyp11B2 mRNA level up to 17-fold. Experiments with the highly selective scavenger receptor class B type I (SR-BI) inhibitor BLT-1 revealed that cholesterol uptake from native and glycoxidized HDL and VLDL for hormone production is considerably mediated by SR-BI. Western blot analysis of extracellular signal-regulated kinase (ERK 1/2) phosphorylation and experiments with the MEK inhibitor U0126 indicated a specific mechanistic role of the ERK cascade in lipoprotein-mediated steroid hormone release. In summary, diabetic dyslipidemia and modification of circulating lipoproteins may promote adrenocortical aldosterone synthesis.

Comparison of the saline infusion test and the fludrocortisone suppression test for the diagnosis of primary aldosteronism.

For the diagnosis of primary aldosteronism (PA), confirmatory testing is mandatory and different function tests can be employed. There are, however, sparse data comparing the fludrocortisone suppression test (FST) and the saline infusion test (SIT). Patients with PA (n=90) or essential hypertension (n=65) were studied. They underwent one or the other test or both of them. Using the DPC Siemens aldosterone radioimmunoassay, we found that the SIT led to a stronger suppression of aldosterone than the FST. Post-test aldosterone-to-renin ratios (ARRs) and the percentage of suppression of aldosterone serum concentrations performed worse. The same results were observed in patients who underwent both FST and SIT. Some patients had divergent results in both tests. For the SIT, a lower cutoff value should be used than for the FST for the adequate identification of patients with unilateral PA. Long-term prospective studies are needed to address the question at what cutoff values patients benefit from subtype differentiation of PA. We discuss here possible explanations for divergent results obtained with both tests.

Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues.

Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors.

Loss of PTEN expression in neuroendocrine pancreatic tumors.

PTEN (phosphatase and tensin homologue deleted from chromosome 10) is a well established tumor suppressor gene, which was cloned to chromosome 10q23. PTEN plays an important role in controlling cell growth, apoptosis, cell adhesion, and cell migration. In various studies, a genetic change as well as loss of PTEN expression by different carcinomas has been described. To date, the role of PTEN as a differentiation marker for neuroendocrine tumors (NET) and for the loss of PTEN expression is still unknown. It is assumed that loss of PTEN expression is important for tumor progression of NETs. We hypothesize that PTEN might be used as a new prognostic marker. We report 38 patients with a NET of the pancreas. Tumor tissues were surgically resected, fixed in formalin, and embedded in paraffin. PTEN expression was evaluated by immunohistochemistry and was correlated with several clinical and pathological parameters of each individual tumor. After evaluation of our immunohistochemistry data using a modified Remmele Score, a widely accepted method for categorizing staining results for reports and statistical evaluation, staining results of PTEN expression were correlated with the clinical and pathological parameters of each individual tumor. Our data demonstrates a significant difference in survival with existence of lymph node or distant metastases. Negative patients show a significant better survival compared with positive patients. Furthermore, we show a significant difference between PTEN expression and WHO or TNM classification. Taken together, our data shows a positive correlation between WHO classification and the new TNM classification of NETs, and loss of PTEN expression as well as survival. These results strongly implicate that PTEN might be helpful as a new prognostic factor.

[Kallmann syndrome]. / Kallmann-Syndrom.

The Kallmann syndrome is a very rare congenital association of gonadotropin-releasing hormone deficiency and hyposmia or anosmia. Clinically it is characterized by low serum concentrations of testosterone and inadequate low levels of luteinizing hormone and follicle-stimulating hormone as well as incomplete sexual maturation, lack of secondary sexual features (facial and body hair growth, deepening of the voice), micropenis and sometimes even cryptorchidism. The reduced or absent sense of smell is typical for the Kallmann syndrome and distinguishes this syndrome from other causes of hypogonadotropic hypogonadism. Additional findings may include synkinesia, hearing loss, unilateral renal aplasia, brachy- or syndactyly, agenesis of corpus callosum, cleft palate and dental agenesis. A 19-year-old man presented to our male infertility clinic with delayed sexual maturation, eunuchoid habitus, micropenis, cryptorchidism, erectile dysfunction and absence of ejaculation, anemia and osteoporosis as well as low serum concentrations of luteinizing hormone, follicle-stimulating hormone and testosterone in combination with hyposmia.

Reproducibility of Elecsys anti-TSHR test results in a lot-to-lot comparison.

Most recently, a new rapid and fully-automated TSH receptor autoantibody (TRAb) assay has been established. This assay system uses the M22 human monoclonal antibody for competing against the patient's TSH receptor autoantibodies (TRAb) to be detected. The aim of our present study was to compare the reproducibility of TRAb values based on measurements with different TSH receptor preparations in a lot-to-lot comparison. For TRAb values > 2 IU/l the relative differences ranged from -9.0 to +10.0%. The mean difference was 0.28 +/- 8%. For TRAb values around the cutoff for positivity (1.75 IU/l) a higher range of relative differences from -20 up to +15% was obtained. The overall mean of differences was -0.8+/-14%. The data clearly demonstrate that the automated TRAb assay has a high stability in regard to TSH receptor preparations.

The effects of the endothelium on adrenal steroidogenesis and growth are mainly mediated by proteins other than endothelin-1.

The endothelium releases factors stimulating the adrenal cortex. It is also known that endothelin-1 (ET-1) promotes generation of cortisol and aldosterone, and proliferation of adrenocortical cells. The aim of the study was to find out whether the effect of the endothelium on adrenocortical cells is dominated by the action of ET-1. The effects of endothelial cell-conditioned medium (ECCM), obtained during growth of human umbilical cord vein endothelial cells, on aldosterone and cortisol release by cells of the adrenocortical cancer cell-line NCI-H295R and the promoter activity of steroidogenic acute-regulatory protein (StAR) were studied. The effect of ECCM on proliferation of human primary normal adrenocortical and NCI-H295R cells was also investigated. Concentration-dependent increases in cortisol release that reached 192.7 ± 62.8 in percent of basal secretion, in aldosterone release that reached 188.2 ± 52.3 in percent of basal secretion, and in proliferation after stimulation with ECCM at concentrations of 10-50% were found. ECCM significantly activated the StAR promoter 3-fold in NCI-H295R cells if the ECCM was not pretreated with pronase. These effects of the endothelium were not reversed after co-incubation with endothelin receptor antagonists and could not be mimicked by incubation with endothelin-1. In conclusion, the cultured endothelial cells secrete a protein that stimulates steroidogenesis in adrenal cells and their growth. It was also shown that the ET-1 does not mediate the effect of ECCM on the NCI-H295R cell line.

The serum sodium to urinary sodium to (serum potassium)2 to urinary potassium (SUSPPUP) ratio in patients with primary aldosteronism.

BACKGROUND: The aldosterone-to-renin ratio (ARR) is an established diagnostic tool in the screening for primary aldosteronism (PA). However, hormonal determinations are time consuming and expensive. Therefore, we studied the effectiveness of the serum sodium to urinary sodium to (serum potassium)(2) to urinary potassium (SUSPPUP) ratio in the diagnosis of PA. DESIGN: This study included 35 patients with PA, 71 patients with essential hypertension to whom this diagnosis could be excluded, 23 normal subjects without hypertension, and 22 patients with primary adrenal insufficiency. We compared the SUSPPUP ratios with the ARR in these patient groups. RESULTS: We show that the ARR distinguished PA from essential hypertension with a sensitivity of 94.2% and a specificity of 92.1% at a cutoff of 33 (ng L(-1): ng L(-1)). It correlated well with the SUSPPUP ratio. The sensitivity and specificity of SUSPPUP was 88.6% and 85.9% at a cutoff of 5.3 (mmol L(-1))(-1), respectively, and thus not as good as the ARR. CONCLUSIONS: The ARR is a good parameter in the screening for PA. The SUSPPUP ratio is a cheap and rapid tool to assess the extent of mineralocorticoid excess and, therefore, can be offered to more patients. In addition, the application of the SUSPPUP ratio can be extended to patients who suffer from other forms of mineralocorticoid hypertension (e.g. with low aldosterone levels).

Role of the novel mTOR inhibitor RAD001 (everolimus) in anaplastic thyroid cancer.

Activation of the phosphatidylinositol-3-kinase (PI3K) signaling cascade is increasingly recognized as a common feature of thyroid follicular neoplasms. Among the PI3K downstream effectors, the main kinase, directly responsible for the increased cell growth and proliferation, is called mammalian target of rapamycin (mTOR). This central kinase might be directly inhibited via rapamycin and its derivatives. The aim of the present study was to examine whether RAD001 (everolimus) can selectively suppress the proliferation of different anaplastic thyroid cancer (ATC) cells. Five different human ATC cell lines were exposed to different concentrations of RAD001. Importantly, we found a dose-dependent growth inhibition in two ATC cell lines at concentrations of 43.5 and 94.5 nM although not as intensive as within the RAD001 responding K562cell line. The other cell lines revealed a GI (50) between 168 to 234 nM. In parallel, quantitative PCR of PCNA displayed a reduced expression of PCNA within the responding cell lines, respectively. In summary, we found a good responding effect in a part of ATC cell lines, which may have a clinical impact.

Nestin as a marker in the classification of adrenocortical tumors.

Expression of the intermediate filament, nestin, was long believed to be restricted to neuroectodermal stem cells. However, nestin expression has recently been detected in several tumors. Since adrenocortical carcinoma, a tumor entity still very difficult to classify, may gain the ability to aberrantly express neuroectodermal proteins including chromogranin A and synaptophysin, we asked the question whether nestin might also be detected in adrenocortical carcinomas, and if so, whether it might serve as a tool for clinical pathology. Therefore, we studied the expression of nestin in normal adrenal glands, adrenocortical adenomas, and adrenocortical cancers using specific immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Immunostaining was nestin-positive in 1 out of 9 normal adrenal glands (11%), 2 out of 20 adrenocortical adenomas (10%), and 13 out of 16 adrenocortical carcinomas (81%). Expression of nestin mRNA could be detected in all microdissected tissues, independently of their grade of dedifferentiation. We conclude that our findings provide further evidence that nestin, as a marker, is not restricted to neuronal stem cells and nestin expression is worth to be studied in adrenocortical tumors.

Adrenal cortical tumors and multiple endocrine neoplasia-related syndromes.

Relatively frequent, adrenal masses include a multitude of different tumor types: uni- or bilateral hyperplasias, adenomas, and the rare entity of adrenocortical carcinomas. With significant progress in our appreciation of their underlying molecular pathomechanisms and from analysis of affected individuals and their families, a number of inherited diseases and tumor syndromes have been linked to adrenocortical tumorigenesis. These syndromes and diseases include the Carney complex, the McCune-Albright syndrome, multiple endocrine neoplasia type 1, familial adenomatosis coli, congenital adrenal hyperplasia, familial forms of primary aldosteronism, the Beckwith-Wiedemann syndrome, and the Li-Fraumeni syndrome. The key to successful management of these syndromes is identification of patients harboring adrenal tumors within the context of hereditary diseases, since diagnostic procedures, therapy and follow-up may significantly differ from the management of sporadic, isolated adrenal tumors. This review explores the underlying genetic defects, diagnosis and therapy of the major heritable tumor syndromes associated with adrenocortical tumorigenesis.

Standard-radical vs. function-preserving surgery of benign nodular goiter: a sonographic and biochemical 10-year follow-up study.

BACKGROUND: The necessary extent of thyroid resection in benign nodular goiter is under debate. The aim of our study was to compare the long-term outcome of different thyroid resection modes with special interest in the incidence of recurrent nodules and the use of oral thyroid hormone medication. MATERIALS AND METHODS: We performed a follow-up examination of 109 patients (23 men and 86 women) having been operated for benign nodular goiter at our department 10 years ago. Unilateral resections and function-preserving resections of at least one thyroid lobe were classified as function-preserving (FP). Total thyroidectomy, Dunhill's operation and bilateral subtotal thyroidectomy were rated as standard-radical (STR). On follow-up, we recorded current oral thyroid hormone medication, thyroid function tests and ultrasound of the neck. RESULTS: Seventy-three patients had FP resection (67%), while 36 were STR-operated (33%). The subsequent medical treatment was performed by dedicated endocrinologists (n = 19), internists (n = 11) or primary-care physicians (n = 59). Twenty patients had no medical attendance. Recurrent nodules were found in 13 cases in the FP group (18.6%) vs. 3 cases in the STR group (2.5%; p < 0.001). In both groups, about 80% of patients used thyroid hormone medication 10 years after operation. CONCLUSION: There was no advantage in thyroid function tests nor lesser medication in the FP group. The risk for recurrent nodules was significantly higher in the FP than in the STR-operated patients.

Association of impaired glucose metabolism in morbid obesity with hypoadiponectinaemia.

BACKGROUND: Increased circulating levels of cytokines and chemokines and decreased adiponectin levels are associated with impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). As obesity is the major risk factor for T2DM it is not clear why many patients with morbid obesity remain normoglycaemic and if this protection can be attributed to a lower grade of inflammation or higher adiponectin levels. MATERIALS AND METHODS: Glucose tolerance of morbidly obese patients (n=2 754, body mass index > or =40 kg/m2) was assessed by oral glucose tolerance tests. In a case-control design we compared levels of eight immune mediators and adiponectin from patients with IGT/T2DM (n=52) and normal glucose tolerance (NGT; n=59). Gene expression in peripheral blood was determined by quantitative RT-PCR, and serum concentrations of immune mediators and adiponectin were measured by ELISA and bead-based multiplex technology. RESULTS: About 54% of the patients in our morbidly obese cohort were normoglycaemic, while 14% were diagnosed with IGT and 32% with T2DM. There was no statistically significant difference in mRNA expression or serum levels of proinflammatory markers. Interestingly, we could demonstrate an association of NGT with higher adiponectin levels (p=0.039). Adiponectin levels were negatively correlated with interleukin (IL)-6 and macrophage chemoattractant protein (MCP)-1, but independent the other immune mediators. CONCLUSIONS: We found an association of lower adiponectin levels with IGT/T2DM, but no further increase in inflammatory markers in morbid obesity. This suggests that in addition to chronic, low-grade inflammation, adiponectin is an important factor in the development of, or protection against, T2DM in obesity.