No evidence for oncogenic mutations in guanine nucleotide-binding proteins of human adrenocortical neoplasms

G-Proteins are membrane-bound heterotrimeric polypeptides that couple receptor signals to second messenger systems such as cAMP. Recently, point mutations at 2 codons of the highly preserved alpha-chain of Gs, the adenyl cyclase-stimulating G-protein, were found in GH-secreting pituitary tumors. These mutations resulted in constitutively activated Gs alpha and high intracellular cAMP levels. In addition, point mutations at similar codons of a different G-protein, G(i) alpha 2, were reported in adrenocortical neoplasms, suggesting a potential role of this isoform in the genesis of these tumors. We reevaluated the frequency of constitutively activating point mutations in the alpha- chain of the stimulatory (Gs alpha) and inhibitory (G(i) alpha 2) G- proteins in human adrenocortical tumors. Seven adrenocortical carcinomas, 2 human adrenocortical tumor cell lines, and 11 adrenocortical adenomas were studied. Genomic DNA was purified from either frozen tumor tissue or paraffin-embedded sections. Using specific primers and the polymerase chain reaction, DNA fragments surrounding codons 201 and 227 (Gs alpha) and 179 and 205 (G(i) alpha 2) were amplified and visualized on a 2% agarose gel. In a second asymmetric polymerase chain reaction, using nested primers, single stranded DNA was generated using 1-10 microL of the initial amplification mixture and directly sequenced using the dideoxy chain termination method of Sanger. We found no mutations at codons 201, 227 and 179, 205 of Gs alpha and G(i) alpha 2, respectively, in the tumors studied. We conclude that previously identified oncogenic point mutations in the stimulatory and inhibitory alpha-chain of G-proteins do not appear to be present at high frequency in adrenal neoplasms. Thus, the mechanism(s) of tumorigenesis in these tumors is different from that in GH-secreting adenomas and may involve oncogenic mutations of other cell constituents

P53 mutations in human adrenocortical neoplasms

The mechanisms of tumorigenesis of adrenocortical neoplasms have not been elucidated as yet. However, loss of heterozygosity at chromosomal locus 17p has been consistently observed in adrenocortical cancer. p53 is a recessive tumor suppressor gene located on chromosome 17p. Mutations in the p53 gene play an important role in the tumorigenesis of diverse types of human neoplasms including breast and colon cancers. More than 90% of all mutations discovered in such tumors have been detected in 4 hot spot areas that lie between exons 5 and 8. In contrast to wild-type p53, mutant p53 accumulates intracellularly and can be easily detected by immunohistochemistry. We therefore investigated the frequency of p53 mutations in human adrenocortical neoplasms using molecular biology and immunohistochemistry techniques. Five patients with adrenocortical adenomas (5 female; ages 39-72 yr), 11 patients with adrenocortical carcinomas (8 female, 3 male; ages 15- 50 yr), and two adrenocortical tumor cell lines were studied. After DNA extraction from frozen tumor tissue or paraffin-embedded material, exons 5 through 8 were amplified using the polymerase chain reaction and directly sequenced by the dideoxy termination method. Immunohistochemistry was performed on paraffin-embedded tumor specimens obtained during adrenalectomy using a monoclonal antibody reacting with both wild-type and mutant p53. Prevalence of mutations was adenomas, 0/5, carcinomas, 3/11, and adrenocortical cell lines, 2/2. Single point mutations were detected in 3 cases (exons 5, 6, and 7, respectively), and rearrangements of exon 7/8 and 8 were found in 2 cases. Immunohistochemistry detected strong nuclear and/or cytoplasmic p53 immunoreactivity in all adrenocortical carcinomas with point mutations of the p53 gene but not in adenomas and carcinomas with the wild-type sequence or with deletion/rearrangement of the p53 gene. We conclude that p53 plays a role in the tumorigenesis of adrenocortical carcinomas but is of less importance to benign adenomas

Clonal Composition of Human Adrenocortical Neoplasms

The mechanisms of tumorigenesis of adrenocortical neoplasms are still not understood. Tumor formation may be the result of spontaneous transformation of adrenocortical cells by somatic mutations. Another factor stimulating adrenocortical cell growth and potentially associated with formation of adrenal adenomas and, less frequently, carcinomas is the chronic elevation of proopiomelanocortin-derived peptides in diseases like ACTH-dependent Cushing's syndrome and congenital adrenal hyperplasia. To further investigate the pathogenesis of adrenocortical neoplasms, we studied the clonal composition of such tumors using X-chromosome inactivation analysis of the highly polymorphic region Xcen-Xp11.4 with the hybridization probe M27ß, which maps to a variable number of tandem repeats on the X-chromsome. In addition, polymerase chain reaction amplification of a phosphoglycerokinase gene polymorphism was performed. After DNA extraction from tumorous adrenal tissue and normal leukocytes in parallel, the active X-chromosome of each sample was digested with the methylation-sensitive restriction enzyme HpaII. A second digestion with an appropriate restriction enzyme revealed the polymorphism of the region Xcen-Xp11.4 and the phosphoglycerokinase locus. Whereas in normal polyclonal tissue both the paternal and maternal alleles are detected, a monoclonal tumor shows only one of the parental alleles. A total of 21 female patients with adrenal lesions were analyzed; 17 turned out to be heterozygous for at least one of the loci. Our results were as follows: diffuse (n = 4) and nodular (n = 1) adrenal hyperplasia in patients with ACTH-dependent Cushing's syndrome, polyclonal pattern; adrenocortical adenomas (n = 8), monoclonal (n = 7), as well as polyclonal (n = 1); adrenal carcinomas (n = 3), monoclonal pattern. One metastasis of an adrenocortical carcinoma showed a pattern most likely due to tumor-associated loss of methylation. In the special case of a patient with bilateral ACTH-independent macronodular hyperplasia, diffuse hyperplastic areas and a small nodule showed a polyclonal pattern, whereas a large nodule was monoclonal. We conclude that most adrenal adenomas and carcinomas are monoclonal, whereas diffuse and nodular adrenal hyperplasias are polyclonal. The clonal composition of ACTH-independent massive macronodular hyperplasia seems to be heterogeneous, consisting of polyclonal and monoclonal areas

Parent-offspring transmission of adipocytokine levels and their associations with metabolic traits

Adipose tissue secreted cytokines (adipocytokines) have significant effects on the physiology and pathology of human metabolism relevant to diabetes and cardiovascular disease. We determined the relationship of the pattern of these circulating hormones with obesity-related phenotypes and whether such pattern is transmitted from parent to offspring. A combined total of 403 individuals from 156 consenting Saudi families divided into initial (119 families with 123 adults and 131 children) and replication (37 families with 58 adults and 91 children) cohorts were randomly selected from the RIYADH Cohort study. Anthropometrics were evaluated and metabolic measures such as fasting serum glucose, lipid profiles, insulin, leptin, adiponectin, resistin, tumor necrosis factor alpha (TNFa), activated plasminogen activator inhibitor 1 (aPAI1), high sensitivity C-reactive protein (hsCRP) and angiotensin II were also assessed. Parent-offspring regressions revealed that with the exception of hsCRP, all hormones measured showed evidence for significant inheritance. Principal component (PC) analysis of standardized hormone levels demonstrated surprising heritability of the three most common axes of variation. PC1, which explained 21% of the variation, was most strongly loaded on levels of leptin, TNFa, insulin, and aPAI1, and inversely with adiponectin. It was significantly associated with body mass index (BMI) and phenotypically stronger in children, and showed a heritability of ,50%, after adjustment for age, gender and generational effects. We conclude that adipocytokines are highly heritable and their pattern of co-variation significantly influences BMI as early as the pre-teen years. Investigation at the genomic scale is required to determine the variants affecting the regulation of the hormones studied

Vitamin D supplementation as an adjuvant therapy for patients with T2DM : an 18-month prospective interventional study

Background Vitamin D deficiency has been associated with impaired human insulin action, suggesting a role in the pathogenesis of diabetes mellitus type 2 (T2DM). In this prospective interventional study we investigated the effects of vitamin D3 supplementation on the metabolic profiles of Saudi T2DM subjects pre- and post-vitamin D supplementation over an 18-month period. Methods T2DM Saudi subjects (men, N = 34: Age: 56.6 ± 8.7 yr, BMI, 29.1 ± 3.3 kg/m2; women, N = 58: Age: 51.2 ± 10.6 yr, BMI 34.3 ± 4.9 kg/m2;) were recruited and given 2000 IU vitamin D3 daily for 18 months. Anthropometrics and fasting blood were collected (0, 6, 12, 18 months) to monitor serum 25-hydroxyvitamin D using specific ELISA, and to determine metabolic profiles by standard methods. Results In all subjects there was a significant increase in mean 25-hydroxyvitamin D levels from baseline (32.2 ± 1.5 nmol/L) to 18 months (54.7 ± 1.5 nmol/L; p < 0.001), as well as serum calcium (baseline = 2.3 ± 0.23 mmol/L vs. 18 months = 2.6 ± 0.1 mmol/L; p = 0.003). A significant decrease in LDL- (baseline = 4.4 ± 0.8 mmol/L vs. 18 months = 3.6 ± 0.8 mmol/L, p < 0.001] and total cholesterol (baseline = 5.4 ± 0.2 mmol/L vs. 18 months = 4.9 ± 0.3 mmol/L, p < 0.001) were noted, as well as a significant improvement in HOMA-β function ( p = 0.002). Majority of the improvements elicited were more prominent in women than men. Conclusion In the Saudi T2DM population receiving oral Vitamin D3 supplementation (2000 IU/day), circulating 25-hydroxyvitamin D levels remained below normal 18 months after the onset of treatment. Yet, this “suboptimal” supplementation significantly improved lipid profile with a favorable change in HDL/LDL ratio, and HOMA-β function, which were more pronounced in T2DM females

Bidirectional Psychoneuroimmune Interactions in the Early Postpartum Period Influence Risk of Postpartum Depression

More than 500,000 U.S. women develop postpartum depression (PPD) annually. Although psychosocial risks are known, the underlying biology remains unclear. Dysregulation of the immune inflammatory response and the hypothalamic–pituitary–adrenal (HPA) axis are associated with depression in other populations. While significant research on the contribution of these systems to the development of PPD has been conducted, results have been inconclusive. This is partly because few studies have focused on whether disruption in the bidirectional and dynamic interaction between the inflammatory response and the HPA axis together influence PPD. In this study, we tested the hypothesis that disruption in the inflammatory-HPA axis bidirectional relationship would increase the risk of PPD. Plasma pro- and anti-inflammatory cytokines were measured in women during the 3rd trimester of pregnancy and on Days 7 and 14, and Months 1, 2, 3, and 6 after childbirth. Saliva was collected 5 times the day preceding blood draws for determination of cortisol area under the curve (AUC) and depressive symptoms were measured using the Edinburgh Postpartum Depression Survey (EPDS). Of the 152 women who completed the EPDS, 18% were depressed according to EDPS criteria within the 6 months postpartum. Cortisol AUC was higher in symptomatic women on Day 14 (p = .017). To consider the combined effects of cytokines and cortisol on predicting symptoms of PPD, a multiple logistic regression model was developed that included predictors identified in bivariate analyses to have an effect on depressive symptoms. Results indicated that family history of depression, day 14 cortisol AUC, and the day 14 IL8/IL10 ratio were significant predictors of PPD symptoms. One unit increase each in the IL8/IL10 ratio and cortisol AUC resulted in 1.50 (p = 0.06) and 2.16 (p = 0.02) fold increases respectively in the development of PPD. Overall, this model correctly classified 84.2% of individuals in their respective groups. Findings suggest that variability in the complex interaction between the inflammatory response and the HPA axis influence the risk of PPD

National strategic framework for research & innovation. 2014-2020

SAFE Professor Michalis Haliassos was a member of the National Council for Research and Technology (ESET) established by the Government of Greece for the period 2010-2013. The council, consisting of eleven scientists from a range of disciplines, has now published their communiqué "National Strategic Framework for Research and Innovation 2014-2020". To promote the advancement of research, technology and innovation in Greece, the strategic plan proposed by the authors seeks to identify areas of existing research strength and excellence that can be further advanced to become engines for progress and growth in Greece, as well as flaws inherent to the present system. The authors stress the need to address current constraints to growth, which include the declining education system; the confusion and weaknesses of R&D governance and management; the discontinuities and inefficiencies of resource allocation and investment; the lack of adaptation to clearly-defined national priorities; and the inadequate opportunities and funding for high-quality research and development to flourish. They stress the need for prioritisation and efficient allocation; stability of the policy frame; predictability of planning; provision of opportunity; recognition of excellence; and responsiveness to current and future needs