Membrane-bound estrogen receptor alpha initiated signaling is dynamin dependent in breast cancer cells.

BACKGROUND: Although membrane-associated estrogen receptors (mERs) have been known to play important role in steroid-induced signal transmission, we still know little about their function in the estrogen-induced proliferation of breast cancer cells. METHODS: In our current work we tried to separate membrane-initiated estrogen receptor signaling from the overall estrogenic effect in MCF-7 breast carcinoma cells. Re-analyzing expression data from multiple microarray experiments, we selected a set of key regulatory genes involved in proliferation regulation and estrogen signaling to monitor estrogen-induced transcription changes. We then compared these expression changes after 17ß-estradiol and a membrane receptor selective estrogen-BSA treatment using quantitative real-time PCR. In order to follow receptor trafficking we used light and electron microscopy. RESULTS: Our quantitative real-time PCR results confirmed that the selective membrane receptor agonist, estrogen-BSA induces similarly pronounced expression changes regarding these genes as 17ß-estradiol. Morphological study revealed that the membrane-bound form of classical estrogen receptor alpha is internalized after ligand binding via dynamin-dependent, caveola-mediated endocytosis. Inhibition of this internalization with dynamin inhibitor, dynasore practically abolished the regulatory effect of E2-BSA, suggesting that interaction and internalization with the scaffold protein is necessary for effective signaling. CONCLUSIONS: The physiological role of plasma membrane estrogen receptor alpha is intensively studied, yet there are still several aspects of it to be resolved. The dynamin-dependent, ligand-mediated internalization of mERs seems to play an important role in estrogen signaling. Our results may serve as another example of how membrane initiated estrogen signaling and nuclear receptor initiated signaling overlap and form an intertwined system.

An unexpected, mild phenotype of glucocorticoid resistance associated with glucocorticoid receptor gene mutation case report and review of the literature.

BACKGROUND: Glucocorticoid resistance is a rare, sporadic or familial condition caused by mutation of the gene encoding the glucocorticoid receptor (GR). Clinically it is characterized by symptoms developed due to local, tissue-specific, or generalized partial insensitivity to glucocorticoids. CASE PRESENTATION: A 31-year-old woman was evaluated because of infertility at the Endocrine Unit of the 2nd Department of Medicine, Semmelweis University. During her laboratory investigations, elevated serum and salivary cortisol were observed which failed to be suppressed after administration of 1 mg dexamethasone. 24 h urinary cortisol was increased, but a normal midnight serum cortisol was detected suggesting a maintained circadian rhythm. Plasma dehydroepiandrosterone-sulfate and androstendione levels were also elevated. Repeated plasma ACTH measurements indicated slightly elevated or normal values. Bone mineral density was normal. All laboratory results confirmed the diagnosis of glucocorticoid resistance. Genetic counseling followed by Sanger sequencing of the coding region of the gene encoding human glucocorticoid receptor was performed and a missense mutation (Arg714Gln, R714Q) in a heterozygous form was detected. Following family screening, the same mutation was found in her clinically-healthy 35-year-old sister who had no fertility problems.This variant was not detected in more than 60 patients and controls tested either for glucocorticoid resistance or Cushing's syndrome in our Laboratory and it was absent in Exome Variant Server, HumanGene Mutation Database and ExAC databases. CONCLUSIONS: Our case fulfils the diagnostic criteria of glucocorticoid resistance, also named Chrousos syndrome. The glucocorticoid receptor gene mutation detected in our patient has been already reported in a 2-year-old child with hypoglycaemia, hypokalaemia, hypertension and premature puberty. These distinct phenotypes may suggest that other factors may modify the functional consequences of the R714Q variant of GR.

[Diagnostic and therapeutical significance of macro-TSH in patients with Hashimoto's thyroiditis]. / A makro-TSH diagnosztikus és terápiás jelentosége Hashimoto-thyreoiditises betegekben.

INTRODUCTION: Structure, importance and incidence and clinical role of macro-TSH not clarified in thyroid diseases. AIM: This study was undertaken to determine the incidence and biological role of macro-TSH in patients with Hashimoto's thyroiditis. METHOD: Blood samples taken from patients with Hashimoto's thyroiditis were screened for the presence of macro-TSH with the polyethylene glycol method and confirmed with protein G agarose absorption test and gel filtration chromatography. Stimulatory capacity of macro-TSH was measured by CHO cells bio-assay. Patients were treated with L-thyroxine (mean 66.5 µg/day) and half of them with selenium (mean 60 µg/day), respectively. RESULTS: 880 patients (728 female, aged 44.8 yr) with Hashimoto's thyroiditis was involved in the study. Macro-TSH was found in the serum of 41 patients (4.6%), the mean TSH 185.4 ± 35 IU/l was before PEG precipitations and after 5.55 ± 1.8 IU/l. Titre of anti-TPO proved to be 445 ± 51 IU/l and gradulally decreased to 212 ± 51 IU/l after one year therapy. Both the precipitation, protein G absorption and gel chromatography supported the presence of anti-TSH antibody in the macro-TSH complex. Stimulatory capacity of macro-TSH on CHO bio-assay was not proved. The macro-TSH was detected in the selenium not treated group for 18 ± 3.2 months, selenium-treated for 12 ± 1.9 months. CONCLUSION: It is concluded that anti-human TSH autoantibodies are a major components of macro-TSH and may cause diagnostic and therapeutical difficulties. The PEG precipitation is a suitable screening method for detection of macro-TSH. Selenium is able to decrease of anti-TPO antibodies and macro-TSH, respectively. When the TSH level is greater than 40.0 IU/l, without the signs of hypothyroidism, the presence of macro-TSH is to be considered. Orv Hetil. 2017; 158(34): 1346-1350.

MEN1 mutations and potentially MEN1-targeting miRNAs are responsible for menin deficiency in sporadic and MEN1 syndrome-associated primary hyperparathyroidism.

Inherited, germline mutations of menin-coding MEN1 gene cause multiple endocrine neoplasia type 1 (MEN1), while somatic MEN1 mutations are the sole main driver mutations in sporadic primary hyperparathyroidism (PHPT), suggesting that menin deficiency has a central role in the pathogenesis of PHPT. MiRNAs are small, noncoding RNAs posttranscriptionally regulating gene expression. Our aim was to investigate both the role of MEN1 mutations and potentially MEN1-targeting miRNAs as the underlying cause of menin deficiency in MEN1-associated and sporadic PHPT tissues. Fifty six PHPT tissues, including 16 MEN1-associated tissues, were evaluated. Diagnosis of MEN1 syndrome was based on identification of germline MEN1 mutations. In silico target prediction was used to identify miRNAs potentially targeting MEN1. Menin expression was determined by immunohistochemistry while expression of miRNAs was analyzed by quantitative real-time PCR. Sporadic PHPT tissues were subjected to somatic MEN1 mutation analysis as well. Lack of nuclear menin was identified in all MEN1-associated and in 28% of sporadic PHPT tissues. Somatic MEN1 mutations were found in 25% of sporadic PHPTs. The sensitivity and specificity of menin immunohistochemistry to detect a MEN1 mutation were 86 and 87%, respectively. Expression levels of hsa-miR-24 and hsa-miR-28 were higher in sporadic compared to MEN1-associated PHPT tissues; however, no difference in miRNA levels occurred between menin-positive and menin-negative PHPT tissues. Menin deficiency is the consequence of a MEN1 mutation in most menin-negative PHPT tissues. Elevated expression of hsa-miR-24 and hsa-miR-28 mark the first epigenetic changes observed between sporadic and MEN1-associated PHPT.

Clinical Characterization of the Pheochromocytoma and Paraganglioma Susceptibility Genes SDHA, TMEM127, MAX, and SDHAF2 for Gene-Informed Prevention.

IMPORTANCE: Effective cancer prevention is based on accurate molecular diagnosis and results of genetic family screening, genotype-informed risk assessment, and tailored strategies for early diagnosis. The expanding etiology for hereditary pheochromocytomas and paragangliomas has recently included SDHA, TMEM127, MAX, and SDHAF2 as susceptibility genes. Clinical management guidelines for patients with germline mutations in these 4 newly included genes are lacking. OBJECTIVE: To study the clinical spectra and age-related penetrance of individuals with mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes. DESIGN, SETTING, AND PATIENTS: This study analyzed the prospective, longitudinally followed up European-American-Asian Pheochromocytoma-Paraganglioma Registry for prevalence of SDHA, TMEM127, MAX, and SDHAF2 germline mutation carriers from 1993 to 2016. Genetic predictive testing and clinical investigation by imaging from neck to pelvis was offered to mutation-positive registrants and their relatives to clinically characterize the pheochromocytoma/paraganglioma diseases associated with mutations of the 4 new genes. MAIN OUTCOMES AND MEASURES: Prevalence and spectra of germline mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes were assessed. The clinical features of SDHA, TMEM127, MAX, and SDHAF2 disease were characterized. RESULTS: Of 972 unrelated registrants without mutations in the classic pheochromocytoma- and paraganglioma-associated genes (632 female [65.0%] and 340 male [35.0%]; age range, 8-80; mean [SD] age, 41.0 [13.3] years), 58 (6.0%) carried germline mutations of interest, including 29 SDHA, 20 TMEM127, 8 MAX, and 1 SDHAF2. Fifty-three of 58 patients (91%) had familial, multiple, extra-adrenal, and/or malignant tumors and/or were younger than 40 years. Newly uncovered are 7 of 63 (11%) malignant pheochromocytomas and paragangliomas in SDHA and TMEM127 disease. SDHA disease occurred as early as 8 years of age. Extra-adrenal tumors occurred in 28 mutation carriers (48%) and in 23 of 29 SDHA mutation carriers (79%), particularly with head and neck paraganglioma. MAX disease occurred almost exclusively in the adrenal glands with frequently bilateral tumors. Penetrance in the largest subset, SDHA carriers, was 39% at 40 years of age and is statistically different in index patients (45%) vs mutation-carrying relatives (13%; P < .001). CONCLUSIONS AND RELEVANCE: The SDHA, TMEM127, MAX, and SDHAF2 genes may contribute to hereditary pheochromocytoma and paraganglioma. Genetic testing is recommended in patients at clinically high risk if the classic genes are mutation negative. Gene-specific prevention and/or early detection requires regular, systematic whole-body investigation.

A unique haplotype of RCCX copy number variation: from the clinics of congenital adrenal hyperplasia to evolutionary genetics.

There is a difficulty in the molecular diagnosis of congenital adrenal hyperplasia (CAH) due to the c.955C>T (p.(Q319*), formerly Q318X, rs7755898) variant of the CYP21A2 gene. Therefore, a systematic assessment of the genetic and evolutionary relationships between c.955C>T, CYP21A2 haplotypes and the RCCX copy number variation (CNV) structures, which harbor CYP21A2, was performed. In total, 389 unrelated Hungarian individuals with European ancestry (164 healthy subjects, 125 patients with non-functioning adrenal incidentaloma and 100 patients with classical CAH) as well as 34 adrenocortical tumor specimens were studied using a set of experimental and bioinformatic methods. A unique, moderately frequent (2%) haplotypic RCCX CNV structure with three repeated segments, abbreviated to LBSASB, harboring a CYP21A2 with a c.955C>T variant in the 3'-segment, and a second CYP21A2 with a specific c.*12C>T (rs150697472) variant in the middle segment occurred in all c.955C>T carriers with normal steroid levels. The second CYP21A2 was free of CAH-causing mutations and produced mRNA in the adrenal gland, confirming its functionality and ability to rescue the carriers from CAH. Neither LBSASB nor c.*12C>T occurred in classical CAH patients. However, CAH-causing CYP21A2 haplotypes with c.955C>T could be derived from the 3'-segment of LBSASB after the loss of functional CYP21A2 from the middle segment. The c.*12C>T indicated a functional CYP21A2 and could distinguish between non-pathogenic and pathogenic genomic contexts of the c.955C>T variant in the studied European population. Therefore, c.*12C>T may be suitable as a marker to avoid this genetic confound and improve the diagnosis of CAH.

Systematic Investigation of Expression of G2/M Transition Genes Reveals CDC25 Alteration in Nonfunctioning Pituitary Adenomas.

Dysregulation of G1/S checkpoint of cell cycle has been reported in pituitary adenomas. In addition, our previous finding showing that deregulation of Wee1 kinase by microRNAs together with other studies demonstrating alteration of G2/M transition in nonfunctioning pituitary adenomas (NFPAs) suggest that G2/M transition may also be important in pituitary tumorigenesis. To systematically study the expression of members of the G2/M transition in NFPAs and to investigate potential microRNA (miRNA) involvement. Totally, 80 NFPA and 14 normal pituitary (NP) tissues were examined. Expression of 46 genes encoding members of the G2/M transition was profiled on 34 NFPA and 10 NP samples on TaqMan Low Density Array. Expression of CDC25A and two miRNAs targeting CDC25A were validated by individual quantitative real time PCR using TaqMan assays. Protein expression of CDC25A, CDC25C, CDK1 and phospho-CDK1 (Tyr-15) was investigated on tissue microarray and immunohistochemistry. Several genes' expression alteration were observed in NFPA compared to normal tissues by transcription profiling. On protein level CDC25A and both the total and the phospho-CDK1 were overexpressed in adenoma tissues. CDC25A correlated with nuclear localized CDK1 (nCDK1) and with tumor size and nCDK1 with Ki-67 index. Comparing primary vs. recurrent adenomas we found that Ki-67 proliferation index was higher and phospho-CDK1 (inactive form) was downregulated in recurrent tumors compared to primary adenomas. Investigating the potential causes behind CDC25A overexpression we could not find copy number variation at the coding region nor expression alteration of CDC25A regulating transcription factors however CDC25A targeting miRNAs were downregulated in NFPA and negatively correlated with CDC25A expression. Our results suggest that among alterations of G2/M transition of the cell cycle, overexpression of the CDK1 and CDC25A may have a role in the pathogenesis of the NFPA and that CDC25A is potentially regulated by miRNAs.

Modulation of the circadian clock by glucocorticoid receptor isoforms in the H295R cell line.

Peripheral clocks are set by different nervous, hormonal and metabolic stimuli, and regulate the circadian expression of several genes. We investigated whether a peripheral clock could be induced in the human adrenocortical cell line H295R and whether glucocorticoid receptor isoforms (GRα and GRß) are involved in this clock system. After synchronization of cells with serum shock, the rhythmic oscillation of clock genes PER1, PER2, REV-ERBα, and ARNTL was confirmed. In addition, H295R cells even without serum shock showed rhythmic expression of PER1, PER2, CRY1 and ARNTL. Glucocorticoid treatment induced a rapid response of PER1, PER2 and CRY1 in a GRα-dependent manner. Continuous glucocorticoid stimulation after 6h caused suppression of REV-ERBα. Administration of a GR antagonist, RU486, disrupted the circadian oscillation of clock genes and prevented the acute changes in PER1, PER2 and CRY1 levels. Overexpression of the GRß isoform alone did not alter the expression of the examined clock genes, but did prevent the GRα-related suppression of REV-ERBα. These alterations occurred independently from ACTH and CRH. Our data demonstrate that a peripheral clock system is present in a human adrenocortical cell line and that periodic oscillations of clock genes are influenced by glucocorticoids, mainly through GRα.

Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer.

Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.

Fluorescence activated cell sorting followed by small RNA sequencing reveals stable microRNA expression during cell cycle progression.

BACKGROUND: Previously, drug-based synchronization procedures were used for characterizing the cell cycle dependent transcriptional program. However, these synchronization methods result in growth imbalance and alteration of the cell cycle machinery. DNA content-based fluorescence activated cell sorting (FACS) is able to sort the different cell cycle phases without perturbing the cell cycle. MiRNAs are key transcriptional regulators of the cell cycle, however, their expression dynamics during cell cycle has not been explored. METHODS: Following an optimized FACS, a complex initiative of high throughput platforms (microarray, Taqman Low Density Array, small RNA sequencing) were performed to study gene and miRNA expression profiles of cell cycle sorted human cells originating from different tissues. Validation of high throughput data was performed using quantitative real time PCR. Protein expression was detected by Western blot. Complex statistics and pathway analysis were also applied. RESULTS: Beyond confirming the previously described cell cycle transcriptional program, cell cycle dependently expressed genes showed a higher expression independently from the cell cycle phase and a lower amplitude of dynamic changes in cancer cells as compared to untransformed fibroblasts. Contrary to mRNA changes, miRNA expression was stable throughout the cell cycle. CONCLUSIONS: Cell cycle sorting is a synchronization-free method for the proper analysis of cell cycle dynamics. Altered dynamic expression of universal cell cycle genes in cancer cells reflects the transformed cell cycle machinery. Stable miRNA expression during cell cycle progression may suggest that dynamical miRNA-dependent regulation may be of less importance in short term regulations during the cell cycle.

Novel SDHB and TMEM127 Mutations in Patients with Pheochromocytoma/Paraganglioma Syndrome.

Pheochromocytomas (Pheo) and paragangliomas (PGL) are rare tumors, with heterogeneous genetic background. In up to 30 % of all, apparently sporadic Pheo/PGL cases germline mutations can be identified in one of the 15 genes representing genetic susceptibility for Pheo/PGL. Malignancy is rare but it frequently associates with SDHB mutations. Our aim was to determine the prevalence of germline SDHx, SDHAF2, MAX and TMEM127 mutations in Hungarian patients with apparently sporadic Pheo/PGLs. Mutation screening of the SDHx, SDHAF2, MAX and TMEM127 genes was performed in 82 Hungarian patients with apparently sporadic Pheo/PGL using PCR and bidirectional Sanger sequencing. Disease-causing germline mutations were identified in 11 patients, of which 4 SDHB and 2 TMEM127 mutations were novel. Earlier development of Pheo/PGL, more malignant phenotype and multiple tumors were observed in genetically positive cases especially in those with SDHB mutations. The presence of bilateral or multiple tumors was the most predictive for identification of a pathogenic mutation. Together with cases harboring germline RET, VHL and NF1 mutations, Hungarian patients with Pheo/PGL exhibit a heterogeneous mutation spectrum, indicating that all of the Pheo/PGL susceptibility genes should be tested. Novel genotype-phenotype associations revealed by our study may contribute to improvement of diagnostic approaches and may help to achieve a better clinical follow up for patients with Pheo/PGL.

Polymorphisms of the GR and HSD11B1 genes influence body mass index and weight gain during hormone replacement treatment in patients with Addison's disease.

OBJECTIVE: Glucocorticoid substitution is essential in patients with chronic primary adrenocortical insufficiency (Addison's disease) and both over-treatment and inadequate dosage have deleterious effects. Individual sensitivity to glucocorticoids is partly genetically determined. CONTEXT: To test the hypothesis whether the well-characterized SNPs of the GR and HSD11B1 genes may modulate the individual sensitivity to exogenous glucocorticoids and may influence clinical and/or laboratory parameters and the glucocorticoid substitution dosage in patients with Addison's disease. PATIENTS AND METHODS: 68 patients with primary adrenocortical insufficiency were involved. Clinical and laboratory data, as well as the dosage of the hormone replacement therapy were collected. Peripheral blood DNA was isolated, and the GR and HSD11B1 SNPs were examined using allele-specific PCR or Taqman assay on Real Time PCR. RESULTS: The allele frequency of the GR N363S polymorphism was higher in patients compared to the control group and the disease appeared significantly earlier in patients harbouring the GR A3669G compared to noncarriers. These patients had higher ACTH level measured at the time of diagnosis. Homozygous BclI carriers had higher body mass index (BMI) and lower total hydrocortisone equivalent supplementation dose needed than heterozygous or noncarriers. The BMI and weight gain during hormone replacement therapy were also higher in carriers of the HSD11B1 rs4844880 treated with glucocorticoids other than dexamethasone. CONCLUSION: The BclI polymorphism of the GR gene and the rs4844880 of the HSD11B1 gene may contribute to weight gain and may affect the individual need of glucocorticoid substitution dose in these patients.

Overexpression of GRß in colonic mucosal cell line partly reflects altered gene expression in colonic mucosa of patients with inflammatory bowel disease.

The glucocorticoid receptor (GR) plays a crucial role in inflammatory responses. GR has several isoforms, of which the most deeply studied are the GRα and GRß. Recently it has been suggested that in addition to its negative dominant effect on GRα, the GRß may have a GRα-independent transcriptional activity. The GRß isoform was found to be frequently overexpressed in various autoimmune diseases, including inflammatory bowel disease (IBD). In this study, we wished to test whether the gene expression profile found in a GRß overexpressing intestinal cell line (Caco-2GRß) might mimic the gene expression alterations found in patients with IBD. Whole genome microarray analysis was performed in both normal and GRß overexpressing Caco-2 cell lines with and without dexamethasone treatment. IBD-related genes were identified from a meta-analysis of 245 microarrays available in online microarray deposits performed on intestinal mucosa samples from patients with IBD and healthy individuals. The differentially expressed genes were further studied using in silico pathway analysis. Overexpression of GRß altered a large proportion of genes that were not regulated by dexamethasone suggesting that GRß may have a GRα-independent role in the regulation of gene expression. About 10% of genes differentially expressed in colonic mucosa samples from IBD patients compared to normal subjects were also detected in Caco-2 GRß intestinal cell line. Common genes are involved in cell adhesion and cell proliferation. Overexpression of GRß in intestinal cells may affect appropriate mucosal repair and intact barrier function. The proposed novel role of GRß in intestinal epithelium warrants further studies.

[Novel methods and their applicability in the evaluation of the genetic background of endocrine system tumours]. / Új módszertani lehetoségek és ezek alkalmazása a hormonális rendszer daganatainak genetikai kivizsgálásában.

The technical developments leading to revolution in clinical genetic testing offer new approaches for patients with cancer. From one mutation or one gene approach the scale of genetic testing moved to whole exome or whole genome scale. It is well known that many tumours are genetically determined and they are part of familial tumour syndromes. In addition, some mutations indicate specific molecular targeted therapies. Although sampling and sample preparation are different for testing germline and somatic mutations, the technical background of the analysis is the same. The aim of clinical genetic testing is to identify patients who are carriers of disease-causing mutations or to test tumour tissue for the presence of genetic alterations which may be targets for therapeutic approaches. In this review the authors summarize novel possibilities offered by next-generation sequencing in clinical genetic testing of patients with endocrine tumours. In addition, the authors review recent guidelines on technical and ethical issues related to these novel methods.

Analysis of Circulating MicroRNAs In Vivo following Administration of Dexamethasone and Adrenocorticotropin.

Purpose. The interaction of hormones of the pituitary-adrenal axis and adrenal cortex-associated circulating microRNAs is mostly unknown. We have studied the effects of dexamethasone and adrenocorticotropin on the expression of five circulating microRNAs (hsa-miR-27a, hsa-miR-200b, hsa-miR-214, hsa-miR-483-5p, and hsa-miR-503) reported to be related to the adrenal cortex in plasma samples. Methods. Expression of microRNAs was studied in plasma samples of 10 individuals examined by 1 mg dexamethasone suppression test and another 10 individuals stimulated by 250 µg tetracosactide (adrenocorticotropin). Total RNA was isolated and microRNA expression was analyzed by real-time reverse transcription quantitative polymerase chain reaction normalized to cel-miR-39 as reference. Results. Only circulating hsa-miR-27a proved to be significantly modulated in vivo by hormonal treatments: its expression was upregulated by dexamethasone whereas it was suppressed by adrenocorticotropin. Secreted hsa-miR-27a was significantly induced by dexamethasone in vitro in NCI-H295R cells, as well. The expression of hsa-miR-483-5p proposed as diagnostic marker for adrenocortical malignancy was not affected by dexamethasone or tetracosactide administration. Conclusions. hsa-miR-27a expression is modulated by hormones of the hypothalamic-pituitary-adrenal axis both in vitro and in vivo. The biological relevance of hsa-miR-27a modulation by hormones is unclear, but the responsiveness of circulating microRNAs to hormones of the pituitary-adrenal axis is noteworthy.

[Analysis of laboratory data of 155 patients with pheochromocytoma-paraganglioma syndrome diagnosed during the past 20 years]. / A laboratóriumi diagnosztika eredményei az elmúlt 20 évben kórismézett 155 phaeochromocytoma/paraganglioma szindrómás beteg adatainak elemzése alapján.

INTRODUCTION: Laboratory diagnosis of pheochromocytoma-paraganglioma syndrome has been markedly improved during the past two decades. AIM: Retrospective assessment of diagnostic utility of urinary catecholamines and their metabolites as well as serum chromogranin A in 155 patients diagnosed at the 2nd Department of Medicine, Semmelweis University. METHOD: Urinary catecholamines and metabolites were measured using high-performance liquid chromatography with electrochemical detection in 155 patients with pheochromocytoma-paraganglioma (of whom 28.4% had hereditary background) and in 170 non-pheochromocytoma patients used as controls. Serum chromogranin A was measured by immunoradiometry. RESULTS: Sensitivity (93.2%) and specificity (87.0%) of urinary fractionated metanephrines were higher than those of urinary catecholamines (90.9% vs. 85.7%, respectively) and serum chromogranin A (88.7% and 77.5%, respectively). Urinary normetanephrine and serum chromogranin A correlated positively with tumor size (r = 0.552, p<0.0001 and r = 0.618, p<0.0001, respectively). CONCLUSIONS: These data confirm the diagnostic utility of urinary catecholamines and their metabolites. Urinary normetanephrine and serum chromogranin A may help to estimate tumour mass and probably tumour progression.

Safety and efficacy of oral octreotide in acromegaly: results of a multicenter phase III trial.

BACKGROUND: A novel oral octreotide formulation was tested for efficacy and safety in a phase III, multicenter, open-label, dose-titration, baseline-controlled study in patients with acromegaly. METHODS: We enrolled 155 complete or partially controlled patients (IGF-1 <1.3 × upper limit of normal [ULN], and 2-h integrated GH <2.5 ng/mL) receiving injectable somatostatin receptor ligand (SRL) for ≥ 3 months. Subjects were switched to 40 mg/d oral octreotide capsules (OOCs), and the dose escalated to 60 and then up to 80 mg/d to control IGF-1. Subsequent fixed doses were maintained for a 7-month core treatment, followed by a voluntary 6-month extension. RESULTS: Of 151 evaluable subjects initiating OOCs, 65% maintained response and achieved the primary endpoint of IGF-1 <1.3 × ULN and mean integrated GH <2.5 ng/mL at the end of the core treatment period and 62% at the end of treatment (up to 13 mo). The effect was durable, and 85 % of subjects initially controlled on OOCs maintained this response up to 13 months. When controlled on OOCs, GH levels were reduced compared to baseline, and acromegaly-related symptoms improved. Of 102 subjects completing the core treatment, 86% elected to enroll in the 6-month extension. Twenty-six subjects who were considered treatment failures (IGF-1 ≥ 1.3 × ULN) terminated early, and 23 withdrew for adverse events, consistent with those known for octreotide or disease related. CONCLUSIONS: OOC, an oral therapeutic peptide, achieves efficacy in controlling IGF-1 and GH after switching from injectable SRLs for up to 13 months, with a safety profile consistent with approved SRLs. OOC appears to be effective and safe as an acromegaly monotherapy.

Cord serum dipeptidyl-peptidase 4 activity in gestational diabetes.

BACKGROUND: Tissue-specific dipeptidyl-peptidase 4 (DPP4) dysregulation has been described in adults with diabetes mellitus. The DPP4 -incretin system has not been studied in foetal life. In this study, DPP4 activity and glucagon-like peptide-1 (GLP-1) levels were assessed in cord blood of neonates born to women with gestational diabetes mellitus (GDM) and nondiabetic controls. MATERIAL AND METHODS: This study has been conducted in two Hungarian and one Austrian centres. PATIENTS: A total of 568 pregnant women were enrolled in the study after their OGTT between the 24th and 28th gestational week. Cord blood samplings with DPP4 activity and GLP-1 level measurements were possible in 270 (DPP4: 159 control, 111 GDM) and 112 (GLP-1: 72 control, 40 GDM) cases. OGTT (24-28th gestational week) and cord blood sampling at delivery were performed. Cord serum DPP4 activity was determined in a continuous monitoring microplate-based kinetic assay, and cord plasma GLP-1 was measured using a fluorescence ELISA method. RESULTS: Cord serum DPP4 activity was lower in GDM [mean (95% CI): 28.07 U/L (26.32-29.82 U/L)] than in controls [31.61 U/L (29.93-33.29 U/L), MWU P = 0.0015]. Cord plasma active GLP-1 levels were close to the lower detection limit and were not altered in GDM (control: mean = 3.43 pM, 95% CI: 3.04-3.82 pM, GDM: mean = 3.61 pM, 95% CI: 2.96-4.28 pM - MWU test P = 0.6). CONCLUSIONS: Decreased cord serum DPP4 activity in gestational diabetes mellitus might be the result of an adaptive foetal response or an early dysregulation in the entero-insular axis with consequences beyond the incretin system. Cord plasma GLP-1 levels may reflect the missing oral intake with a limited glucose sensing of L cells via the circulation in foetal life.

Antioxidant effect of the active metabolites of tibolone.

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10(-7), 10(-8), 10(-9 )M) of either estradiol, tibolone, 3α-hydroxytibolone, 3ß-hydroxytibolone, Δ(4)-tibolone, 3α-sulfated-tibolone, 3α-17ß-disulfated-tibolone, 3ß-sulfated-tibolone or 3ß-17ß-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine-xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10(-9 )M of steroids. Using 10(-8 )M concentration: estradiol (80.9 ± 2.5%); 3ß-sulfated-tibolone (83.3 ± 4.7%); 3ß-17ß-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10(-7 )M, 3ß-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine-xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17ß-disulfated-tibolone (71.9 ± 2.5%), 3ß-sulfated-tibolone (73.9 ± 5.0%), and 3ß-17ß-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

MicroRNAs in adrenal tumors: relevance for pathogenesis, diagnosis, and therapy.

Several lines of evidence support the relevance of microRNAs in both adrenocortical and adrenomedullary (pheochromocytomas) tumors. Significantly differentially expressed microRNAs have been described among benign and malignant adrenocortical tumors and different forms of pheochromocytomas that might affect different pathogenic pathways. MicroRNAs can be exploited as markers of malignancy or disease recurrence. Besides tissue microRNAs, novel data show that microRNAs are released in body fluids, and blood-borne microRNAs can be envisaged as minimally invasive markers of malignancy or prognosis. MicroRNAs might even serve as treatment targets that could expand the rather-limited therapeutic repertoire in the field of adrenal tumors. In this review, we present a critical synopsis of the recent observations made in the field of adrenal tumor-associated microRNAs regarding their pathogenic, diagnostic, and potential therapeutic relevance.