Relacorilant or surgery improved hemostatic markers in Cushing syndrome.

PURPOSE: Glucocorticoid-mediated hypercoagulability can persist in patients with endogenous Cushing syndrome (CS) after curative surgery and may transiently worsen early postoperatively. These studies aimed to characterize coagulation markers at baseline in patients with CS and the impact of relacorilant or remission post-surgery in an open-label, phase 2 study (NCT02804750) and a retrospective, longitudinal, surgical cohort study. METHODS: In the relacorilant study, 34 patients received relacorilant (100-200 mg/day for up to 12 weeks or 250-400 mg/day for up to 16 weeks) and had postbaseline data. Coagulation markers were assessed before and during treatment. In the surgical study, conducted at "Federico II" University of Naples, Italy, coagulation markers were assessed in 30 patients before surgery and after biochemical remission. RESULTS: In the relacorilant study, significant mean changes from baseline to last observed visit were reported in factor VIII (- 18.9%, P = 0.022), activated partial thromboplastin time (aPTT) (+ 1.5 s, P = 0.046), and platelet count (- 68.8*109/L, P < 0.0001), whereas von Willebrand factor was unchanged. In the surgical study, the mean time to hemostasis assessment was 6.2 months. Significant mean changes from baseline to hemostasis assessment were reported in factor VIII (- 24.2%, P = 0.044), von Willebrand factor (- 20.6%, P = 0.018), and aPTT (+ 2.0 s, P = 0.031), whereas platelet count was unchanged. CONCLUSIONS: Several coagulation markers improved in patients with CS after 3-4 months of relacorilant treatment and within an average of 6 months after surgery. Relacorilant's positive effects on coagulation markers support further investigation of its use preoperatively in patients with CS or in patients who are not eligible for surgery. CLINICAL TRIAL REGISTRATION NUMBER: NCT0280475 (registration date: 15 June 2016).

Autoimmune polyendocrine syndrome type 1: an Italian survey on 158 patients.

BACKGROUND: Autoimmune Polyglandular Syndrome type 1 (APS-1) is a rare recessive inherited disease, caused by AutoImmune Regulator (AIRE) gene mutations and characterized by three major manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CH) and Addison's disease (AD). METHODS: Autoimmune conditions and associated autoantibodies (Abs) were analyzed in 158 Italian patients (103 females and 55 males; F/M 1.9/1) at the onset and during a follow-up of 23.7 ± 15.1 years. AIRE mutations were determined. RESULTS: The prevalence of APS-1 was 2.6 cases/million (range 0.5-17 in different regions). At the onset 93% of patients presented with one or more components of the classical triad and 7% with other components. At the end of follow-up, 86.1% had CH, 77.2% AD, 74.7% CMC, 49.5% premature menopause, 29.7% autoimmune intestinal dysfunction, 27.8% autoimmune thyroid diseases, 25.9% autoimmune gastritis/pernicious anemia, 25.3% ectodermal dystrophy, 24% alopecia, 21.5% autoimmune hepatitis, 17% vitiligo, 13.3% cholelithiasis, 5.7% connective diseases, 4.4% asplenia, 2.5% celiac disease and 13.9% cancer. Overall, 991 diseases (6.3 diseases/patient) were found. Interferon-ω Abs (IFNωAbs) were positive in 91.1% of patients. Overall mortality was 14.6%. The AIRE mutation R139X was found in 21.3% of tested alleles, R257X in 11.8%, W78R in 11.4%, C322fsX372 in 8.8%, T16M in 6.2%, R203X in 4%, and A21V in 2.9%. Less frequent mutations were present in 12.9%, very rare in 9.6% while no mutations in 11% of the cases. CONCLUSIONS: In Italy, APS-1 is a rare disorder presenting with the three major manifestations and associated with different AIRE gene mutations. IFNωAbs are markers of APS-1 and other organ-specific autoantibodies are markers of clinical, subclinical or potential autoimmune conditions.

Treatment of skeletal impairment in patients with endogenous hypercortisolism: when and how?

Guidelines for the management of osteoporosis induced by endogenous hypercortisolism are not available. Both the American College of Rheumatology and the International Osteoporosis Foundation recommend to modulate the treatment of exogenous glucocorticoid-induced osteoporosis (GIO) based on the individual fracture risk profile (calculated by FRAX) and dose of glucocorticoid used, but it is difficult to translate corticosteroid dosages to different degrees of endogenous hypercortisolism, and there are no data on validation of FRAX stratification method in patients with endogenous hypercortisolism. Consequently, it is unclear whether such recommendations may be adapted to patients with endogenous hypercortisolism. Moreover, patients with exogenous GIO take glucocorticoids since suffering a disease that commonly affects bone. On the other hand, the correction of coexistent risk factors, which may contribute to increase the fracture risk in patients exposed to glucocorticoid excess, and the removal of the cause of endogenous hypercortisolism, may lead to the recovery of bone health. Although the correction of hypercortisolism and of possible coexistent risk factors is necessary to favor the normalization of bone turnover with recovery of bone mass; in some patients, the fracture risk could not be normalized and specific anti-osteoporotic drugs should be given. Who, when, and how the patient with endogenous hypercortisolism should be treated with bone-active therapy is discussed.

Proteomic profiles in hyperandrogenic syndromes.

BACKGROUND: Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. METHODS: Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18-35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. RESULTS: Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. CONCLUSION: Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

Modulation of proteomic profile in H295R adrenocortical cell line induced by mitotane.

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chloro-phenyl) ethane (o,p'-DDD), is a compound that represents the effective agent in the treatment of the adrenocortical carcinoma (ACC), able to block cortisol synthesis. In this type of cancer, the biological mechanism induced by this treatment remains still unknown. In this study, we have already shown a greater impairment in the first steps of the steroidogenesis and recognized a little effect on cell cycle. We also evaluated the variation of proteomic profile of the H295R ACC cell line, either in total cell extract or in mitochondria-enriched fraction after treatment with mitotane. In total cell extracts, triose phosphate isomerase, alpha-enolase, D-3-phosphoglycerate dehydrogenase, peroxiredoxin II and VI, heat shock protein 27, prohibitin, histidine triad nucleotide-binding protein, and profilin-1 showed a different expression. In the mitochondrial fraction, the following proteins appeared to be down regulated: aldolase A, peroxiredoxin I, heterogenous nuclear ribonucleoprotein A2/B1, tubulin-beta isoform II, heat shock cognate 71 kDa protein, and nucleotide diphosphate kinase, whereas adrenodoxin reductase, cathepsin D, and heat shock 70 kDa protein 1A were positively up-regulated. This study represents the first proteomic study on the mitotane effects on ACC. It permits to identify some protein classes affected by the drug involved in energetic metabolism, stress response, cytoskeleton structure, and tumorigenesis.

Mitotane increases the radiotherapy inhibitory effect and induces G2-arrest in combined treatment on both H295R and SW13 adrenocortical cell lines.

Mitotane, 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane (o,p'-DDD) is an agent with adrenotoxic effect, which is able to block cortisol synthesis. This drug and radiotherapy are used also in adrenal cancer treatment even if their biological action in this neoplasia remains unknown. We investigated the effects of o,p'-DDD and ionizing radiations (IR) on cell growth inhibition and cell cycle perturbation in H295R and SW13 adrenocortical cancer cells. Both cell lines were irradiated at a 6 Gy dose and were treated with o,p'-DDD 10(-5) M separately and with IR/o,p'-DDD in combination. This combination treatment induced an irreversible inhibition of cell growth in both adrenocortical cancer cells. Cell cycle analysis showed that IR alone and IR/o,p'-DDD in combination induced the cell accumulation in the G2 phase. At 120 h after IR, the cells were able to recover the IR-induced G2 block while cells treated with IR/o,p'-DDD were still arrested in G2 phase. In order to study the molecular mechanism involved in the G2 irreversible arrest, we have considered the H295R cell line showing the highest inhibition of cell proliferation associated with a noteworthy G2 arrest. In these cells, cyclin B1 and Cdk2 proteins were examined by western blot and Cdk2 kinase activity measured by assay kit. The H295R cells treated with IR/o,p'-DDD shared an increase in cyclin B1 amount as the coimmunoprecipitation of Cdc2-cyclin B1 complex. The kinase activity also shows an increase in the treated cells with combination therapy. Moreover, in these cells, sequence analysis of p53 revealed a large deletion of exons 8 and 9. The same irreversible block on G2 phase, induced by IR/o,p'-DDD treatment, happened in H295R cells with restored wild-type p53 suggesting that this mechanism is not mediated by p53 pathway.

Proteomic analysis of peripheral T lymphocytes, suitable circulating biosensors of strictly related diseases.

T lymphocytes and/or their subpopulations from peripheral blood may represent molecular sensors to be used for the evaluation of gene expression modification in physiological and pathological conditions, providing a unique and easily available biological model for integrated studies of gene expression in humans. In this study, a proteomic approach was applied to evaluate the association between changes in T cell protein expression patterns and specific diseased conditions. In particular, two hyperandrogenic syndromes were studied, sharing many clinical and biochemical signs: polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH). Comparison of proteomic maps of T lymphocytes derived from patients affected by PCOS or CAH with those derived from healthy subjects showed that 14 proteins are expressed differentially in both PCOS and CAH, 15 exclusively in PCOS and 35 exclusively in CAH. Seventeen of these proteins have been identified by mass spectrometry analysis. Furthermore, proteomic data mining by hierarchical clustering was performed, highlighting T lymphocytes competence as a living biosensor system.

Increased metastatic lymph node 64 and CYP17 expression are associated with high stage prostate cancer.

The metastatic lymph node 64 (MLN64), which is localized in the human chromosome 17, encodes a protein with strong homology with steroidogenic acute regulatory protein. Its overexpression in human breast carcinomas and MLNs led to the hypothesis that this protein could be involved in intraneoplastic steroidogenesis. In the present study, we investigated the expression of MLN64 in prostate cancer, another hormone-dependent tumor, and compared its expression with that of CYP17, the gene encoding for the key enzyme of androgen synthesis. We investigated by RT-PCR the expression of MLN64 and CYP17 in 60 prostatic tumors and compared their expression with the stage of disease and the appearance of relapses in a follow-up of 24 months. We found MLN64 and CYP17 expressed in all samples examined, with significantly higher expression in neoplastic tissues with respect to normal tissues (NTs). Moreover, only in neoplastic but not in NTs, a positive linear correlation was found between MLN64 and CYP17 gene expression. MLN64 and CYP17 expression seems to correlate with high stage, high Gleason score and short relapse-free time. These data, for the first time, demonstrate the presence of MLN64 and CYP17 expression in both normal and neoplastic prostatic tissues. The biological role of MLN64 in human prostate and, particularly, in neoplastic tissue is still unclear. Our findings concerning MLN64 and CYP17 gene expression and their significant positive correlation in human prostate cancer may suggest their possible role in intraneoplastic autonomous steroidogenesis.

A multicenter experience on the prevalence of ARMC5 mutations in patients with primary bilateral macronodular adrenal hyperplasia: from genetic characterization to clinical phenotype.

ARMC5 mutations have recently been identified as a common genetic cause of primary bilateral macronodular adrenal hyperplasia (PBMAH). We aimed to assess the prevalence of ARMC5 germline mutations and correlate genotype with phenotype in a large cohort of PBMAH patients. A multicenter study was performed, collecting patients from different endocrinology units in Italy. Seventy-one PBMAH patients were screened for small mutations and large rearrangements in the ARMC5 gene: 53 were cortisol-secreting (two with a family history of adrenal hyperplasia) and 18 were non-secreting cases of PBMAH. Non-mutated and mutated patients' clinical phenotypes were compared and related to the type of mutation. A likely causative germline ARMC5 mutation was only identified in cortisol-secreting PBMAH patients (one with a family history of adrenal hyperplasia and ten apparently sporadic cases). Screening in eight first-degree relatives of three index cases revealed four carriers of an ARMC5 mutation. Evidence of a second hit at somatic level was identified in five nodules. Mutated patients had higher cortisol levels (p = 0.062), and more severe hypertension and diabetes (p < 0.05). Adrenal glands were significantly larger, with a multinodular phenotype, in the mutant group (p < 0.01). No correlation emerged between type of mutation and clinical parameters. ARMC5 mutations are frequent in cortisol-secreting PBMAH and seem to be associated with a particular pattern of the adrenal masses. Their identification may have implications for the clinical care of PBMAH cases and their relatives.

Regional variations of insulin-like growth factor I (IGF-I), IGF-II, and receptor type I in benign prostatic hyperplasia tissue and their correlation with intraprostatic androgens.

Benign prostatic hyperplasia (BPH) is an androgen-dependent disease; it originates exclusively in the inner prostate, which includes tissue surrounding the urethra. Stromal-epithelial interaction has a pivotal role in the regulation of the development and growth of the prostate, and locally produced peptide growth factors are considered important mediators of this interaction. Insulin-like growth factor I (IGF-I) and IGF-II, acting mainly through type 1 IGF receptor (IGFR1), have mitogenic and antiapoptotic effects on epithelial and stromal prostatic cells. In this study the expression of IGF-I, IGF-II, and IGFR1 messenger ribonucleic acid (mRNA), the immunoreactive content of IGF-I (irIGF-I) and IGF-II (irIGF-II) were determined in periurethral, intermediate, and subcapsular regions of BPH tissue to verify their possible regional variation; a correlation to the tissue levels of dihydrotestosterone (DHT) and 3 alpha-androstanediol (3 alpha Diol) was also determined to verify their possible androgen dependence. Prostates were removed by suprapubic prostatectomy from 14 BPH patients and sectioned in the periurethral, intermediate, and subcapsular regions. Gene expression of IGF-I, IGF-II, and IGFR1 was evaluated by semiquantitative RT-PCR, using beta-actin as a control. irIGF-I was measured by RIA, and irIGF-II was measured by IRMA after acidification and chromatography on Sep-Pak C(18) cartridges. DHT and 3 alpha Diol concentrations were evaluated by RIA after extraction and purification on Celite microcolumns. IGF-II and IGFR1, but not IGF-I, mRNA was higher in the periurethral than in the intermediate (P < 0.05) and subcapsular (P < 0.01) region. Also, prostatic levels of irIGF-II, expressed as picomoles per g tissue, were higher in the periurethral (20.84 +/- 1.84) than in the intermediate (14.81 +/- 2.11; P < 0.05) and subcapsular (10.88 +/- 1.21; P < 0.001) region. No significant differences were found in irIGF-I content. Considering prostatic androgen levels, DHT and 3alphaDiol presented a regional variation, with the highest concentrations in the periurethral region. IGF-II mRNA and irIGF-II levels were positively correlated with both DHT and 3 alpha Diol content. These results demonstrate that in BPH tissue a greater IGF-II activity is present in the periurethral region, the site of origin of BPH. Moreover, we can hypothesize that the tissue androgen content may modulate prostatic production of IGF-II, acting at the transcriptional and probably the posttranscriptional level. Therefore, even though further studies will need to confirm this hypothesis, DHT may increase IGF-II activity, mainly in the periurethral region, which, in turn, induces, through IGFR1, benign proliferation of both epithelial and stromal cells, characteristic of BPH.

[Molecular analysis of the gene of steroidogenic acute regulatory protein (StAR) in adrenal incidentaloma]. / Analisi molecolare del gene della proteina di regolazione acuta della steroidogenesi (StAR) negli incidentalomi surrenalici.

The steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating cholesterol entry into the inner compartment of mitochondria. Mutations (either transitions or transversions) of StAR gene have been described as a cause of lethal forms of congenital lipoid adrenal hyperplasia. Adrenal incidentalomas are frequently discovered during radiologic examinations performed in patients with diagnosis for other diagnostic problems. Enzymatic defects along the steroidogenetic cascade are observed with high frequency in these patients. Aim of the present study was to asses the involvement of alteration of the StAR gene in incidentally discovered adrenal masses (incidentalomas). The mutational analysis of 32 incidentalomas demonstrated a "missense" mutation in exon five of the gene in one of the tumors analysed. This is the first evidence of an alteration of the StAR gene in adrenal incidentalomas.

AME position statement on adrenal incidentaloma.

OBJECTIVE: To assess currently available evidence on adrenal incidentaloma and provide recommendations for clinical practice. DESIGN: A panel of experts (appointed by the Italian Association of Clinical Endocrinologists (AME)) appraised the methodological quality of the relevant studies, summarized their results, and discussed the evidence reports to find consensus. RADIOLOGICAL ASSESSMENT: Unenhanced computed tomography (CT) is recommended as the initial test with the use of an attenuation value of ≤10 Hounsfield units (HU) to differentiate between adenomas and non-adenomas. For tumors with a higher baseline attenuation value, we suggest considering delayed contrast-enhanced CT studies. Positron emission tomography (PET) or PET/CT should be considered when CT is inconclusive, whereas fine needle aspiration biopsy may be used only in selected cases suspicious of metastases (after biochemical exclusion of pheochromocytoma). HORMONAL ASSESSMENT: Pheochromocytoma and excessive overt cortisol should be ruled out in all patients, whereas primary aldosteronism has to be considered in hypertensive and/or hypokalemic patients. The 1 mg overnight dexamethasone suppression test is the test recommended for screening of subclinical Cushing's syndrome (SCS) with a threshold at 138 nmol/l for considering this condition. A value of 50 nmol/l virtually excludes SCS with an area of uncertainty between 50 and 138 nmol/l. MANAGEMENT: Surgery is recommended for masses with suspicious radiological aspects and masses causing overt catecholamine or steroid excess. Data are insufficient to make firm recommendations for or against surgery in patients with SCS. However, adrenalectomy may be considered when an adequate medical therapy does not reach the treatment goals of associated diseases potentially linked to hypercortisolism.

Myc down-regulation affects cyclin D1/cdk4 activity and induces apoptosis via Smac/Diablo pathway in an astrocytoma cell line.

OBJECTIVES: We investigated the antiproliferative effect of Myc down-regulation via cell proliferation inhibition, cell cycle perturbation and apoptosis in two human astrocytoma models (T98G and ADF) steadily expressing an inducible c-myc Anti-sense RNA. MATERIALS AND METHODS: Cell growth experiments were performed using the trypan blue dye exclusion test and cell cycle analysis was evaluated by flow cytometry. Cell cycle molecules were detected by Western blot analysis, co-immunoprecipitation and reverse transcription-polymerase chain reaction assays. RESULTS: We showed that Myc down-regulation in astrocytoma cells led to G1 accumulation and an inhibition of cell proliferation characterized by S phase delay. Co-immunoprecipitation experiments detected formation of inactive cyclin D1/cdk4 complexes as evaluated by presence of an active unphosphorylated form of retinoblastoma protein, the best characterized target substrate for cyclin D1/cdk4 complex, in ADF pINDc-myc anti-sense 7 cells. We also found that either p57Kip2 "apice" or p27Kip1 "apice" inhibitors bound to cyclin D1/cdk4 complex, thus, suggesting that they cooperated to inhibit the activity of cyclin D1/cdk4. Moreover, c-Myc down-regulation led to activation of the apoptotic mitochondrial pathway, characterized by release of cytochrome c and Smac/Diablo proteins and by reduction of c-IAP levels through activation of proteasome-mediated protein degradation system. CONCLUSIONS: Our results suggest that c-Myc could be considered as a good target for the study of new approaches in anticancer astrocytoma treatment.

Human immunodeficiency virus type 1 tat protein modulates fibronectin expression in thymic epithelial cells and impairs in vitro thymocyte development.

Infection of both lymphoid and stromal components of the thymus by human immunodeficiency virus type 1 (HIV-1) suggests that impairment of lymphocyte differentiation from early T cells progenitors in the thymus may contribute to the HIV-induced T cell depletion. Cross-talk between immature thymocyte and thymic epithelium through cell-to-cell adhesion mediated by fibronectin/receptor interaction plays a central role in driving T cell development. HIV-1 tat protein, like fibronectin, contains an RGD sequence involved in the interaction with fibronectin receptor. We demonstrated that gene transfer-mediated tat expression in thymic stroma is able to influence the in vitro maturation of T cell progenitors as tat-expressing epithelial cells have a decreased ability to drive the generation of CD4+8+ thymocytes from CD4-8- precursors. Furthermore, tat-expressing cells produce more fibronectin and display upregulation of VLA-5 cell surface receptor levels compared to control cells, while alpha v expression was unchanged. Cellular distribution of fibronectin is also influenced by tat. Fibronectin is distributed in the whole cell surface and along cell processes in control cells whereas it is mainly concentrated in the intracytoplasmic area in tat-expressing cells. Therefore, expression of tat in thymic epithelial cells impairs thymocyte maturation and modulates fibronectin expression: this suggests a crucial role of this viral protein in regulating the T lymphocyte differentiation program through modulation of intrathymic lympho-stromal interactions.

Expression of trKB neurotrophin receptor during T cell development. Role of brain derived neurotrophic factor in immature thymocyte survival.

The relationships between the nervous and the immune systems raise the question of whether neurotrophic factors, in addition to the regulation of neural cell ontogeny, may influence lymphocyte development. We report in this work that the pattern of neurotrophin receptor expression depends on the developmental stage of T cells. The presence of nerve growth factor receptor trkA could not be detected in any of the thymocyte subsets, whereas brain-derived neurotrophic factor (BDNF) receptor trkB was expressed in all thymocyte subpopulations. Interestingly, both trkB mRNA and protein expression inversely correlated with the maturation stage and the differentiation potential of thymocytes, being more expressed in CD4-8- immature thymocytes and progressively declining in CD8+ and CD4+ single-positive and CD4+8+ more mature thymocytes. The developmentally regulated expression of trkB is further shown by the inhibition or enhancement of trkB expression induced by signals that either trigger or impair the transition from immature to more differentiated stages of the thymocyte developmental pathway. Signals generated following the interaction of BDNF with trkB receptor resulted in the stimulation of trkB autophosphorylation and in the up-regulation of the expression of the c-fos gene in CD4-8- cells and enhanced thymocyte survival. Finally, BDNF is expressed in thymic stroma and is further up-regulated by signals generated by the thymocyte/stromal cell interaction. These data suggest that BDNF may be a novel survival factor for thymocyte precursors and support the presence of developmentally regulated feedback mechanisms based on autocrine/paracrine neurotrophin/receptor interactions that may be involved in the thymocyte differentiation process.