Memoria para optar al Título Profesional de Médico Veterinario / El Cadmio (Cd2+) es un contaminante ampliamente distribuido en el ambiente; la exposición de la población ocurre principalmente a través del humo de tabaco. Se han descrito efectos adversos en la salud, algunos de los cuales son dependientes del sexo. Específicamente, en crías expuestas durante la gestación, disminuye el peso de nacimiento (PN) lo que se asocia a una exposición fetal aumentada a glucocorticoides (GC). Los GC actúan a través de sus receptores específicos (GR) localizados en los tejidos blancos; en el hígado, la unión GC-GR activa la expresión de genes involucrados en el metabolismo de carbohidratos (CH) y lípidos. Entre éstos se encuentra el gen de la enzima fosfoenol piruvato carboxiquinasa (PEPCK, limitante en el proceso de gluconeogénesis) y el de la enzima acetil Co-A oxidasa (AOX, limitante en el proceso de β-oxidación peroxisomal). En este estudio, se postuló que el Cd2+ administrado durante la gestación induce en las crías neonatas una alteración en la expresión hepática del GR y consecuentemente, de sus genes blancos, PEPCK y AOX, con una concomitante alteración en los niveles hepáticos de glicógeno, triacilglicerol (TAG) y colesterol, de manera diferenciada en machos y hembras. El modelo de estudio utilizado fue ratas de la cepa Wistar tratadas con 10 ppm de Cd2+ (como CdCl2 en el agua de bebida) desde el destete hasta el cruzamiento, y luego con 50 ppm de Cd2+ durante toda la gestación. Posteriormente, en el hígado de las crías neonatas se determinó la expresión de GR (proteína, mediante Western Blot y mRNA por qRT-PCR), PEPCK (mRNA) y AOX (mRNA). También se analizó el contenido de glicógeno, TAG y colesterol. Los resultados indican que GR hepático aumenta su expresión en hembras y en machos disminuye. También hay efectos diferenciados en los niveles de mRNA para PEPCK, y AOX. Se concluye que la exposición a Cd2+ durante el desarrollo induce alteraciones tempranas en el patrón de expresión de genes relacionados con el sistema GC y metabolismo de CH y lípidos. Los efectos son dependientes del sexo y pueden inducir una reprogramación fetal a largo plazo explicando el desarrollo de ciertas patologías / Fondecyt No. 1071110

Nous avons développé des nanoparticules d’une prodrogue de glucocorticoïde, la dexaméthasone palmitate (DXP) à visée thérapeutique dans le traitement de la polyarthrite rhumatoïde (PR). Cette maladie auto-immune est caractérisée par une inflammation articulaire, une érosion osseuse et cartilagineuse et une dérégulation du système immunitaire. Parmi les traitements indiqués dans la PR, l'utilisation des glucocorticoïdes est limitée par leurs effets secondaires importants induits par leur pharmacocinétique défavorable. Afin de traiter la PR par voie intraveineuse, la formulation de nanoparticules PEGylées semble indispensable afin d’échapper au phénomène d’opsonisation et d’espérer obtenir une accumulation spécifique au niveau des articulations inflammées. Pour cela, nous avons développé des nanoparticules de DXP (DXP-NP) stabilisées par le DSPE-PEG2000.Les caractéristiques physico-chimiques des nanoparticules obtenues ont été évaluées ainsi que leur stabilité au cours du temps. La structure interne des nanoparticules définie comme amorphe ainsi que leur très fort taux de charge prouvent ainsi l’impact du DSPE-PEG2000 dans l’organisation moléculaire des DXP-NP. In vivo, l’étude de la pharmacocinétique et de la biodistribution des DXP-NP suite à leur administration intraveineuse a démontré une circulation prolongée du système. Dans un modèle murin de polyarthrite rhumatoïde, les DXP-NP ont démontré leur accumulation spécifique dans les articulations inflammées en corrélation avec une supériorité thérapeutique significative en comparaison avec la molécule libre hydrosoluble. Des études histologiques ainsi que l’évaluation du traitement sur l’apparition d’effets indésirables complètent l’étude in vivo. / We developed nanoparticles of a glucocorticoid prodrug, dexamethasone palmitate (DXP) for the treatment of rheumatoid arthritis (RA). Joint inflammation, bone and cartilage erosion and dysregulation of the immune system characterize this autoimmune disease. Among the treatments indicated in RA, the use of glucocorticoids is hampered by their side effects induced by their unfavorable pharmacokinetics. To treat RA intravenously, the formulation of PEGylated nanoparticles seems essential in order to escape from opsonization and to obtain a specific accumulation in the joints inflamed. Therefore, we developed DXP nanoparticles (DXP-NPs) stabilized by the DSPE-PEG2000.The physicochemical characteristics of the nanoparticles obtained were evaluated as well as their stability over time. The amorphous internal structure of the nanoparticles and their very high drug loading thus prove the impact of DSPE-PEG2000 in the molecular organization of DXP-NPs. In vivo, the study of the pharmacokinetics and biodistribution of DXP-NPs following intravenous administration demonstrated prolonged circulation of the system. In a mouse model of rheumatoid arthritis, DXP-NPs their demonstrated specific accumulation in inflamed joints in correlation with significant therapeutic superiority in comparison with the water-soluble free molecule. Histological studies as well as adverse events evaluation supplemented the in vivo study.

[ES] Nuestra investigación se centra en comprender los mecanismos moleculares que median las acciones de los glucocorticoides (GCs) en la fisiopatología de la piel mediante el análisis funcional del receptor de GCs (GR) y el receptor de mineralocorticoides (MR), dos proteínas altamente relacionadas estructural y funcionalmente, que actúan como factores de transcripción dependientes de ligando. Nuestros datos previos demuestran que GR juega un papel central en el desarrollo de la piel; en la edad adulta, tanto GR como MR actúan como mediadores anti-inflamatorios en enfermedades cutáneas (Sevilla et al. 2013; Boix et al. 2016). No obstante, desconocíamos si los receptores ejercían funciones cooperativas o antagónicas en la epidermis. Esta tesis doctoral se ha centrado en la generación y caracterización de ratones con inactivación específica en la epidermis de GR y MR (ratones double knock-out o DKO). Al nacer, los DKO mostraron un fenotipo cutáneo con diferenciación epidérmica defectuosa y un estado inflamatorio único caracterizado por infiltrados inmunes epiteliales y alteraciones en la expresión génica, similar a las lesiones psoriáticas. Este fenotipo fue mucho más severo que el de los KO individuales (ratones GR epidermal KO o GREKO y MR epidermal KO o MREKO), pero se resolvió espontáneamente a partir del día post-natal 3. En la edad adulta, la piel DKO mostró un aumento en el grosor epidérmico, similar al de los KO individuales. Todos los ratones KO mostraron una mayor susceptibilidad a la inflamación aguda respecto a los controles (CO), que no se contrarrestó de forma efectiva por un tratamiento tópico con GCs. Además, los ratones DKO mostraron una mayor susceptibilidad a la psoriasis inducida por imiquimod respecto a los KO individuales. El aumento de la respuesta inflamatoria en los DKO era consistente con un aumento significativo de la actividad de AP-1 y NF-kappaB en queratinocitos DKO respecto a los CO o KO individuales. En conjunto, nuestros datos demuestran que GR y MR epidérmicos actúan de manera cooperativa para contrarrestar la inflamación de la piel, durante el desarrollo y la edad adulta, y que ambos son necesarios para una respuesta transcripcional óptima y una actividad terapéutica de los GCs. Los tratamientos prolongados con dosis farmacológicas de GCs producen defectos como la atrofia cutánea, similar a la que tiene lugar durante el envejecimiento cronológico, que correlaciona con un aumento de los niveles locales endógenos de GCs. Este trabajo ha abordado las consecuencias fenotípicas de la pérdida epidérmica de MR durante el envejecimiento cronológico y los mecanismos involucrados. Los ratones MREKO de 13 meses de edad fueron resistentes a la atrofia epidérmica pero mostraron un menor grosor dérmico y depósito de colágeno, en parte debido a una disminución de la actividad SMAD2/3 respecto a la piel de ratones CO. Además, el tejido adiposo subcutáneo (dWAT) se engrosó 2.5 veces en MREKO vs CO a los 13 meses, con hiperplasia e hipertrofia de adipocitos. Estos cambios se desencadenaron, al menos en parte, a través de alteraciones en la señalización mediada por GCs, y la activación de WNT/beta-catenina inducida por señales paracrinas epidérmicas que condujeron al aumento de expresión de Pparg. Estos resultados demuestran un papel crucial de MR epidérmico en la regulación del cross-talk entre compartimientos durante el envejecimiento cronológico de la piel. / [CA] La nostra investigació se centra en comprendre els mecanismes moleculars que regulen les accions dels glucocorticoides (GCs) en la fisiopatologia de la pell mitjançant l'anàlisi funcional del receptor de GCs (GR) i el receptor de mineralocorticoides (MR), dues proteïnes altament relacionades estructural i funcionalment, que actuen com a factors de transcripció dependents de lligant. Els nostres resultats previs demostren que GR juga un paper central en el desenvolupament de la pell; en l'edat adulta, tant GR com MR actuen com a mediadors antiinflamatoris en malalties cutànies (Sevilla et al. 2013; Boix et al. 2016). No obstant, desconeixíem si els receptors exercien funcions cooperatives o antagòniques en l'epidermis. Aquesta tesi doctoral s'ha centrat en la generació i caracterització de ratolins amb inactivació específica en l'epidermis de GR i MR (ratolins double knock-out o DKO). En néixer, els DKO van mostrar un fenotip cutani amb diferenciació epidèrmica defectuosa i un estat inflamatori únic caracteritzat per infiltrats immunes epitelials i alteracions en l'expressió gènica, similar a les lesions psoriàtiques. Aquest fenotip va ser molt més sever que el dels KO individuals (ratolins GR epidermal KO o GREKO i MR epidermal KO o MREKO), però es va resoldre espontàniament a partir del dia post-natal 3. En l'edat adulta, la pell DKO va mostrar un augment en el gruix epidèrmic, similar al dels KO individuals. Tots els ratolins KO van mostrar una major susceptibilitat a la inflamació aguda en comparació als controls (CO), que no va ser contrarestada de manera efectiva per un tractament tòpic amb GCs. A més, els ratolins DKO van mostrar una major susceptibilitat a la psoriasis induïda per imiquimod respecte als KO individuals. L'augment de la resposta inflamatòria en els DKO era consistent amb un augment significatiu de l'activitat d'AP-1 i NF-kappaB en queratinòcits DKO respecte als CO o KO individuals. En conjunt, les nostres dades demostren que GR i MR epidèrmics actuen de manera cooperativa per contrarestar la inflamació de la pell, durant el desenvolupament i l'edat adulta, i que tots dos són necessaris per a una resposta transcripcional òptima i una activitat terapèutica dels GCs. Els tractaments prolongats amb dosis farmacològiques de GCs produeixen defectes com l'atròfia cutània, similar a la que té lloc durant l'envelliment cronològic, que correlaciona amb un augment dels nivells locals endògens de GCs. Aquest treball ha abordat les conseqüències fenotípiques de la pèrdua epidèrmica de MR durant l'envelliment cronològic i els mecanismes involucrats. Els ratolins MREKO de 13 mesos d'edat van ser resistents a l'atròfia epidèrmica però van mostrar un menor gruix dèrmic i dipòsit de col¿lagen, en part a causa d'una disminució de l'activitat SMAD2/3 respecte a la pell de ratolins CO. A més, el teixit adipós subcutani (dWAT) es va engrossir 2.5 vegades en MREKO vs CO als 13 mesos, amb hiperplàsia i hipertròfia d'adipòcits. Aquests canvis es van desencadenar, almenys en part, a través d'alteracions en la senyalització mediada per GCs, i l'activació de WNT/beta-catenina induïda per senyals paracrines epidèrmiques que van conduir a l'augment d'expressió de Pparg. Aquests resultats demostren un paper crucial de MR epidèrmic en la regulació del cross-talk entre compartiments durant l'envelliment cronològic de la pell. / [EN] Our research focuses on understanding the molecular mechanisms that mediate the actions of glucocorticoids (GCs) in skin pathophysiology through functional analysis of the GC receptor (GR) and the mineralocorticoid receptor (MR), two highly related structural and functionally proteins, which act as ligand-dependent transcription factors. Our previous data show that GR plays a central role in skin development; in adulthood, both GR and MR act as anti-inflammatory mediators in skin diseases (Sevilla et al. 2013; Boix et al. 2016). However, we did not know if the receptors exerted cooperative or antagonistic functions in the epidermis. This doctoral thesis has focused on the generation and characterization of mice with specific inactivation in the epidermis of GR and MR (double knock-out or DKO mice). At birth, DKO show a skin phenotype with defective epidermal differentiation and a unique inflammatory state characterized by epithelial immune infiltrates and alterations in gene expression, similar to psoriatic lesions. This phenotype was much more severe than that of individual KO (GR epidermal KO or GREKO and MR epidermal KO or MREKO mice), but resolved spontaneously from postnatal day 3. In adulthood, DKO skin showed an increase in epidermal thickness, similar to that of individual KO. All KO mice showed greater susceptibility to acute inflammation compared to controls (CO), which was not effectively counteracted by topical treatment with GCs. Furthermore, DKO mice show a greater susceptibility to imiquimod-induced psoriasis relative to individual KO. The increased inflammatory response in DKO was consistent with a significant increase in AP-1 and NF-kappaB activity in DKO keratinocytes relative to CO or individual KO. Taken together, our data show that epidermal GR and MR act cooperatively to counteract skin inflammation, during development and adulthood, and that both are required for optimal transcriptional response and therapeutic activity of GCs. Prolonged treatments with pharmacological doses of GCs produce defects such as cutaneous atrophy, similar to that which occurs during chronological aging, which correlates with an increase in endogenous local levels of GCs. This work has addressed the phenotypic consequences of epidermal loss of MR during chronological aging and the mechanisms involved. The 13-month-old MREKO mice were resistant to epidermal atrophy but displayed reduced dermal thickness and collagen deposition, in part due to a decrease in SMAD2 3 activity relative to the skin of CO mice. In addition, the subcutaneous adipose tissue (dWAT) thickened 2.5 times in MREKO vs CO at 13 months, with hyperplasia and hypertrophy of adipocytes. These changes were triggered, at least in part, through alterations in GC-mediated signaling, and the activation of WNT/beta-catenin induced by epidermal paracrine signals that led to increased expression of Pparg. These results show a crucial role for epidermal MR in the regulation of the cross-talk between compartments during chronological skin aging. / Este trabajo ha sido realizado con el apoyo económico de los proyectos de investigación que se enumeran a continuación: SAF2014-59474-R, SAF2017-88046-R. Judit Bigas Corominas ha disfrutado de una beca predoctoral FPI (BES2015-072722) otorgada por el Ministerio de Economía y Competitividad, asociada al proyecto SAF2014-59474-R. Agradecemos el apoyo de COST ADMIRE BM-1301 y NuRCaMeIn (SAF2015-71878-REDT y SAF2017-90604-REDT). / Bigas Corominas, J. (2020). Contribuciones relativas de los receptores de glucocorticoides y mineralocorticoides en la biología cutánea [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/156214 / TESIS

El propósito de la investigación fue determinar si la administración intramuscular en el glúteo de 4 mg Dexametasona antes y la administración intramuscular en el masetero de 4 mg de Dexametasona después de la extracción de terceras molares inferiores provocaba una mayor disminución de la inflamación frente a la administración intramuscular de 4 mg de Dexametasona antes de la cirugía, la administración intramuscular en el glúteo de 4 mg de Dexametasona en el masetero después de la cirugía y frente al grupo control. Para esto se administró la Dexametasona en 28 pacientes (16 mujeres y 12 varones) del departamento de Estomatología del Centro Médico Naval de la Marina de Guerra del Perú, con diagnóstico de terceras molares inferiores impactadas los cuales fueron divididos en 4 grupos: el grupo de administración intramuscular en el glúteo de 4 mg Dexametasona antes y administración intramuscular en el masetero de 4 mg de Dexametasona después de la extracción de terceras molares inferiores, el grupo de administración intramuscular en el glúteo de 4 mg de Dexametasona antes de la cirugía de terceras molares inferiores y el grupo de administración intramuscular de 4 mg de Dexametasona intramuscular en el masetero terminada la cirugía y el grupo CONTROL (pacientes a los cuales no se les administró Dexametasona). Para evaluar la inflamación ésta se dividió en tres subvariables: edema, trismus y dolor que fue evaluado en los grupos de Estudio y el grupo Control. El edema fue determinado por la variación de la medición horizontal de la intersección de los puntos anatómicos (tragus-pogonion de tejidos blandos, gonion-ángulo externo del ojo). Las mediciones se realizaron los tres días posteriores a la extracción y el séptimo día. El Trismus fue determinado evaluando la máxima apertura interincisal con mediciones diarias los siete días posteriores a la extracción de las terceras molares inferiores. El dolor fue determinado usando la Escala Visual Análoga y cuantificando el número de analgésicos tomados por los pacientes. La medición del dolor se realizó los tres días posteriores a la extracción de las terceras molares inferiores. Los resultados mostraron que no existe diferencias estadísticas significativas en la disminución del edema, trismus y dolor entre el grupo de administración intramuscular en el glúteo de 4 mg Dexametasona antes y administración intramuscular local en el masetero de 4 mg de Dexametasona terminada la extracción de terceras molares inferiores frente los grupos a quienes recibieron la administración intramuscular en el glúteo de 4 mg de Dexametasona antes de la cirugía y el grupo de administración intramuscular de 4 mg de Dexametasona en el masetero terminada la cirugía. La administración intramuscular en el glúteo de 4 mg Dexametasona antes y de 4 mg de Dexametasona intramuscular en el masetero terminada la extracción de terceras molares inferiores, y la administración de 4 mg de Dexametasona intramuscular en el glúteo antes de la extracción de terceras molares inferiores provocaron una mayor disminución de la inflamación frente al grupo control siendo esta diferencia altamente significativa. Finalmente de acuerdo a los resultados encontrados el empleo la Dexametasona en cirugías de terceras molares inferiores producen menor inflamación postcirugía con respecto a los pacientes que no reciben Dexametasona. El uso de la Dexametasona es una herramienta útil para la prevención de complicaciones inflamatorias postcirugía de terceras molares inferiores y otros tipos de cirugía orales. / --- “Appraisal of the Anti-inflammatory effects of glucocorticoids in patients that need inferior third molars surgery” The purpose of the research was to determinate if the intramuscular (IM) administration in gluteus muscle of 4mg Dexamethasone before and the IM administration in masseter muscle of 4mg Dexamethasone after inferior third molar’s extraction provoked greater decrease than the IM administration in gluteus muscle of 4mg of Dexamethasone only before of surgeon, the IM administration of 4 mg Dexamethasone in masseter only after of surgeon and front of Control group. The Dexamethasone was administered in 28 patients (16 woman and 12 man) of Stomatology Department at Naval Medical Center, with Impact inferior third molar diagnosis whose were divided in four groups: three groups in the Study Group; group of patients with IM administration in gluteus muscle of 4mg Dexamethasone before and the IM administration of 4mg Dexamethasone in Masseter muscle after of inferior third molar’s extraction, the group of IM administration in gluteus muscle of 4 mg Dexamethasone only before the surgeon, the group of IM administration of 4 mg. Dexametasona in masseter muscle finished the surgeon and Control Group (patients who hadn’t been administered Dexamethasone). The evaluation of inflammation was divided in three sub variable: edema, trismus and pain these were evaluated in Study Group and Control Group. Edema was determinated by horizontal measurement’s variation of the anatomic point’s intersection (Tragus- Pogonion of soft tissue, Gonion-external angle of eyes). The measure was doing until the three day and the seven day after the extraction. Trismus was determinated evaluating the interincisal opening maxim with measurements daily for seven days after of inferior third molar’s extraction. The pain was determinated wearing the Analogous Visual Scale and counting the number of analgesic taken by patients, the pain’s measurement was done three days later of inferior third molar’s extraction. The results showed that it doesn’t exist significant statistics difference in decreases of edema, trismus, and pain among the group of IM administration in gluteus muscle of 4mg Dexamethasone before and the IM administration in masseter muscle of 4mg Dexamethasone after of inferior third molar’s extraction, the Group which has received IM administration in gluteus muscle of 4 mg Dexamethasone only before the surgeon and the group of IM administration in masseter muscle of 4 mg Dexamethasone finished the surgeon only. The group of IM administration in gluteus muscle of 4mg. Dexamethasone before and the IM administration in masseter muscle of 4mg Dexamethasone after of inferior third molar’s extraction; and the IM administration in gluteus muscle of 4 mg Dexamethasone only before the surgeon provoked a greater decrease of inflammation front of Control group with significant high difference. Finally the results found the use of Dexamethasone in inferior third molar’s surgeon produced less inflammation post surgeon than patients who hadn’t been receive Dexamethasone. The use of Dexamethasone is an useful tool to prevention of inflammatory complications post surgeon of inferior third molars and other kinds of oral surgeons. / Tesis

Introdução: A deficiência da 21-hidroxilase (21-OHD) é um frequente erro herdado do metabolismo que resulta no comprometimento da síntese do cortisol e/ou aldosterona e aumento da produção de andrógenos. A doença é caracterizada por uma diversidade fenotípica, variando desde virilização pré-natal da genitália externa de fetos femininos e pós-natal em ambos os sexos, com ou sem perda de sal, até quadros assintomáticos. Em seu tratamento é necessária reposição com glicocorticoide para se evitar a insuficiência adrenocortical e os sinais de virilização. Um fino ajuste na dose diária do glicocorticoide é essencial para se evitar sub ou supertratamento, com o objetivo de preservar o potencial de estatura final e fertilidade. Entretanto, tem sido observada maior frequência de obesidade e outras comorbidades metabólicas nestes pacientes; porém, a prevalência destas complicações ainda não é conhecida, bem como se estariam associadas à exposição ao glicocorticoide e/ou com fatores genéticos. Objetivos: avaliar a frequência de obesidade e de síndrome metabólica (SM) em pacientes com 21OHD; caracterizar a distribuição alélica dos polimorfismos dos genes do receptor de glicocorticoide (NR3C1) e da enzima 11beta-hidroxiesteróide desidrogenase tipo I (HSD11B1), e correlacionar a distribuição destes polimorfismos com a presença das complicações metabólicas. Métodos: Foram selecionados 109 pacientes (60 PS/49 VS), sendo 41 crianças e adolescentes (idade média 11,4 ± 3,9 anos) e 68 adultos (idade média 28,4 ± 9 anos) em tratamento com glicocorticoide e com adequado controle hormonal. Pacientes com a forma PS também receberam fludrocortisona. Adequado controle foi caracterizado por concentração normal de atividade plasmática de renina e de andrógenos de acordo com o sexo e idade nos últimos 2 anos. A obesidade nos adultos foi definida pelo IMC >= 30 kg/m² e em crianças e adolescentes pelo IMC acima do percentil 95. Síndrome metabólica foi definida segundo o critério do National Cholesterol Education Program em adultos e crianças. História familiar de hipertensão arterial, diabetes, dislipidemia, obesidade e/ou doença cardiovascular também foi avaliada. Foram mensuradas glicemia, lipoproteínas, triglicérides, colesterol total e insulina. Os alelos BclI, A3669G, ER22/23EK e N363S do gene NR3C1 e o alelo 4436InsA do gene HSD11B1 foram genotipados e as análises de associação com os fenótipos foram realizadas por meio dos testes Chi-quadrado, t-studant e análise de regressão. As análises de correlação foram feitas utilizando o teste de correlação de Pearson. Resultados: Obesidade foi observada em 31,7% das crianças e 23,5% dos adultos. Síndrome metabólica foi observada em 14,6% das crianças e 7,3% dos adultos. A prevalência dos componentes da SM foi maior no grupo dos obesos quando comparada a de pacientes não obesos (crianças e adultos). Não houve correlação significante entre o IMC, sexo, forma clínica da 21-OHD, duração da terapia e dose de GC. História familiar positiva para obesidade, hipertensão, dislipidemia e doença cardiovascular foi mais frequente nos pacientes obesos quando comparada a de pacientes não obesos, em adultos e crianças. Os polimorfismos BclI, A3669G e 4436InsA foram identificados em 23,2%, 9,7% e 14,6% dos alelos das crianças, respectivamente, e nos adultos em 26,4%, 9,6% e 18,4% dos alelos, respectivamente. A variante A3669G foi associada à maiores concentrações de LDL-c em crianças quando comparada aos carreadores do alelo selvagem. Os pacientes adultos carreadores do polimorfismo BclI apresentaram maior IMC, circunferência abdominal e PAS quando comparados aos carreadores do alelo selvagem. Não observamos diferenças estatisticamente significantes no perfil metabólico entre pacientes carreadores e não carreadores do polimorfismo 4436InsA (adultos e crianças). Conclusão: observamos que pacientes 21-OHD possuem maior prevalência de obesidade, e o grupo pediátrico maior prevalência de SM em relação à população de referência, sendo ambas independentes da dose de glicocorticoide e do tempo do tratamento. A presença de perfil metabólico adverso esteve associada à obesidade e à predisposição genética, tais como história familiar e variantes genéticas do receptor de glicocorticoide / Introduction: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) is a common autosomal recessive disorder that leads to decreased glucocorticoid secretion, with or without mineralocorticoid deficiency, and increased androgen production. The disease is characterized by phenotypic variability, including a severe form with prenatal virilization of the external genitalia in female fetuses and postnatal virilization in both sexes, with or without salt loss. Current therapy aims to provide adequate glucocorticoid (GC) replacement and to suppress the abnormal androgen secretion; mineralocorticoid replacement aims to control the renal salt balance to avoid adrenal crisis. Nevertheless, these therapeutic goals are difficult to achieve in practice due to the complexity of replicating the physiologic cortisol circadian rhythm. Increased prevalence of obesity, insulin resistance, hypertension and adverse lipid profile have been observed among CAH patients under GC therapy; however, the extent of its prevalence and also whether it is associated with the GC dose or with genetic factors are not known. Objectives: to evaluate the obesity and metabolic syndrome (SM) frequencies in 21-OHD patients; to characterize the allelic distribution of the NR3C1 and HSD11B1 polymorphisms, and to correlate with the metabolic profile. Methods: One hundred and nine patients (60SW/49SV) were selected, 41 being children and adolescents (mean age 11.4 ± 3.9 yrs) and 68 adults (mean age 28.4 ± 9 yrs) all of whom received GC treatment and had adequate hormonal control. SW patients also received fludrocortisone. Adequate hormonal control was characterized by normal plasmatic rennin activity and androgen levels according to age and sex for at least two years. Blood fasting was used to obtain glucose, lipoproteins, triglycerides, total cholesterol and insulin levels. Obesity in the adult group was defined by BMI >= 30 kg/m², and in the young group by BMI > 95th percentile. Metabolic syndrome was defined by the NCEP ATPIII criteria. Family history of the hypertension, diabetes, dyslipidemia, obesity and/or cardiovascular disease was also evaluated. The BclI, A3669G, ER22/23EK and N363S alleles of the NR3C1 gene and 4436InsA of the HSD11B1 gene were genotyped and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Correlation analyses were performed by Pearson correlation test. Results: obesity was observed in 31.7% of children and 23.5% of adults. SM was observed in 14.6% of young and 7.3% of adult patients. SM prevalence was higher in the obese group than the nonobese group (children and adults). There was no significant correlation between GC dose and BMI, sex, clinical form or treatment duration. Prevalence of family history of obesity, hypertension, dyslipidemia and cardiovascular disease was higher in the obese than in non-obese patients (children and adults). The BclI, A3669G and 4436InsA polymorphisms were found in 23.2%, 9.7% and 14.6% of the alleles in children, respectively and in 26.4%, 9.6% and 18.4% of the alleles in adults. The A3669G variant was associated to increased LDL-c levels in comparison with noncarriers in the young group. The BclI adult carriers presented higher BMI, abdominal circumference and systolic blood pressure in comparison with noncarriers. Statistically significant differences were not observed in the metabolic profile between carriers and non-carriers of the 4436InsA polymorphism (children and adults). Conclusion: in the present study, which analyzed the clinical and metabolic profile of 21-OHD patients, high obesity prevalence, independent of GC dose and treatment duration, was observed. Adverse metabolic profile was mainly associated with obesity and genetic predisposition, such as family history and NR3C1 polymorphisms

Introdução: A deficiência da 21-hidroxilase (21-OHD) é um frequente erro herdado do metabolismo que resulta no comprometimento da síntese do cortisol e/ou aldosterona e aumento da produção de andrógenos. A doença é caracterizada por uma diversidade fenotípica, variando desde virilização pré-natal da genitália externa de fetos femininos e pós-natal em ambos os sexos, com ou sem perda de sal, até quadros assintomáticos. Em seu tratamento é necessária reposição com glicocorticoide para se evitar a insuficiência adrenocortical e os sinais de virilização. Um fino ajuste na dose diária do glicocorticoide é essencial para se evitar sub ou supertratamento, com o objetivo de preservar o potencial de estatura final e fertilidade. Entretanto, tem sido observada maior frequência de obesidade e outras comorbidades metabólicas nestes pacientes; porém, a prevalência destas complicações ainda não é conhecida, bem como se estariam associadas à exposição ao glicocorticoide e/ou com fatores genéticos. Objetivos: avaliar a frequência de obesidade e de síndrome metabólica (SM) em pacientes com 21OHD; caracterizar a distribuição alélica dos polimorfismos dos genes do receptor de glicocorticoide (NR3C1) e da enzima 11beta-hidroxiesteróide desidrogenase tipo I (HSD11B1), e correlacionar a distribuição destes polimorfismos com a presença das complicações metabólicas. Métodos: Foram selecionados 109 pacientes (60 PS/49 VS), sendo 41 crianças e adolescentes (idade média 11,4 ± 3,9 anos) e 68 adultos (idade média 28,4 ± 9 anos) em tratamento com glicocorticoide e com adequado controle hormonal. Pacientes com a forma PS também receberam fludrocortisona. Adequado controle foi caracterizado por concentração normal de atividade plasmática de renina e de andrógenos de acordo com o sexo e idade nos últimos 2 anos. A obesidade nos adultos foi definida pelo IMC >= 30 kg/m² e em crianças e adolescentes pelo IMC acima do percentil 95. Síndrome metabólica foi definida segundo o critério do National Cholesterol Education Program em adultos e crianças. História familiar de hipertensão arterial, diabetes, dislipidemia, obesidade e/ou doença cardiovascular também foi avaliada. Foram mensuradas glicemia, lipoproteínas, triglicérides, colesterol total e insulina. Os alelos BclI, A3669G, ER22/23EK e N363S do gene NR3C1 e o alelo 4436InsA do gene HSD11B1 foram genotipados e as análises de associação com os fenótipos foram realizadas por meio dos testes Chi-quadrado, t-studant e análise de regressão. As análises de correlação foram feitas utilizando o teste de correlação de Pearson. Resultados: Obesidade foi observada em 31,7% das crianças e 23,5% dos adultos. Síndrome metabólica foi observada em 14,6% das crianças e 7,3% dos adultos. A prevalência dos componentes da SM foi maior no grupo dos obesos quando comparada a de pacientes não obesos (crianças e adultos). Não houve correlação significante entre o IMC, sexo, forma clínica da 21-OHD, duração da terapia e dose de GC. História familiar positiva para obesidade, hipertensão, dislipidemia e doença cardiovascular foi mais frequente nos pacientes obesos quando comparada a de pacientes não obesos, em adultos e crianças. Os polimorfismos BclI, A3669G e 4436InsA foram identificados em 23,2%, 9,7% e 14,6% dos alelos das crianças, respectivamente, e nos adultos em 26,4%, 9,6% e 18,4% dos alelos, respectivamente. A variante A3669G foi associada à maiores concentrações de LDL-c em crianças quando comparada aos carreadores do alelo selvagem. Os pacientes adultos carreadores do polimorfismo BclI apresentaram maior IMC, circunferência abdominal e PAS quando comparados aos carreadores do alelo selvagem. Não observamos diferenças estatisticamente significantes no perfil metabólico entre pacientes carreadores e não carreadores do polimorfismo 4436InsA (adultos e crianças). Conclusão: observamos que pacientes 21-OHD possuem maior prevalência de obesidade, e o grupo pediátrico maior prevalência de SM em relação à população de referência, sendo ambas independentes da dose de glicocorticoide e do tempo do tratamento. A presença de perfil metabólico adverso esteve associada à obesidade e à predisposição genética, tais como história familiar e variantes genéticas do receptor de glicocorticoide / Introduction: Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) is a common autosomal recessive disorder that leads to decreased glucocorticoid secretion, with or without mineralocorticoid deficiency, and increased androgen production. The disease is characterized by phenotypic variability, including a severe form with prenatal virilization of the external genitalia in female fetuses and postnatal virilization in both sexes, with or without salt loss. Current therapy aims to provide adequate glucocorticoid (GC) replacement and to suppress the abnormal androgen secretion; mineralocorticoid replacement aims to control the renal salt balance to avoid adrenal crisis. Nevertheless, these therapeutic goals are difficult to achieve in practice due to the complexity of replicating the physiologic cortisol circadian rhythm. Increased prevalence of obesity, insulin resistance, hypertension and adverse lipid profile have been observed among CAH patients under GC therapy; however, the extent of its prevalence and also whether it is associated with the GC dose or with genetic factors are not known. Objectives: to evaluate the obesity and metabolic syndrome (SM) frequencies in 21-OHD patients; to characterize the allelic distribution of the NR3C1 and HSD11B1 polymorphisms, and to correlate with the metabolic profile. Methods: One hundred and nine patients (60SW/49SV) were selected, 41 being children and adolescents (mean age 11.4 ± 3.9 yrs) and 68 adults (mean age 28.4 ± 9 yrs) all of whom received GC treatment and had adequate hormonal control. SW patients also received fludrocortisone. Adequate hormonal control was characterized by normal plasmatic rennin activity and androgen levels according to age and sex for at least two years. Blood fasting was used to obtain glucose, lipoproteins, triglycerides, total cholesterol and insulin levels. Obesity in the adult group was defined by BMI >= 30 kg/m², and in the young group by BMI > 95th percentile. Metabolic syndrome was defined by the NCEP ATPIII criteria. Family history of the hypertension, diabetes, dyslipidemia, obesity and/or cardiovascular disease was also evaluated. The BclI, A3669G, ER22/23EK and N363S alleles of the NR3C1 gene and 4436InsA of the HSD11B1 gene were genotyped and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Correlation analyses were performed by Pearson correlation test. Results: obesity was observed in 31.7% of children and 23.5% of adults. SM was observed in 14.6% of young and 7.3% of adult patients. SM prevalence was higher in the obese group than the nonobese group (children and adults). There was no significant correlation between GC dose and BMI, sex, clinical form or treatment duration. Prevalence of family history of obesity, hypertension, dyslipidemia and cardiovascular disease was higher in the obese than in non-obese patients (children and adults). The BclI, A3669G and 4436InsA polymorphisms were found in 23.2%, 9.7% and 14.6% of the alleles in children, respectively and in 26.4%, 9.6% and 18.4% of the alleles in adults. The A3669G variant was associated to increased LDL-c levels in comparison with noncarriers in the young group. The BclI adult carriers presented higher BMI, abdominal circumference and systolic blood pressure in comparison with noncarriers. Statistically significant differences were not observed in the metabolic profile between carriers and non-carriers of the 4436InsA polymorphism (children and adults). Conclusion: in the present study, which analyzed the clinical and metabolic profile of 21-OHD patients, high obesity prevalence, independent of GC dose and treatment duration, was observed. Adverse metabolic profile was mainly associated with obesity and genetic predisposition, such as family history and NR3C1 polymorphisms

Orientador: Chung Man Chin / Banca: Renato Farina Menegon / Banca: Cleverton Roberto de Andrade / Banca: Eduardo René Perez Gonzalez / Banca: Jean Leandro dos Santos / Resumo: Os glicocorticoides (GCs) são fármacos utilizados amplamente na terapêutica devido suas atividades anti-inflamatória, imunossupressora e antiangiogênica. Possuem a capacidade de reduzir a transcrição de uma série de enzimas/proteínas inflamatórias como a ciclooxigenase-2 (COX-2), lipoxigenases, fosfolipase A2, óxido nítrico sintase induzida (iNOS) e citocinas pró-inflamatórias como o fator de necrose tumoral alfa (TNF-alfa), super expresso em diversas doenças inflamatórias. Porém o seu uso é limitado devido as diversas reações adversas. Derivados ftalimídicos também têm sido relatados na literatura com importante atividade anti-inflamatória. Dessa forma, no presente trabalho, planejou-se através da estratégia de hibridação molecular, novos derivados anti-inflamatórios esteroides moduladores da citocina TNF-alfa, úteis no tratamento de doenças inflamatórias crônicas. Nesse contexto, foram sintetizados e caracterizados seis compostos derivados da prednisolona e budesonida da série Lapdesf GL-FT 1-6. Os compostos finais foram avaliados quanto à atividade anti-inflamatória em modelo de colite ulcerativa distal, atividade imunossupressora e determinação da viabilidade celular necessária para o ensaio de doseamento da citocina TNF-alfa. Além disso, os compostos GLFT 2 e 3 foram avaliados quanto à atividade anti-inflamatória em modelo de edema de pata. No ensaio de colite ulcerativa os compostos Lapdesf GL-FT 5 e 6 foram iguais estatisticamente ao controle negativo e apresentaram at... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Glucocorticoids (GCs) are drugs widely used in therapy due their anti-inflammatory, immunosuppressive and antiangiogenic activities. They have the ability to reduce the transcription of a series of inflammatory enzymes / proteins such as cyclooxygenase-2 (COX2), lipoxygenases, phospholipase A2, induced nitric oxide synthase (iNOS) and proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), over expressed in several inflammatory diseases. However, its use is limited due to the various adverse reactions. Phthalimide derivatives have also been reported in the literature with important antiinflammatory activity. Thus, in the present work, new steroid anti-inflammatory derivatives as TNF-alpha cytokine modulators, useful in the treatment of chronic inflammatory diseases, have been designed through the molecular hybridization strategy. In this context, six compounds prednisolone and budesonide derivatives, Lapdesf GL-FT series, were synthesized and characterized. The final compounds were evaluated for anti-inflammatory activity in a model of distal ulcerative colitis, immunosuppressive activity and cell viability assay required for the TNF-alpha assay. In addition, GLFT 2 and 3 compounds were evaluated for anti-inflammatory activity in paw edema model. In the ulcerative colitis test, Lapdesf GL-FT 5 and 6 compounds were statistically equal to the negative control and showed anti-inflammatory activity with regression of the ulceration caused by the induction with acetic acid. Compound 5 also showed improvement in clinical signs such as weight gain and increased survival of animals and total regression of ulceration in 83.3% of treated animals. GLFT 1 and 3 compounds were statistically different from the positive and negative control groups. They presented antiinflammatory activity with... (Complete abstract click electronic access below) / Doutor

Objetivo: Uma série de casos sugerindo uma possível associação de pancreatite aguda (PA) e síndrome de ativação macrofágica (SAM) em lúpus eritematoso sistêmico pediátrico (LESP) foi reportada em dez crianças no nosso serviço, no entanto, não existem dados relativos à comparação entre PA e SAM em grandes populações de LESP e LES adulto (LESA). Métodos: Este estudo incluiu 362 pacientes LESP e 1.830 pacientes LESA. SAM foi diagnosticada de acordo com os critérios diagnósticos preliminares e PA de acordo com a presença de dor abdominal e/ou vômitos associados a um aumento de enzimas pancreáticas e/ou alterações radiológicas pancreáticas nos exames de ultrassonografia e/ou tomografia abdominal. Dados demográficos, características clínicas, SLEDAI-2K, SLICC/ACR-DI e tratamento foram avaliados. Resultados: A frequência de PA foi significantemente aumentada no LESP em comparação ao LESA [12/362 (3,3%) vs. 20/1830 (1,1%), p=0,003], com similar duração da PA nos dois grupos [22 (6-60) vs. 15 (4-90), dias, p=0,534]. As frequências de SAM (85% vs 30%, p=0,003) e óbito (31% vs. 0%; p=0,017) foram significantemente elevadas em crianças com PA comparadas com adultos com PA. Na análise dos pacientes com PA e SAM em comparação com os com somente PA sem SAM demonstrou que a idade dos pacientes com PA e SAM foi significantemente menor em comparação com aqueles sem SAM [15 (8,8- 55) vs. 33,5 (10,2-45,7) anos, p=0,007]. As frequências de febre (94% vs. 37%, p=0,001), leucopenia (82% vs. 19%, p=0,0001), trombocitopenia (65% vs. 19%, p=0,013), hipertrigliceridemia (87% vs. 42%, p=0,037) e hiperferritinemia (93% vs. 37%, p=0,011) foram significantemente aumentadas nos pacientes com PA e SAM comparados aos pacientes com somente PA. A concomitância de febre e hiperferritinemia foi significantemente mais freqüente no primeiro grupo (86% vs. 12%, p=0,0015). Conclusões: Este estudo forneceu novos dados que evidenciaram que SAM ocorreu na maioria dos LESP com PA com uma maior mortalidade em comparação com LESA. Além disso, foram identificados em pacientes com PA e SAM, um conjunto de parâmetros clínicos e laboratoriais associado com as duas complicações / Objective: We previously reported a case series of acute pancreatitis (AP) and macrophage activation syndrome (MAS) in childhood (cSLE) patients, however there are no data regarding the comparison of AP and MAS in large populations of cSLE and adult SLE (aSLE). Methods: This study included 362 cSLE and 1,830 aSLE patients. MAS was diagnosed according to preliminary diagnostic guidelines and AP according to the presence of abdominal pain or vomiting associated to an increase of pancreatic enzymes and/or pancreatic radiological abnormalities. Demographic data, clinical features, SLEDAI-2K, SLICC/ACR-DI and treatment were assessed. Results: Higher and significant frequency of AP in cSLE compared to aSLE patients [12/362(3.3%) vs. 20/1830(1.1%), p=0.003], with similar AP duration [22(6- 60) vs. 15(4-90) days, p=0.534]. MAS (85% vs. 30%, p=0.003) and death by MAS complication (31% vs. 0%, p=0.017) were significantly higher in children with AP compared with aSLE with AP. Further analysis of patients with AP and MAS compared with AP without MAS demonstrated that age in MAS patients was significantly lower compared with those without this complication [15(8.8-55) vs. 33.5(10.2-45.7) years, p=0.007]. The frequencies of fever (94% vs. 37%,p=0.001), leucopenia (82% vs. 19%,p=0.0001), thrombocytopenia (65% vs. 19%,p=0.013), hypertriglyceridemia (87% vs. 42%,p=0.037) and hyperferritinemia (93% vs. 37%,p=0.011) were also more frequently observed in AP patients with MAS compared in AP patients without MAS. Fever and hyperferritinemia concomitantly were more frequent in the former group (86% vs. 12%, p=0.0015). Conclusions: This study provides novel data demonstrating that MAS occur in the majority of cSLE with AP with a higher mortality compared to aSLE. In addition, we identified in AP patients, a cluster of MAS clinical and laboratorial parameters more associated with this complication

Objetivo: Uma série de casos sugerindo uma possível associação de pancreatite aguda (PA) e síndrome de ativação macrofágica (SAM) em lúpus eritematoso sistêmico pediátrico (LESP) foi reportada em dez crianças no nosso serviço, no entanto, não existem dados relativos à comparação entre PA e SAM em grandes populações de LESP e LES adulto (LESA). Métodos: Este estudo incluiu 362 pacientes LESP e 1.830 pacientes LESA. SAM foi diagnosticada de acordo com os critérios diagnósticos preliminares e PA de acordo com a presença de dor abdominal e/ou vômitos associados a um aumento de enzimas pancreáticas e/ou alterações radiológicas pancreáticas nos exames de ultrassonografia e/ou tomografia abdominal. Dados demográficos, características clínicas, SLEDAI-2K, SLICC/ACR-DI e tratamento foram avaliados. Resultados: A frequência de PA foi significantemente aumentada no LESP em comparação ao LESA [12/362 (3,3%) vs. 20/1830 (1,1%), p=0,003], com similar duração da PA nos dois grupos [22 (6-60) vs. 15 (4-90), dias, p=0,534]. As frequências de SAM (85% vs 30%, p=0,003) e óbito (31% vs. 0%; p=0,017) foram significantemente elevadas em crianças com PA comparadas com adultos com PA. Na análise dos pacientes com PA e SAM em comparação com os com somente PA sem SAM demonstrou que a idade dos pacientes com PA e SAM foi significantemente menor em comparação com aqueles sem SAM [15 (8,8- 55) vs. 33,5 (10,2-45,7) anos, p=0,007]. As frequências de febre (94% vs. 37%, p=0,001), leucopenia (82% vs. 19%, p=0,0001), trombocitopenia (65% vs. 19%, p=0,013), hipertrigliceridemia (87% vs. 42%, p=0,037) e hiperferritinemia (93% vs. 37%, p=0,011) foram significantemente aumentadas nos pacientes com PA e SAM comparados aos pacientes com somente PA. A concomitância de febre e hiperferritinemia foi significantemente mais freqüente no primeiro grupo (86% vs. 12%, p=0,0015). Conclusões: Este estudo forneceu novos dados que evidenciaram que SAM ocorreu na maioria dos LESP com PA com uma maior mortalidade em comparação com LESA. Além disso, foram identificados em pacientes com PA e SAM, um conjunto de parâmetros clínicos e laboratoriais associado com as duas complicações / Objective: We previously reported a case series of acute pancreatitis (AP) and macrophage activation syndrome (MAS) in childhood (cSLE) patients, however there are no data regarding the comparison of AP and MAS in large populations of cSLE and adult SLE (aSLE). Methods: This study included 362 cSLE and 1,830 aSLE patients. MAS was diagnosed according to preliminary diagnostic guidelines and AP according to the presence of abdominal pain or vomiting associated to an increase of pancreatic enzymes and/or pancreatic radiological abnormalities. Demographic data, clinical features, SLEDAI-2K, SLICC/ACR-DI and treatment were assessed. Results: Higher and significant frequency of AP in cSLE compared to aSLE patients [12/362(3.3%) vs. 20/1830(1.1%), p=0.003], with similar AP duration [22(6- 60) vs. 15(4-90) days, p=0.534]. MAS (85% vs. 30%, p=0.003) and death by MAS complication (31% vs. 0%, p=0.017) were significantly higher in children with AP compared with aSLE with AP. Further analysis of patients with AP and MAS compared with AP without MAS demonstrated that age in MAS patients was significantly lower compared with those without this complication [15(8.8-55) vs. 33.5(10.2-45.7) years, p=0.007]. The frequencies of fever (94% vs. 37%,p=0.001), leucopenia (82% vs. 19%,p=0.0001), thrombocytopenia (65% vs. 19%,p=0.013), hypertriglyceridemia (87% vs. 42%,p=0.037) and hyperferritinemia (93% vs. 37%,p=0.011) were also more frequently observed in AP patients with MAS compared in AP patients without MAS. Fever and hyperferritinemia concomitantly were more frequent in the former group (86% vs. 12%, p=0.0015). Conclusions: This study provides novel data demonstrating that MAS occur in the majority of cSLE with AP with a higher mortality compared to aSLE. In addition, we identified in AP patients, a cluster of MAS clinical and laboratorial parameters more associated with this complication

Les glucocorticoïdes (GC), généralement sécrétés par le cortex surrénalien, exercent de très nombreuses fonctions dans l’organisme, via leur liaison au récepteur des glucocorticoïdes (GR). Les rares mutations inactivatrices du GR déjà décrites, sont responsables d’un syndrome de résistance aux GC et peuvent conduire à une hypertension artérielle (HTA), une hyperplasie surrénalienne (HBS), un hirsutisme et une obésité. Dans ce travail, nous avons caractérisé fonctionnellement 13 variants hétérozygotes du GR (expression, transactivation, localisation subcellulaire,...). Six variants du GR, découverts par séquençage à haut débit (NGS) ne sont pas pathogènes alors que 7 mutations hétérozygotes originales délétères ont été identifiées dans le cadre du protocole hospitalier de recherche clinique (Muta-GR). Ce PHRC a permis de préciser une prévalence à 5% de mutations inactivatrices du GR dans une cohorte de 100 patients avec HBS associée à une HTA et/ou un hypercortisolisme biologique sans signe clinique de Cushing.Une haploinsuffisance du GR, démontrée par la diminution d’induction par la déxamethasone du gène cible FKBP5, a été mise en évidence dans les fibroblastes cutanés de certains patients porteurs de mutations inactivatrices du GR. Ces patients présentent souvent un hypercorticisme avec hypokaliémie, aldostérone et rénine basse, signant un pseudohyperaldostéronisme. Nous avons démontré que le gène HSD11B2 codant pour l’enzyme 11β-HSD2, assurant l’inactivation des GC, est une cible directe du GR comme démontré par transfection transitoire de gène-rapporteur, RT-qPCR, LC/MSMS et ChIP. L’établissement des modèles de knock-in de mutations GR par stratégie Crispr/cas9 dans des lignées cellulaires préadipocytaires ou corticosurrénaliennes humaines s’est soldé par un échec. Ce travail devrait faciliter la sélection des patients chez qui la recherche de mutation inactivatrice du GR doit être faite et invite à un suivi régulier de ces patients. / Glucocorticoids (GC) regulate many essential biological functions by activating the glucocorticoid receptor (GR). GR loss-of function mutations are responsible for GC resistance syndrome, often associated with high blood pressure, hirsutism, bilateral adrenal hyperplasia (BAH) and obesity. Herein, functional characterization of 13 GR variants is presented (expression and binding studies, transactivation assays, subcellular localization) 6 variants were discovered with next-generating sequencing and had no functional impact on GR signaling while 7 GR loss-of-function mutations were mainly discovered during the National Clinical Hospital Research Program, Muta-GR. This PHRC discloses a 5% prevalence of GR loss-of-function mutations in a cohort of 100 patients with BAH, biological hypercortisolism and/or hypertension without Cushing signs. A GR haploinsuffisiency was demonstrated by a reduced dexamethasone-induced FKBP5 expression in skin fibroblasts of some patients harbouring GR loss-of-function mutations. These patients often presented with hypercorticism, hypokalemia, low renin and aldosterone levels, consistent with a pseudohypermineralocorticism. We showed that HSD11B2 encoding the 11β-HSD2 enzyme inactivating GC, is a direct GR target gene by transient transfection of reporter gene, RT-qPCR, LC/MSMS and ChIP. We failed to introduce GR loss-of-function mutations in human preadipocytes and adrenocortical cells by Crispr/Cas 9 technology. This work should facilitate selection of patients in whom GR mutation may be search, enabling an appropriate follow-up.