Publications

Inflammation normally results in enhanced synthesis and secretion of hypothalamic corticotropin-releasing hormone (CRH) which, in turn, exerts antiinflammatory effects by virtue of increased adrenal glucocorticoid production.CRH and CRH binding sites are also expressed in the peripheral nervous and immune systems.In the periphery, CRH has pleiotropic effects on immune/inflammatory systems.We demonstrated CRH expression in the joints of Lewis rats with experimental arthritis and in synovial tissues of patients with rheumatoid arthritis.These data support an autocrine/paracrine immunomodulatory role for CRH.

Increased extraglandular aromatization has been reported as the cause of familial gynecomastia. We studied a kindred with aromatase excess inherited in an autosomal dominant manner, in which affected males had heterosexual precocity and/or gynecomastia, and affected females had isosexual precocity and/or macromastia. The propositus was a 9-yr-old boy with gynecomastia. His 7.5-yr-old sister had precocious puberty, and their father and paternal grandmother had peripubertal gynecomastia and macromastia, respectively. Serum concentrations of gonadal and adrenal steroid hormones were determined before and after the administration of corticotropin and/or hCG. Aromatase activity was determined by [3H]delta4-androstenedione to [3H]estrone conversion by cultured skin fibroblasts and/or Epstein-Barr virus-transformed lymphocytes and was detected by immunohistochemistry and/or Western analysis. Linkage was examined with a polymorphism of the aromatase (P450arom) gene. The P450arom messenger ribonucleic acid was analyzed by rapid amplification of complementary DNA (cDNA) ends, ribonuclease protection assay, and RT-PCR. hCG testing demonstrated a high rate of conversion of delta4-androstenedione to estrone and of testosterone to estradiol in the propositus and his father. Treatment of the propositus and his sister was initiated with an aromatase inhibitor (testolactone) and a GnRH analog, which successfully delayed skeletal and pubertal development in both children. Markedly increased aromatase activity was found in the patients' fibroblasts and Epstein-Barr virus-transformed lymphocytes. The P450arom polymorphism segregated with the disease in the family. A new 5'-splice variant was present in the patients' P450arom messenger ribonucleic acid, thus identifying yet another first exon of this gene, which appears to be aberrantly expressed in this family. In conclusion, a family with the aromatase excess syndrome is described, in which the condition was inherited in an autosomal dominant manner, led to feminizing manifestations in both sexes, and was associated with the aberrant utilization of a novel transcript of the P450arom gene.

The aim of this study was to examine the associations between multiple indices of stress, inflammation and metabolism vs. body composition parameters in 121 (43 boys, 78 girls) children and adolescents, aged 5-15 y. Subjects were divided into two groups: normal weight (N) (N = 40, BMI z-score = -0.1923 ± 0.6), and overweight/obese (OB) (N = 81, BMI z-score = 2.1947 ± 1.4). All subjects completed the State-Trait Anxiety Inventory for Children (STAIC) and Children's Depression Inventory, and underwent cortisol measurements in hair, diurnal series of saliva, and morning serum. Circulating concentrations of high sensitivity C-reactive protein (hsCRP) and other inflammation biomarkers were also obtained. Body composition analysis was performed with a clinically validated, advanced bioimpedance apparatus (BIA), while heart rate variability (HRV) was measured as a stress biomarker by photoplethysmography (PPG). The OB group had a higher STAIC-state score, waist-to-hip ratio, skeletal muscle mass, and total and abdominal fat mass, and a lower percent fat-free mass (FFM) and bone density than the N group. HRV did not differ between the groups. In the entire population, percent fat mass correlated strongly with circulating hsCRP (r = 0.397, p = 0.001), ferritin, and other inflammatory biomarkers, as well as with indices of insulin resistance. A strong correlation between serum hsCRP and hair cortisol was also observed (r = 0.777, p < 0.001), suggesting interrelation of chronic stress and inflammation. Thus, body fat accumulation in children and adolescents was associated with an elevation in clinical and laboratory biomarkers of stress, inflammation, and insulin resistance. BIA-ACC and PPG can be utilized as a direct screening tool for assessing overweight- and obesity -related health risks in children and adolescents.

Schmid metaphyseal chondrodysplasia (SMCD; MIM 156500) is an autosomal dominant disorder of the skeleton that is manifested in early childhood by short stature,coxa vara,and a waddling gait. Patients with SMCD have mutations in the gene that codes for the α-1 chain of collagen X (COL10A1); however, mutation analysis of this gene is hampered by its size. We studied a family with SMCD: the mother, a 36-year-old woman with a height of 149 cm, had mild bilateralcoxa vara.Her two sons presented with short stature, bowed legs, andcoxa varain early childhood. DNA was extracted from peripheral lymphocytes from the three patients and subjected to PCR amplification by COL10A1 gene-specific primers. In addition to single-strand conformational polymorphism (SSCP) analysis of the COL10A1 gene, we used a novel method, dideoxy fingerprinting (ddF). The genetic defect in this family was found to be a previously unreported missense mutation (T-to-C transition) at nucleotide 2011. This change resulted in a Ser-to-Pro substitution at position 671 of the carboxy-terminus of the COL10A1 protein. In addition, the two boys, but not the mother, were found to carry a trinucleotide (CCC) deletion at position 2048 of the 3′ untranslated region, a polymorphism of the COL10A1 gene. We conclude that ddF can be used in the analysis of the COL10A1 gene along with SSCP. The S671P substitution is novel, but located in the same region with the other reported COL10A1 mutations, confirming type X collagen as the locus for this disease.

Cancer is considered one of the dominant life-threatening diseases in children. Working in the field of pediatric oncology, although rewarding, can be a source of stress and emotional burden for health care providers. The aim of this systematic review was to summarise the evidence regarding the occupational stress of health care providers working with pediatric cancer patients. Extensive search of the Pubmed and Scopus databases was performed to identify studies relevant to the topic. Initial search retrieved 657 studies. The reviewing investigators, after applying the inclusion/exclusion criteria, extracted data to critically appraise the quality of evidence. The final step of search concluded in 23 studies of heterogeneous design. Results revealed two main domains of which occupational stress derived from i) the interaction of the health care provider with the patient and the family, and ii) several organisational factors, such as hierarchical structures, experience, workload, and low organisational support. Literature on the stress of pediatric oncology staff is limited. The rather small sample sizes of the studies, the heterogeneity of methodological design, the lack of assessment from a sociological point of view, as well as the limited psychometric instruments adapted to pediatric oncology staff, make the validity of the results questionable. Further research is warranted to obtain a more accurate view of the field, to identify a cause-effect relation between work-related stress and pediatric oncology staff, and, more importantly, to guide future recommendations on support systems and stress management training within pediatric oncology settings.

Heat shock proteins (hsps) and glucocorticoids are produced in response to many common stressors. In addition, hsps interact physically with the intracellular glucocorticoid receptor (GR) and facilitate its activation by the hormone. To study the effect of stress on the hsp70/90 system and the potential association of the system with the hypothalamic-pituitary-adrenal (HPA) axis, we subjected 3 month-old male Harlan-Sprague Dawley rats to single or repeated (once daily for 6 consecutive days) immobilizations, and measured hsp70/90 steady-state levels in a panel of tissues, as well as circulating ACTH and corticosterone concentrations before, during and after immobilization. Single or repeated immobilizations had, as expected, a profound stimulatory effect on the HPA axis but did not influence the steady-state levels of hsp70/90 in any of the gross brain regions (pituitary, hypothalamus, hippocampus or brain cortex) or most peripheral tissues (thymus, adrenal glands, testes) examined. The hsp90 levels, however, were markedly and reversibly decreased in the liver and spleen both by single and repeated immobilizations. The potential inhibitory effect of glucocorticoids on liver and spleen hsp90 was investigated in bilaterally adrenalectomized rats treated with placebo or oral pharmacologic doses of corticosterone for 1 week. Neither adrenalectomy nor corticosterone treatment had an effect on the hsp70/90 system, suggesting that factors other than glucocorticoids mediate the effect of stress on hsp90 concentrations in the liver and spleen. We conclude that acute and chronic stress are associated with a reversible reduction of hsp90 in the liver and spleen, and this appears independent of glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

CRH, the principal regulator of the hypothalamic-pituitary-adrenal axis and modulator of autonomic nervous system activity, also participates in the regulation of appetite and energy expenditure. Antalarmin, a pyrrolopyrimidine compound, antagonizes CRH type 1 receptor-mediated effects of CRH, including pituitary ACTH release, stress behaviors, and acute inflammation. We administered antalarmin chronically to evaluate its effects on hypothalamic-pituitary-adrenal axis function and metabolic status. Adult male rats were treated twice daily with 20 mg/kg of ip antalarmin or placebo over 11 days. The animals were weighed; plasma ACTH, corticosterone, leptin, and blood glucose levels were determined; and morphometric analyses were performed to determine adrenal size and structure, including sizing, histochemistry, immunohistochemistry, and electron microscopy. Leptin messenger RNA expression in peripheral fat was analyzed by Northern blot. Antalarmin decreased plasma ACTH (mean ± sd, 2.62 ± 0.063 pg/ml) and corticosterone concentrations (10.21 ± 1.80 μg/dl) compared with those in vehicle-treated rats [respectively, 5.3 ± 2.0 (P < 0.05) and 57.02 ± 8.86 (P < 0.01)]. Antalarmin had no significant effect on body weight, plasma leptin, or blood glucose concentrations or fat cell leptin messenger RNA levels. The width of the adrenal cortex of animals treated with antalarmin was reduced by 31% compared with that in controls without atrophy of the gland. On the ultrastructural level, adrenocortical cells were in a hypofunctional state characterized by reduced vascularization, increased content of lipid droplets, and tubulovesicular mitochondria with fewer inner membranes. The apoptotic rate was increased in the outer zona fasciculata of animals treated with the antagonist (26.6 ± 3.58%) compared with that in placebo-treated controls (6.8 ± 0.91%). We conclude that chronic administration of antalarmin does not affect body weight, carbohydrate metabolism, or leptin expression, whereas it reduces adrenocortical function mildly, without anatomical, clinical, or biochemical evidence of causing adrenal atrophy. These results are promising for future uses of such an antagonist in the clinic.

The guanine nucleotide exchange factor Brx is essential for osmotic stress–mediated expression of nfat5 in lymphocytes.

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.

Primary generalized glucocorticoid resistance is a rare genetic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. We review the clinical aspects, molecular mechanisms, and implications of this disorder.We conducted a systematic review of the published, peer-reviewed medical literature using MEDLINE (1975 through February 2008) to identify original articles and reviews on this topic.We have relied on the experience of a number of experts in the field, including our extensive personal experience.The clinical spectrum of primary generalized glucocorticoid resistance is broad, ranging from asymptomatic to severe cases of hyperandrogenism, fatigue, and/or mineralocorticoid excess. The molecular basis of the condition has been ascribed to mutations in the human glucocorticoid receptor (hGR) gene, which impair glucocorticoid signal transduction and reduce tissue sensitivity to glucocorticoids. A consequent increase in the activity of the hypothalamic-pituitary-adrenal axis compensates for the reduced sensitivity of peripheral tissues to glucocorticoids at the expense of ACTH hypersecretion-related pathology. The study of functional defects of natural hGR mutants enhances our understanding of the molecular mechanisms of hGR action and highlights the importance of integrated cellular and molecular signaling mechanisms for maintaining homeostasis and preserving normal physiology.