[Analysis of BRAF and NRAS mutations in thyroid tumors in patients from Argentina]. / Análisis de mutaciones BRAF y NRAS de tumores tiroideos en pacientes de Argentina.

INTRODUCTION: Molecular alterations in follicular cells in the BRAF or NRAS genes have been reported to be associated with the process of carcinogenesis. Our aim was to determine the mutational frequency of BRAF and NRAS in fine-needle aspiration (FNA) specimens in our population. METHODS: The mutational status of BRAF (codon 600) and NRAS (codon 61) was analysed by qPCR in 193 FNA specimens from suspicious nodules and compared with pathological data of 115 patients. RESULTS: BRAF mutation was identified in 40 samples (74.1%) of FNAs classified as Bethesda VI (n = 54). In samples histologically diagnosed as classic papillary thyroid carcinoma (cPTC, n = 47), mutation was observed in 70% of cases, while in other subtypes the prevalence was lower (p = 0.013). In FNA specimens of follicular lesions (n = 36), positivity for NRAS was found in 50% of the follicular carcinomas (FTCs), but only in 6.7% of adenomas. Finally, there was a significant correlation between BRAF and PTC with lymph-node metastasis (p = 0.014) and increased relative risk of recurrence based on the Argentine Intersociety Consensus (RR = 6.77, p = 0.022). No significant differences were found between BRAF mutation and other features of aggressiveness in PTC. CONCLUSION: BRAF and NRAS mutations are observed in a significant number of PTCs and FTCs, in our population. There is a significant correlation between BRAF mutation and lymph-node metastasis.

Introducción: Se ha descrito que alteraciones moleculares de las células foliculares tiroideas en el gen BRAF o en NRAS están asociadas con el proceso de carcinogénesis. Nuestro objetivo fue conocer la frecuencia mutacional de BRAF y NRAS a partir de muestras de punción aspirativa con aguja fina (PAAF) en nuestra población. Métodos: Se analizó por qPCR el estado mutacional de BRAF (codón 600) y NRAS (codón 61) de 193 muestras obtenidas por PAAF de nódulos sospechosos y se comparó con los datos de la anatomía patológica de 115 pacientes. Resultados: La mutación BRAF se identificó en 40 muestras (74.1%) de las punciones categorizadas como Bethesda VI (n = 54). En las muestras que se correspondieron con carcinoma papilar de tiroides (CPT) variante clásica por histología (n = 47), el 70% presentó la mutación, mientras que en los otros subtipos la prevalencia fue más baja (p = 0.013). En muestras de lesión folicular (n = 36), el 50% de los carcinomas foliculares resultaron positivos para NRAS pero solo el 6.7% de los adenomas presentaron esta variación. La presencia de mutación BRAF y CPT se asociaron con metástasis en los ganglios linfáticos (p = 0.014) y mayor riesgo relativo de recurrencia según el Consenso Argentino Intersocietario (RR = 6.77, p = 0.022). No hubo diferencias significativas entre la mutación de BRAF y otras características de agresividad en CPT. Conclusión: La mutación de BRAF y NRAS se observa en un número significativo de CPT y carcinoma folicular, respectivamente, en nuestra población. La mutación BRAF se correlaciona significativamente con metástasis en los ganglios linfáticos.

Análisis de mutaciones BRAF y NRAS de tumores tiroideos en pacientes de Argentina / Analysis of BRAF and NRAS mutations in thyroid tumors in patients from Argentina

Resumen Introducción : Se ha descrito que alteraciones molecu lares de las células foliculares tiroideas en el gen BRAF o en NRAS están asociadas con el proceso de carcinogé nesis. Nuestro objetivo fue conocer la frecuencia muta cional de BRAF y NRAS a partir de muestras de punción aspirativa con aguja fina (PAAF) en nuestra población. Métodos : Se analizó por qPCR el estado mutacional de BRAF (codón 600) y NRAS (codón 61) de 193 mues tras obtenidas por PAAF de nódulos sospechosos y se comparó con los datos de la anatomía patológica de 115 pacientes. Resultados : La mutación BRAF se identificó en 40 muestras (74.1%) de las punciones categorizadas como Bethesda VI (n = 54). En las muestras que se correspon dieron con carcinoma papilar de tiroides (CPT) variante clásica por histología (n = 47), el 70% presentó la muta ción, mientras que en los otros subtipos la prevalencia fue más baja (p = 0.013). En muestras de lesión folicular (n = 36), el 50% de los carcinomas foliculares resultaron positivos para NRAS pero solo el 6.7% de los adenomas presentaron esta variación. La presencia de mutación BRAF y CPT se asociaron con metástasis en los gan glios linfáticos (p = 0.014) y mayor riesgo relativo de recurrencia según el Consenso Argentino Intersocietario (RR = 6.77, p = 0.022). No hubo diferencias significativas entre la mutación de BRAF y otras características de agresividad en CPT. Conclusión : La mutación de BRAF y NRAS se observa en un número significativo de CPT y carcinoma folicular, respectivamente, en nuestra población. La mutación BRAF se correlaciona significativamente con metástasis en los ganglios linfáticos.

Abstract Introduction : Molecular alterations in follicular cells in the BRAF or NRAS genes have been reported to be associated with the process of carcinogenesis. Our aim was to determine the mutational frequency of BRAF and NRAS in fine-needle aspiration (FNA) specimens in our population. Methods : The mutational status of BRAF (codon 600) and NRAS (codon 61) was analysed by qPCR in 193 FNA specimens from suspicious nodules and compared with pathological data of 115 patients. Results : BRAF mutation was identified in 40 samples (74.1%) of FNAs classified as Bethesda VI (n = 54). In samples histologically diagnosed as classic papillary thyroid carcinoma (cPTC, n = 47), mutation was observed in 70% of cases, while in other subtypes the prevalence was lower (p = 0.013). In FNA specimens of follicular lesions (n = 36), positivity for NRAS was found in 50% of the follicular carcinomas (FTCs), but only in 6.7% of adenomas. Finally, there was a significant correlation between BRAF and PTC with lymph-node metastasis (p = 0.014) and increased relative risk of recurrence based on the Argentine Intersociety Consensus (RR = 6.77, p = 0.022). No significant differences were found between BRAF mutation and other features of aggressiveness in PTC. Conclusion : BRAF and NRAS mutations are observed in a significant number of PTCs and FTCs, in our popu lation. There is a significant correlation between BRAF mutation and lymph-node metastasis.

Early life stress induces visual dysfunction and retinal structural alterations in adult mice.

Early life stress (ELS) is defined as a period of severe and/or chronic trauma, as well as environmental/social deprivation or neglect in the prenatal/early postnatal stage. Presently, the impact of ELS on the retina in the adult stage is unknown. The long-term consequences of ELS at retinal level were analyzed in an animal model of maternal separation with early weaning (MSEW), which mimics early life maternal neglect. For this purpose, mice were separated from the dams for 2 h at postnatal days (PNDs) 4-6, for 3 h at PNDs 7-9, for 4 h at PNDs 10-12, for 6 h at PNDs 13-16, and weaned at PND17. At the end of each separation period, mothers were subjected to movement restriction for 10 min. Control pups were left undisturbed from PND0, and weaned at PND21. Electroretinograms, visual evoked potentials, vision-guided behavioral tests, retinal anterograde transport, and retinal histopathology were examined at PNDs 60-80. MSEW induced long-lasting functional and histological effects at retinal level, including decreased retinal ganglion cell function and alterations in vision-guided behaviors, likely associated to decreased synaptophysin content, retina-superior colliculus communication deficit, increased microglial phagocytic activity, and retinal ganglion cell loss through a corticoid-dependent mechanism. A treatment with mifepristone, injected every 3 days between PNDs 4 and16, prevented functional and structural alterations induced by MSEW. These results suggest that retinal alterations might be included among the childhood adversity-induced threats to life quality, and that an early intervention with mifepristone avoided ELS-induced retinal disturbances.

Evaluation of stress, burnout and hair cortisol levels in health workers at a University Hospital during COVID-19 pandemic.

In the critical context of COVID-19 pandemic, healthcare workers are on the front line, participating directly in the care, diagnosis, and treatment of patients with COVID-19. This exposes them to a higher risk of developing chronic stress, psychological distress, and any other mental health symptoms. OBJECTIVE: to evaluate stress and burnout in a health workers population and, in addition, to measure hair cortisol concentration as a current biomarker of stress. MATERIALS AND METHODS: 234 health workers from Hospital de Clínicas "José de San Martín", Buenos Aires University, were included in this study. In this population hair samples were obtained from the posterior vertex as close to the scalp as possible and the individuals completed the following surveys: perceived stress, social support, burnout scale, life event scale, and sociodemographic data. Hair cortisol was measured by an automated chemiluminescent method. The studied population was divided into three groups considering those individuals below the healthy reference sample range (< 40 pg/mg hair), within the healthy reference range (40-128 pg/mg hair) and above the reference range (> 128 pg/mg hair). This study used a transversal and observational design. RESULTS: Our results show that 40% of the studied population presented hair cortisol values outside of the healthy reference range. In the whole studied population, a direct correlation was found between hair cortisol concentration and perceived stress as well as between hair cortisol concentration and the emotional exhaustion component of burnout (r = 0.142, p = 0.030; r = 0.143, p = 0.029, respectively). 12% of the studied population showed Burnout (52% doctors and residents, 19% nurses, 19% administrative personnel). Higher values in hair cortisol levels were found in the group with burnout versus individuals without burnout (p = 0.034). Finally, a mediation analysis was performed, finding that depersonalization is a mediating variable in the relationship between self-perceived stress and hair cortisol level (F = 4.86, p = 0.0086; indirect effect IC: 0.0987-1.8840). CONCLUSION: This is the first study in which a stress biomarker such as hair cortisol is evaluated in this population and in this context. Healthcare workers are subjected to increased levels of stress and burnout. High depersonalization, emotional exhaustion, and decreased personal sense of accomplishment characterize this population. It is the responsibility of the health authorities to implement strategies to manage this psychological emergency.

Hair Cortisol Measurement by an Automated Method.

We present the development of the first procedure for hair cortisol measurement through an automated method. Hair samples were obtained from 286 individuals. After cortisol extraction, samples were measured in a Siemens Immulite 2000 (Gwynedd, UK) automated chemoluminiscent immunoassay analyzer. Normal reference values were obtained from hair cortisol levels measured in 213 healthy individuals with low levels of stress. Hair cortisol concentration median was 55 pg/mg hair (2.5-97.5 percentile (40-128)) in healthy individuals with low levels of stress and 250 pg/mg hair (range 182-520) in stressed individuals. No significant differences were observed in hair cortisol levels between subjects with and without dye (40 (40-107) and 40 (40-155) pg/mg hair, respectively; p = 0.128). The novel procedure presented here shows an adequate analytical performance.

Impaired HPA axis function in diabetes involves adrenal apoptosis and phagocytosis.

PURPOSE: The aim of the present study was to analyze the involvement of oxidative stress and inflammation in the modulation of glucocorticoid production in the adrenal cortex of diabetic rats. METHODS: Male Wistar rats were treated with or without streptozotocin (STZ, an insulinopenic model of diabetes) and either α-lipoic (90 mg/kg ip.), α-tocopherol (200 mg/kg po.) or with STZ and supplemented with insulin (STZ + INS: 2.5U/day) for 4 weeks. Oxidative/nitrosative stress parameters and antioxidant enzymes were determined in adrenocortical tissues. Apoptosis and macrophage activation were evaluated by immunohistochemistry (TUNEL and ED1+). Basal and ACTH-stimulated corticosterone production were assessed by RIA and plasma ACTH levels were determined by an immunometric assay. RESULTS: Diabetic rats showed a diminished response to exogenous ACTH stimulation along with higher basal corticosterone and lower plasma ACTH levels. In the adrenal cortex we determined an increase in the levels of lipoperoxides, S-nitrosothiols, nitric oxide synthase activity and nitro-tyrosine modified proteins while catalase activity and heme oxygenase-1 expression levels were also elevated. Antioxidant treatments were effective in the prevention of these effects, and in the increase in the number of apoptotic and phagocytic (ED1+) cells detected in diabetic rats. No changes were observed in the STZ + INS group. CONCLUSIONS: Generation of oxidative/nitrosative stress in the adrenal cortex of diabetic rats leads to the induction of apoptosis and the activation of adrenocortical macrophages and is associated with an elevated basal corticosteronemia and the loss of the functional capacity of the gland.

Prostate Cancer, High Cortisol Levels and Complex Hormonal Interaction.

Prostate cancer (PCa) is one of the most common diseases in men. It is important to assess prognostic factors and whether high cortisol levels and complex hormonal interactions could be responsible for PCa development. We evaluated the relationship between cortisol, leptin and estrogens in 141 men, 71 with PCa and the remaining 70 constituting a low risk group (LRG). They were recruited for this study from a total of 2906 middleaged men (ages 4570 years) who completed an evaluation for prostatic diseases at the Urology Division, Hospital de Clinicas "Jose de San Martin", University of Buenos Aires, in May 2009. In this cross sectional study, cortisol, PSA, totaltestosterone, freetestosterone, bioavailable testosterone, LH and estradiol were measured in serum. We observed increased cortisol levels in PCa patients as compared to LRG cases (p=0.004,). Leptin and estradiol levels were also higher in PCa patients (p=0.048; p<0.0001, respectively). Logistic regression analysis indicated that serum cortisol (OR: 1.110 (95% CI 1.0161.213), p=0.022), estradiol (OR: 1.044 (95% CI 1.0081.081), p=0.016) and leptin (OR: 1.248 (95% CI 1.0481.487), p=0.013) explained 27% of the variance of dependent variables, even after adjusting for age, smoking, BMI and waist circumference. We found increased cortisol levels in PCa patients as compared to LRG, as well as an altered circulating hormonal profile.

El ejercicio físico moderado evita la aparición de insulinorresistencia y de alteraciones morfofuncionales adrenocorticales en ratas tratadas con una dieta rica en sacarosa / Moderate exercise prevents insulin resistance and changes in adrenocortical morphology and function in rats fed a sucrose-enriched diet

Introducción: niveles elevados de glucocorticoides se asocian a las alteraciones somáticas y bioquímicas presentes en los pacientes y en animales con insulinorresistencia (IR). Hemos demostrado previamente que la IR inducida por una dieta rica en sacarosa (DRS) induce cambios morfológicos y funcionales a nivel adrenocortical y que estas alteraciones pueden evitarse mediante la administración simultánea de un agonista PPAR-γ. Objetivos: en el presente estudio evaluamos el impacto de un protocolo de ejercicio moderado sobre las alteraciones morfológicas y funcionales adrenocorticales asociadas con el desarrollo de IR inducida por una DRS administrada durante siete semanas. Metodología. Resultados: los animales (ratas Wistar macho adultas) tratados con la DRS (agregado de sacarosa al 30% en el agua de bebida) mostraron un incremento del peso corporal y de los panículos adiposos, así como de los niveles séricos de glucosa, insulina y triglicéridos. La respuesta glucémica a la administración de insulina i.p. se vio claramente menoscabada. Se observó una infiltración lipídica de la corteza adrenal, con aumento de la expresión de proteínas esteroidogénicas y marcadores de inflamación (IL-1ß, TNF-α, iNOS, COX-2) y un incremento marcado de la corticosteronemia basal. El protocolo de ejercicio consistió en correr en una cinta continua adaptada especialmente durante un máximo de 7 min/día. Este ejercicio moderado previno la aparición de los cambios somáticos y bioquímicos característicos del estado de IR y la infiltración lipídica adrenocortical, revirtiendo además los cambios inflamatorios y normalizando la corticosteronemia. Conclusiones: nuestros resultados subrayan el rol deletéreo del consumo exagerado de carbohidratos simples conteniendo fructosa y sugieren que el ejercicio moderado podría tener efectos adicionales cuando se emplea en el tratamiento de la IR

Moderate Exercise Prevents Functional Remodeling of the Anterior Pituitary Gland in Diet-Induced Insulin Resistance in Rats: Role of Oxidative Stress and Autophagy.

A sustained elevation of glucocorticoid production, associated with the establishment of insulin resistance (IR) could add to the deleterious effects of the IR state. The aim of this study is to analyze the consequences of long-term feeding with a sucrose-rich diet (SRD) on Pomc/ACTH production, define the underlying cellular processes, and determine the effects of moderate exercise (ME) on these parameters. Animals fed a standard chow with or without 30% sucrose in the drinking water were subjected to ME. Circulating hormone levels were determined, and pituitary tissues were processed and analyzed by immunobloting and quantitative real-time PCR. Parameters of oxidative stress (OxS), endoplasmic reticulum stress, and autophagy were also determined. Rats fed SRD developed a decrease in pituitary Pomc/ACTH expression levels, increased expression of antioxidant enzymes, and induction of endoplasmic reticulum stress and autophagy. ME prevented pituitary dysfunction as well as induction of antioxidant enzymes and autophagy. Reporter assays were performed in AtT-20 corticotroph cells incubated in the presence of palmitic acid. Pomc transcription was inhibited by palmitic acid-dependent induction of OxS and autophagy, as judged by the effect of activators and inhibitors of both processes. Long-term feeding with SRD triggers the generation of OxS and autophagy in the pituitary gland, which could lead to a decline in Pomc/ACTH/glucocorticoid production. These effects could be attributed to an increase in fatty acids availability to the pituitary gland. ME was able to prevent these alterations, suggesting additional beneficial effects of ME as a therapeutic strategy in the management of IR.

Hair cortisol: A new tool for evaluating stress in programs of stress management.

AIMS: Longitudinal and experimental studies have shown that chronic stress contributes to the onset and progression of different diseases. Although it is not possible to eliminate stress completely, people can learn to manage it by participating in different kinds of stress management interventions. This study examined the effectiveness of stress management interventions on neuroendocrine responses in stressed students and health professionals, by measuring hair cortisol in comparison to salivary cortisol. MAIN METHODS: Salivary and hair cortisol measurements were performed in 37 subjects (31women, 6 men; mean age 34.0±10.6) who attended to a Coping Stress and Quality of Care Program at the University of Buenos Aires. Cortisol was measured at the beginning and at the end of the program. The State-Trait Anxiety Inventory STAI was used to evaluate state and trait anxiety. KEY FINDINGS: In subjects who completed the program, no differences were observed in salivary cortisol levels between the first and the last session. However, in these subjects, hair cortisol obtained in the last session was significantly lower than hair cortisol in the first session. SIGNIFICANCE: Hair cortisol appears to be a better biomarker than salivary cortisol for evaluation of the effectiveness of a stress reduction program and it seems to be a better indicator of stress system dysregulation as well.

Life events are positively associated with luteinizing hormone in middle age adult men: role of cortisol as a third variable.

Previous studies have tested the relationship between chronic stress and sex hormones, but inconsistent results have been found. One possibility is that this association may depend on other biological factors. This study examined the relationship between stressful life events (LE) and sex hormones in men, and whether cortisol is involved in this relationship. From a total number of 2906 men who completed a screening for the early detection of prostate cancer, 139 healthy men (mean ± SD age, 57.8 ± 5.7 years) were included in this study. Participants were assessed with the Holmes and Rahe questionnaire in relation to their experience of LE during the previous 1-5 years. Salivary and serum cortisol was measured at 08:00-09:00 h, as well as luteinizing hormone (LH), total testosterone, epinephrine (E) and norepinephrine (NE). LE weight sum and LE number positively correlated with LH (r = 0.293, p = 0.004; r = 0.220, p = 0.031, respectively). In a multiple regression analysis, LE-sum explained an additional and significant 10.4% of the variance in LH levels, after statistically controlling for the effects of age, waist circumference (WC) and BMI (F(1,90) = 6.61, p < 0.05). Importantly, cortisol interacted with LE in relation to total testosterone. In men with high cortisol values (≥15.4 µg/dl), there was a statistically significant positive relationship between LE number and total testosterone levels (p = 0.05), while LE were unrelated to total testosterone in men with low cortisol. LE correlated with sex hormones, predicting LH values, and in men with high cortisol levels shows a possible moderator effect of cortisol on the relationship between LE and total testosterone.

Crosstalk between nitric oxide synthases and cyclooxygenase 2 in the adrenal cortex of rats under lipopolysaccharide treatment.

The effect of lipopolysaccharide on the modulation of steroid production by adrenal cells has been recently acknowledged. The purpose of this study was to determine the in vivo effects of LPS on adrenal cyclooxygenase 2 (COX-2) expression, analyze its crosstalk with the nitric oxide synthase (NOS) system, and assess its involvement on the modulation of glucocorticoid production. Male Wistar rats were injected with LPS and with specific inhibitors for NOS and COX activities. PGE2 and corticosterone levels were determined by RIA. Protein levels were analyzed by immunoprecipitation and western blotting. Transfection assays were performed in murine adrenocortical Y1 cells. Results show that LPS treatment increases PGE2 production and COX-2 protein levels in the rat adrenal cortex. Systemic inhibition of COX-2 blunted the glucocorticoid response to ACTH, as well as the increase in NOS activity and the NOS-2 expression levels induced by LPS. Conversely, NOS inhibition prevented the LPS-dependent increase in PGE2 production, COX-2 protein levels, and the nitrotyrosine modification of COX-2 protein. Treatment of adrenocortical cells with a NO-donor significantly potentiated the LPS-dependent increase in NFκB activity and COX-2 expression levels. In conclusion, our results show a significant crosstalk between COX-2 and NOS in the adrenal cortex upon LPS stimulation, in which each activity has a positive impact on the other. In particular, as both the activities differently affect adrenal steroid production, we hypothesize that this kind of fine modulation enables the gland to adjust steroidogenesis to prevent either an excessive or an insufficient response to the endotoxin challenge.

Insulin sensitization with a peroxisome proliferator-activated receptor γ agonist prevents adrenocortical lipid infiltration and secretory changes induced by a high-sucrose diet.

It has been hypothesized that deviations in glucocorticoid secretion and/or action may contribute to somatic and biochemical changes observed in patients with and animal models of insulin resistance (IR). In this study, we analyzed changes in rat adrenocortical function and morphology associated with the development of IR, generated in male adult rats by the addition of 30% sucrose to the drinking water. Caloric intake, body and adipose tissue weights, and biochemical parameters associated with IR were determined. Expression levels of Star, Cyp11A1, Mc2r, Pparγ (Pparg), and Cd36 were evaluated by real-time PCR, histochemical analysis of the adrenal cortex was performed using Masson's trichrome and Sudan III staining, and corticosterone levels were measured by RIA. After 7 weeks of sucrose administration, higher serum glucose, insulin, and triglyceride levels and an altered glycemic response to an i.p. insulin test were detected. Adrenal glands showed a neutral lipid infiltration. An increase in Star, Cyp11A1, Mc2r, Pparg and Cd36 and a decrease in Mc2r levels were also found. Furthermore, sucrose-treated animals exhibited higher basal corticosterone levels and a blunted response to ACTH injection. Noteworthy, the adrenocortical (functional and histological) abnormalities were prevented in sucrose-treated rats by the simultaneous administration of an insulin-sensitizing PPARγ agonist. In conclusion, sucrose-induced IR affects adrenocortical morphology and function possibly via the generation of adipokines or lipid metabolites within the adrenal gland. These abnormalities are prevented by the administration of a PPARγ agonist by mechanisms involving both extra- and intra-adrenal effects.

Characterization of L-arginine transport in adrenal cells: effect of ACTH.

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.