RESUMO
We asked whether plasma concentrations of endothelin-1 (ET-1) or adrenomedullin (ADM) are altered by different activity states of the renin-angiotensin-aldosterone system (RAAS). Levels of ET-1 and ADM were studied in patients with primary aldosteronism (n = 15), essential hypertension (n = 15), and adrenal insufficiency (n = 7). Effects of fludrocortisone, dexamethasone, or spironolactone treatment on ET-1 and ADM levels were also analyzed. Plasma ET-1 and ADM concentrations did not differ significantly between the patient groups. After fludrocortisone, dexamethasone, or spironolactone treatment, both ET-1 and ADM did not change significantly. The data support the hypothesis that the RAAS is not directly linked with the ET-1/ADM system.
Assuntos
Adrenomedulina/sangue , Anti-Inflamatórios/uso terapêutico , Dexametasona/uso terapêutico , Endotelina-1/sangue , Fludrocortisona/uso terapêutico , Hipertensão/tratamento farmacológico , Espironolactona/uso terapêutico , Insuficiência Adrenal/tratamento farmacológico , Adulto , Biomarcadores/sangue , Diuréticos/uso terapêutico , Feminino , Humanos , Hiperaldosteronismo/tratamento farmacológico , Hipertensão/sangue , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Sistema Renina-Angiotensina/efeitos dos fármacosRESUMO
The Wnt-signaling pathway regulates ß-cell functions. It is not known how the expression of endogenous Wnt-signaling molecules is regulated in ß-cells. Therefore, we investigated the effect of antidiabetic drugs and glucose on the expression of Wnt-signaling molecules in ß-cells. Primary islets were isolated and cultured. The expression of Wnt-signaling molecules (Wnt-4, Wnt-10b, Frizzled-4, LRP5, TCF7L2) and TNFα was analyzed by semiquantitative PCR and Western blotting. Transient transfections were carried out and proliferation assays of INS-1 ß-cells performed using [(3)H]thymidine uptake and BrdU ELISA. Insulin secretion was quantified. A knockdown (siRNA) of Wnt-4 in ß-cells was carried out. Exendin-4 significantly increased the expression of Wnt-4 in ß-cells on the mRNA level (2.8-fold) and the protein level (3-fold) (P < 0.001). The effect was dose dependent, with strongest stimulation at 10 nM, and it was maintained after long-term stimulation over 4 wk. Addition of exd-(9-39), a GLP-1 receptor antagonist, abolished the effect of exendin-4. Treatment with glucose, insulin, or other antidiabetic drugs had no effect on the expression of any of the examined Wnt-signaling molecules. Functionally, Wnt-4 antagonized the activation of canonical Wnt-signaling in ß-cells. Wnt-4 had no effect on glucose-stimulated insulin secretion or insulin gene expression. Knocking down Wnt-4 decreased ß-cell proliferation to 45% of controls (P < 0.05). In addition, Wnt-4 and exendin-4 treatment decreased the expression of TNFaα mRNA in primary ß-cells. These data demonstrate that stimulation with exendin-4 increases the expression of Wnt-4 in ß-cells. Wnt-4 modulates canonical Wnt signaling and acts as regulator of ß-cell proliferation and inflammatory cytokine release. This suggests a novel mechanism through which GLP-1 can regulate ß-cell proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos/farmacologia , Peçonhas/farmacologia , Proteína Wnt4/genética , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Exenatida , Regulação da Expressão Gênica/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/farmacologia , Hipoglicemiantes/farmacologia , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/fisiologia , Metformina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/farmacologia , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/metabolismo , Receptores de Glucagon/fisiologia , Rosiglitazona , Tiazolidinedionas/farmacologia , Tolbutamida/farmacologia , Regulação para Cima/efeitos dos fármacos , Proteína Wnt4/antagonistas & inibidores , Proteína Wnt4/metabolismoRESUMO
OBJECTIVE: Diabetes mellitus type 2 (DM2) is a risk factor for coronary heart disease (CHD). While there is a clear correlation of fasting blood glucose (FBG) and 2 h post-challenge blood glucose values (2h-BG) with microvascular complications, the risk for CHD conferred by glucose dysregulation antecedent to DM2 is less clear. Therefore, we investigated associations of FBG and 2h-BG values with the prevalence of CHD assessed by coronary angiography as the most sensitive diagnostic tool. RESEARCH DESIGN AND METHODS: Coronary angiography was performed in 1394 patients without known DM. Capillary blood glucose was analyzed before and 2 h after an oral glucose tolerance test. Associations between FBG as well as 2h-BG levels and the risk for CHD were assessed by logistic regression analysis. RESULTS: 1064 (75%) of patients were diagnosed with CHD. 204 (15%) were diagnosed with so far unknown DM2, 274 (20%) with isolated impaired fasting glucose (IFG), 188 (13%) with isolated impaired glucose tolerance (IGT) and 282 (20%) with both, IGT and IFG. We found a continuous increase in the risk for CHD with fasting and post-challenge blood glucose values even in the subdiabetic range. This correlation did however not suggest clear cut-off values. The increase in risk for CHD reached statistical significance at FBG levels of > 120 mg/dl (Odds Ratio of 2.7 [1.3-5.6] and 2h-BG levels > 140 mg/dl (141-160 mg/dl OR 1.8 [1.1-2.9], which was however lost after adjusting for age, sex and BMI. CONCLUSIONS: In our study population we found a continuous increased risk for CHD at fasting and 2h-BG levels in the sub-diabetic glucose range, but no clear cut-off values for cardiovascular risk.
Assuntos
Glicemia/metabolismo , Estenose Coronária/diagnóstico por imagem , Estenose Coronária/epidemiologia , Hiperglicemia/complicações , Idoso , Angiografia Coronária , Progressão da Doença , Feminino , Humanos , Hiperglicemia/sangue , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Prevalência , Estudos Retrospectivos , Fatores de RiscoRESUMO
Cushing's disease rarely appears as a consequence of hereditary disease. However, familial diseases with diminished glucocorticoid feedback are associated with secondary hypercorticotropinism and have been shown to give rise to pituitary adenomas. We here describe the rare case of a 30-year old female patient with congenital adrenal hyperplasia who also showed clinical signs and a typical history of hypercortisolism that was specified as Cushing's disease. After removal of a pituitary microadenoma, serum-cortisol levels fell below normal and the symptoms improved. However, after four years the menstrual cycle was irregular again and ACTH levels were in the upper range of normal. A corticotropin challenge showed a minor cortisol response but a marked increase in 17-hydroxyprogesterone serum concentrations. Genetic analysis revealed a homozygous mutation in exon 7 of the CYP21A2 gene (CTG>TTG, p.V281L). We conclude that a marked ACTH drive was able to override insufficient 21-hydroxylation and even to cause hypercortisolism. Although we describe a rare case, the impairment of the glucocorticoid feedback system in the context of congenital adrenal hyperplasia and other diseases may contribute to the development of secondary hypercorticotropinism as well as corticotropin producing adenomas.
Assuntos
Hiperplasia Suprarrenal Congênita/complicações , Hipersecreção Hipofisária de ACTH/complicações , Adenoma/complicações , Adenoma/diagnóstico , Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Adulto , Feminino , Humanos , Hipersecreção Hipofisária de ACTH/diagnóstico , Neoplasias Hipofisárias/complicações , Neoplasias Hipofisárias/diagnóstico , Esteroide 21-Hidroxilase/genéticaRESUMO
The adrenal gland contains a well-organized network of blood vessels, and adrenocortical cells are situated in close proximity to endothelial cells. Recently, several new mechanisms have been characterized that control the release of aldosterone by adrenocortical cells, including the involvement of endothelial-cell-derived factors. Interestingly, a CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), which is necessary for adrenal development, has been linked to aldosterone synthesis. We have therefore examined the effects of endothelial-cell-conditioned medium (ECCM), as produced during the incubation of human umbilical vein endothelial cells for 24 h, on the promoter activity and mRNA and protein expression of CITED2 in adrenocortical cells as represented by the NCI-H295R cell line. We have found a dose-dependent effect of ECCM on CITED2 promoter activity; this peaks at 480%. Activation of the CITED2 promoter occurs in parallel to an increase in CITED2 messenger RNA (as quantified by real-time polymerase chain reaction) and protein. The stimulatory effect of ECCM can be reversed by blocking mitogen-activated protein kinase activity with the MEK1-inhibitor PD98059. We conclude that products secreted by endothelial cells control not only steroidogenesis, but also factors that are important for adrenocortical development, thereby highlighting the role of cellular interactions within adrenocortical development and physiology.
Assuntos
Glândulas Suprarrenais/metabolismo , Células Endoteliais/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Glândulas Suprarrenais/citologia , Linhagem Celular Tumoral , Células Endoteliais/citologia , Humanos , Imuno-Histoquímica , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Transativadores/genéticaRESUMO
PURPOSE: No relevant breakthrough has yet been achieved in the identification of tumor antigens in many neuroendocrine cancer types that exist, such as malignant gastrinoma, insulinoma, or medullary thyroid carcinoma. The aim of this study was to proof the concept of dendritic cell immunization with a tumor cell-specific polypeptide hormone as a target molecule in a transgenic mouse model for medullary thyroid carcinoma (Ret/Cal mice). EXPERIMENTAL DESIGN: Ret/Cal mice were repeatedly immunized for up to 6 months with amino acid-modified (xenogenic) calcitonin-pulsed dendritic cells. Xenogenic calcitonin was chosen for immunization due to its higher immunogenicity as compared with murine calcitonin. RESULTS: Lymph nodes from control protein-immunized mice did not show any macroscopic abnormalities, whereas tumor peptide-treated mice revealed in general profoundly enlarged lymph nodes. In tetramer analysis of paratumorous lymph nodes, 1.9% to 3.1% of all infiltrating CD8(+) T cells were specific for one of three tumor epitopes tested. Analysis of the activated IFN-gamma-secreting component in splenic cells revealed an average of 2.8% tumor epitope-specific CD8(+) cells. Immunohistochemistry revealed strong CD8(+) tumor infiltration in calcitonin-vaccinated mice. In addition, these cells also showed strong in vitro lysis capacity at up to 63.3%. Most importantly, calcitonin-immunized mice revealed largely diminished tumor outgrowth (-74.3%) compared with control mice (P < 0.0001). Likewise, serum calcitonin levels in calcitonin-vaccinated Ret/Cal mice were lower than in the control group. CONCLUSION: These results have a major effect, as they are the first to establish a role for xenogenic polypeptide hormones as target molecules for immunotherapy in endocrine malignancies.
Assuntos
Vacinas Anticâncer/química , Células Dendríticas/citologia , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/terapia , Hormônios Peptídicos/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Carcinoma Medular/terapia , Feminino , Interferon gama/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peptídeos/química , Neoplasias da Glândula Tireoide/metabolismoRESUMO
Objective Insulinomas are rare pancreatic endocrine tumors characterized by hypoglycemia. Guidelines by the Endocrine Society (ES), the European (ENETS) and the North American (NANETS) Neuroendocrine Tumor Societies provide divergent diagnostic criteria. This study compared the diagnostic accuracy of these different criteria during the 72-h fasting test. Design Retrospective cohort study. Methods From 2000 to 2014, 64 patients with a suspected insulinoma underwent a 72-h fasting test and were included in the analysis. This study assessed the diagnostic sensitivity, specificity and accuracy based on venous blood glucose and corresponding insulin levels measured by electrochemiluminescence immunoassay (ECLIA). Results Based on 64 individuals (18 with, 46 without insulinoma), the ES criteria provided a diagnostic sensitivity of 0.94 (0.73-1.00), specificity of 0.89 (0.76-0.96) and accuracy of 0.91 (0.81-0.96). ENETS/NANETS criteria reached a diagnostic sensitivity of 0.78 (0.52-0.94), specificity of 1.00 (0.92-1.00) and accuracy of 0.94 (0.85-0.98). Conclusions These results point to a higher diagnostic sensitivity with less specificity for diagnosing insulinoma using ES criteria and a higher specificity at lower sensitivity by using ENETS/NANETS criteria. Before considering these results when applying the different criteria in clinical practice, the results should be confirmed in further studies comprising larger cohorts.
Assuntos
Insulinoma/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Glicemia/análise , Jejum , Feminino , Humanos , Insulina/sangue , Insulinoma/sangue , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/sangue , Guias de Prática Clínica como Assunto , Sensibilidade e EspecificidadeRESUMO
Up to now, no relevant tumor antigen has been identified in medullary thyroid carcinoma (MTC). The aim of the present study was to prove the concept of an immunization with an amino acid-modified calcitonin (CT) for the treatment of MTC in a transgenic mouse model. Amino acid-modified (human) CT has been chosen for vaccination because of its higher binding affinity to the murine H2-Kb-MHC molecule. Mice were immunized over 6 months with monthly injections of amino acid-modified CT-pulsed dendritic cells. For enumeration of tumor epitope-specific CD8+ cytotoxic T cells, tetramer analyses were performed. CT peptide-treated mice revealed a mean 0.73 +/- 0.45 and 0.91 +/- 0.59% positive cells, depending on the two tetramers tested, whereas no increase was seen in control protein-immunized mice (0.08-0.12% tetramer-positive cells). Importantly, the subset of CT-specific CD8+ T cells also showed a high expression of interferon-gamma. In line with these results, CT-immunized mice also showed an intratumor infiltration with CD8+ T lymphocytes. Importantly, we also found a diminished tumor outgrowth of -57% and a decrease of the serum CT levels (2.0 +/- 0.1 pg/ml) compared with control protein-immunized Ret/Cal mice (3.0 +/- 0.4 pg/ml). In summary, we show that amino acid-modified CT is recognized from the immune system leading to a specific antitumor immune response and a diminished tumor outgrowth in transgenic MTC mice. The results are of potential importance because they might be applicable to patients with metastatic spread of a MTC.
Assuntos
Antígenos de Neoplasias/imunologia , Calcitonina/imunologia , Carcinoma Medular/terapia , Neoplasias da Glândula Tireoide/terapia , Aminoácidos/química , Animais , Especificidade de Anticorpos , Linfócitos T CD8-Positivos/imunologia , Calcitonina/química , Carcinoma Medular/imunologia , Carcinoma Medular/patologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Imunização/métodos , Imunoterapia Ativa , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infiltração de Neutrófilos/imunologia , Neoplasias da Glândula Tireoide/imunologia , Neoplasias da Glândula Tireoide/patologia , Carga TumoralAssuntos
Anticoncepcionais Orais/uso terapêutico , Dexametasona/farmacologia , Hidrocortisona/urina , Adolescente , Adulto , Anticoncepcionais Orais/sangue , Relação Dose-Resposta a Droga , Interações Medicamentosas , Etinilestradiol/uso terapêutico , Reações Falso-Positivas , Feminino , Humanos , Hidrocortisona/sangue , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
CONTEXT: Gastric neuroendocrine tumors are rare neoplasms that originate from gastric enterochromaffin-like (ECL) cells in the oxyntic mucosa. Gastrin and its derivates have been reported to regulate epithelial cell proliferation, migration, and differentiation. Mutations in the epithelial cadherin (E-cadherin) gene have been shown to be associated with the occurrence of diffuse gastric carcinomas in affected families. OBJECTIVE: In this study we investigated the histopathological and molecular findings in the gastrointestinal wall of a patient with multiple endocrine neoplasia type 1 with malignant duodenal gastrinoma and multiple gastric ECL cell tumors, who additionally developed a signet-ring cell carcinoma of the stomach. DESIGN AND PATIENT: Biopsies from the gastrointestinal tract of a patient with multiple endocrine neoplasia type 1 were immunostained for vesicular monoamine transporter-2 and E-cadherin. Nonamidated gastrin products were measured in the serum of the patient using antibodies that react with progastrin, Gly-extended, and amidated gastrins. Genetic analyses were performed to exclude germ-line mutations within the E-cadherin gene. RESULTS: Immunohistochemical studies of gastric ECL cell tumors showed a largely diminished E-cadherin expression in comparison to gastric surface mucosa cells and a loss of E-cadherin expression in the cells of the signet-ring carcinoma. Detailed biochemical measurements revealed progastrin concentrations that were approximately 20%, and Gly-gastrin concentrations that were approximately 10% the amidated gastrin concentrations in plasma. Molecular analyses revealed no E-cadherin germ-line mutation. CONCLUSION: Our immunohistochemical studies might suggest that the gastrinoma-associated excessive progastrin tissue concentrations led to diminished expression of E-cadherin within the gastric mucosa and promoted tumor development of a signet-ring cell carcinoma.
Assuntos
Carcinoma de Células em Anel de Sinete/complicações , Gastrinas/sangue , Neoplasia Endócrina Múltipla Tipo 1/complicações , Neoplasias Gástricas/complicações , Síndrome de Zollinger-Ellison/complicações , Caderinas/genética , Carcinoma de Células em Anel de Sinete/diagnóstico por imagem , Carcinoma de Células em Anel de Sinete/genética , Carcinoma de Células em Anel de Sinete/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Neoplasia Endócrina Múltipla Tipo 1/diagnóstico por imagem , Neoplasia Endócrina Múltipla Tipo 1/patologia , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Ultrassonografia , Síndrome de Zollinger-Ellison/diagnóstico por imagem , Síndrome de Zollinger-Ellison/genética , Síndrome de Zollinger-Ellison/patologiaRESUMO
The putative transcriptional corepressor ETO/MTG8 has been extensively studied due to its involvement in a chromosomal translocation causing the t(8;21) form of acute myeloid leukemia. Despite this, the role of ETO in normal physiology has remained obscure. Here we show that ETO is highly expressed in preadipocytes and acts as an inhibitor of C/EBPbeta during early adipogenesis, contributing to its characteristically delayed activation. ETO prevents both the transcriptional activation of the C/EBPalpha promoter by C/EBPbeta and its concurrent accumulation in centromeric sites during early adipogenesis. ETO expression rapidly reduces after the initiation of adipogenesis, and this is essential to the normal induction of adipogenic gene expression. These findings define, for the first time, a molecular role for ETO in normal physiology as an inhibitor of C/EBPbeta and a novel regulator of early adipogenesis.
Assuntos
Tecido Adiposo/crescimento & desenvolvimento , Tecido Adiposo/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteína 1 Parceira de Translocação de RUNX1 , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , TransfecçãoRESUMO
Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of ß-catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect ß-catenin activity in adrenocortical NCIH295R cells. Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of ß-catenin and an increase in ß-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway. Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced ß-catenin-dependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM. Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate ß-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.
Assuntos
Córtex Suprarrenal/citologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , beta Catenina/metabolismo , Linhagem Celular , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Luciferases/metabolismo , Proteínas Quinases/metabolismo , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Adrenal catecholamines and steroids are important regulators of the stress response, immune function, blood pressure, and energy homeostasis. Historically, the two cell populations within the adrenal gland-the steroid-producing adrenocortical cells and the catecholamine-producing chromaffin cells-have been regarded as two independent endocrine systems. Research on adrenal physiology and pathophysiology has therefore largely focused on the individual understanding of each cell type. However, adrenal cortex and medulla appear to be interwoven and show multiple contact zones without separation by connective tissue or interstitial membranes. In vitro studies, animal models, and the analysis of human adrenal pathophysiology have demonstrated critical importance of cortical-chromaffin crosstalk for adrenal function and disease. Thus, chromaffin cells regulate steroid-hormone release by the adrenal cortex and steroids induce catecholamine production in the medulla. Consequently, disorders of the adrenal cortex have been shown to affect chromaffin cell function and vice versa. Mouse models of adrenal cortical dysfunction, such as the targeted disruption of the 21-hydroxylase- or the CRHR1 genes, show alterations in chromaffin cell function, while disruption of tyrosine hydroxylase, a key enzyme in catecholamine synthesis, impairs adrenal cortical function. Accordingly, patients with congenital adrenal hyperplasia (CAH) and Addison's disease show reduced catecholamine biosynthesis. Immense progress in characterizing the mechanisms of chromaffin-cortical interactions has been achieved in recent years. Here, we summarize the current view on intraadrenal communication with respect to adrenal pathophysiology.
Assuntos
Córtex Suprarrenal/citologia , Córtex Suprarrenal/fisiologia , Medula Suprarrenal/fisiologia , Comunicação Celular/fisiologia , Células Cromafins/fisiologia , Doenças das Glândulas Suprarrenais/fisiopatologia , Animais , Humanos , Receptor Cross-Talk/fisiologiaRESUMO
Peroxisome proliferator-activated receptor gamma is a member of the nuclear receptor superfamily involved in adipocyte differentiation and glucose homeostasis. There is evidence that peroxisome proliferator-activated receptor gamma may also act as a tumor suppressor. Here, we demonstrate expression of peroxisome proliferator-activated receptor gamma in benign melanocytic naevi, different variants of primary cutaneous melanomas, and melanoma metastases. Peroxisome proliferator-activated receptor gamma protein and peroxisome proliferator-activated receptor gamma1 mRNA were also detected in human melanoma cell lines. The peroxisome proliferator-activated receptor gamma specific agonists 15-deoxy-Delta12,14-prostaglandin J2, troglitazone, and rosiglitazone dose-dependently inhibited cell proliferation in four melanoma cell lines, whereas a specific agonist of peroxisome proliferator-activated receptor alpha had no such effect. At a concentration of 50 microM rosiglitazone, the most potent peroxisome proliferator-activated receptor gamma agonist tested suppressed cell growth by approximately 90%. Apoptosis could be induced in melanoma cell lines by incubation with tumor-necrosis-factor-related apoptosis-inducing ligand. In contrast, the growth inhibitory effect of peroxisome proliferator-activated receptor gamma activation was independent of apoptosis and seemed to occur primarily through induction of cell cycle arrest. Our data indicate that melanoma cell growth may be modulated through peroxisome proliferator-activated receptor gamma.
Assuntos
Melanoma , Prostaglandina D2/análogos & derivados , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Neoplasias Cutâneas , Tiazolidinedionas , Fatores de Transcrição/agonistas , Fatores de Transcrição/metabolismo , Antineoplásicos/farmacologia , Apoptose , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Cromanos/farmacologia , Relação Dose-Resposta a Droga , Fibrinolíticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fatores Imunológicos/farmacologia , Prostaglandina D2/farmacologia , RNA Mensageiro/análise , Receptores Citoplasmáticos e Nucleares/genética , Rosiglitazona , Tiazóis/farmacologia , Fatores de Transcrição/genética , Troglitazona , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
BACKGROUND: The impact of excessive iodine intake on the development of autoimmune thyroiditis (AIT) is still under debate. Transgenic, antibody-devoid TAZ10 mice spontaneously develop AIT due to autoreactive thyroperoxidase-specific T cells. In this model, development of AIT is determined by a T cell infiltration of the thyroid gland leading to an elevation of serum thyrotropin (TSH) levels and significant weight gain. In the present study we investigated the impact of moderate and high iodine supplementation on the course of disease in these mice, which are immunologically prone to AIT. METHODS: In addition to normal nutrition, mice were supplemented for 20 weeks with 2.5 µg versus 5 µg iodine per milliliter drinking water, which corresponds to a human daily iodine supplementation of 150 µg, 315 µg, and 615 µg iodine. AIT-defining parameters (weight gain, elevation of serum TSH levels, cellular infiltration of the thyroid) and immunologic effects were analyzed. RESULTS: No significant differences were displayed when comparing weight and serum TSH levels in the iodine-supplemented versus control groups. Increased thyroid infiltrates with CD8⺠T cells were detected by fluorescein-activated cell sorter (FACS) and immunofluorescence staining in mice supplemented with elevated iodine amounts (315 µg and 615 µg iodine per day, respectively). Immunologic monitoring revealed selective changes in immune cell frequencies (CD8⺠and regulatory T cells, natural killer [NK] cells) and cytokine production (interferon-γ, interleukin-1α, and interleukin-17), however, without affecting the overall immune balance. CONCLUSION: Our results demonstrate that elevated iodine supplementation has no physical impact on the course of disease in transgenic, antibody-devoid TAZ10 mice, which are immunologically prone to AIT.
Assuntos
Suplementos Nutricionais , Imunidade Celular , Fatores Imunológicos/uso terapêutico , Iodo/uso terapêutico , Células Th1/imunologia , Glândula Tireoide/imunologia , Tireoidite Autoimune/dietoterapia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Citocinas/sangue , Citocinas/metabolismo , Feminino , Fatores Imunológicos/administração & dosagem , Iodo/administração & dosagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Masculino , Camundongos Transgênicos , Tamanho do Órgão , Organismos Livres de Patógenos Específicos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Células Th1/metabolismo , Células Th1/patologia , Glândula Tireoide/metabolismo , Glândula Tireoide/patologia , Tireoidite Autoimune/imunologia , Tireoidite Autoimune/metabolismo , Tireoidite Autoimune/patologia , Tireotropina/sangue , Aumento de PesoRESUMO
Hedgehog (Hh)-signaling pathway is important in embryonic development. Activation of Hh-signaling is associated with tumorigenesis. Recent studies demonstrate that Hh-signaling is involved in the development of the adrenal gland in mice and is important in regulating adrenal proliferation. We studied the expression of Sonic hedgehog (SHH), Smoothened (SMO), Patched1 (PTCH1) and GLI family zinc finger 1 (GLI1) in human adrenal and in adrenocortical tumors using immunohistochemistry and semi-quantitative reverse transcriptase-polymerase chain reaction. Modulation of GLI1 and SMO messenger ribonucleic acid (mRNA) expression was investigated with forskolin. The role of Hh-signaling was studied in NCI-H295R cells and in an immortalized primary cell line using the Hh-agonist smoothened agonist (SAG) and the Hh-antagonist cyclopamine. The Hh-pathway components SHH, GLI1, PTCH1 and SMO were detectable in all adrenal glands. While in cortisol-producing adenomas (CPA), Hh-signaling expression levels were comparable to that in normal adrenal cortex, a much higher mRNA expression of GLI1, SMO and SHH was observed in non-producing adenomas (NPA). Interestingly, stimulation of cultured adrenal cells with forskolin led to a decrease in expression of GLI1 and SMO mRNAs. Antagonism of Hh-signaling resulted in a lower proliferation rate of adrenocortical cells, while Hh-agonism had no significant effect on adrenal cell proliferation. Our data show Hh-signaling activity in adult adrenal glands. Activation of the PKA pathway results in lower expression of Hh-signaling proteins. This might explain the lower expression of the Hh components GLI1 and SMO in CPA in comparison to the higher expression in NPA. Hh-signaling might be involved in the tumorigenesis of NPA.
Assuntos
Adenoma/metabolismo , Neoplasias das Glândulas Suprarrenais/metabolismo , Proliferação de Células , Proteínas Hedgehog/metabolismo , Córtex Suprarrenal/metabolismo , Córtex Suprarrenal/patologia , Linhagem Celular Tumoral , Expressão Gênica , Proteínas Hedgehog/genética , Humanos , Receptores Patched , Receptor Patched-1 , Cultura Primária de Células , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Receptor Smoothened , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Proteína GLI1 em Dedos de ZincoRESUMO
Progressive loss of pancreatic ß-cell mass is a crucial feature of type 2 diabetes mellitus. As ß-cells express very low amounts of the antioxidant enzymes catalase and glutathione peroxidase (GPx), they appear to be particularly vulnerable to oxidative damage in the pathogenesis of diabetes. Here, we investigated the pancreatic expression pattern and regulation of selenoprotein P (Sepp1), which may serve as an additional antioxidant enzyme inside and outside of cells. Sepp1 was detected in rodent pancreas by immunofluorescence and real-time RT-PCR. Regulation of Sepp1 biosynthesis in INS-1 rat insulinoma cells was investigated by real-time RT-PCR, luciferase gene reporter assay, and immunoblotting. Sepp1 and Gpx1 gene expressions in rat pancreas were 58 and 22% respectively of the liver values. Pancreatic Sepp1 expression was restricted to the endocrine tissue, with Sepp1 being present in the α- and ß-cells of mouse islets. In INS-1 insulinoma cells, Sepp1 expression was stimulated by the selenium compound sodium selenate and diminished in the presence of high glucose (16.7 vs 5â mM) concentrations. Sepp1 mRNA stability was also lowered at 16.7â mM glucose. Moreover, Sepp1 mRNA levels were decreased in isolated murine islets cultured in high-glucose (22 âmM) medium compared with normal glucose (5.5 âmM) medium. Pancreatic Sepp1 expression was elevated upon treatment of mice with the ß-cell toxin streptozotocin. This study shows that pancreatic islets express relatively high levels of Sepp1 that may fulfill a function in antioxidant protection of ß-cells. Downregulation of Sepp1 expression by high glucose might thus contribute to glucotoxicity in ß-cells.
Assuntos
Ilhotas Pancreáticas/metabolismo , Selenoproteína P/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Regulação para Baixo , Imunofluorescência , Genes Reporter , Glucose/administração & dosagem , Camundongos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Selenoproteína P/biossíntese , Selenoproteína P/genética , Estreptozocina/farmacologiaRESUMO
Several studies have reported a positive relationship of the body fat mass and bone density. However, it is not clear whether adipocyte-derived signaling molecules directly act on osteoblasts or osteoclasts. Therefore, we investigated the effect of fat cell-secreted factors on the proliferation and differentiation of preosteoblasts and the molecular mechanisms involved. This stimulation led to an increased proliferation of MC3T3-E1 and primary preosteoblastic cells (2.8-fold and 1.5-fold, respectively; p<0.0001), which could be reduced with inhibitors of protein tyrosine kinases, FGFR1 and PI3K. Concordantly, we found human adipocytes to secrete bFGF and bFGF to mimic the effect of adipocyte-secreted factors. The ratio of OPG/RANKL secretion in primary human preosteoblasts increased 9-fold (mRNA and protein) when stimulated with adipocyte-secreted factors. Moreover, osteoblasts which were prestimulated with adipocyte-secreted factors inhibited the formation of osteoclasts. In conclusion, human adipocytes secrete factors that directly act on preosteoblasts and alter their crosstalk with osteoclasts. These in vitro findings reflect the higher bone mass in obese people and attribute it to effects of adipocyte-secreted factors on bone formation.
Assuntos
Adipócitos/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Meios de Cultivo Condicionados , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/fisiologia , Osteoprotegerina/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Ligante RANK/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismoRESUMO
CONTEXT: Hashimoto's thyroiditis (HT) is a common autoimmune disease leading to thyroid destruction due to lymphocytic infiltration. Only rare data are available regarding the recognition of specific cellular antigens, e.g. of thyroperoxidase (TPO) and thyroglobulin (Tg). OBJECTIVE: The aim of this study was to quantify and characterize TPO- and Tg-epitope-specific CD8-positive T cells of HT patients. DESIGN: Six different human leukocyte antigen (HLA)-A2 restricted, TPO- or Tg-specific tetramers were synthesized and used for measuring CD8-positive T cells in HT patients and controls. RESULTS: The frequency of peripheral TPO- and Tg-specific CD8-positive T cells was significantly higher in HLA-A2-positive HT patients (2.8 ± 9.5%) compared with HLA-A2-negative HT patients (0.5 ± 0.7%), HLA-A2-positive nonautoimmune goiter patients (0.2 ± 0.4%), and HLA-A2-positive healthy controls (0.1 ± 0.2%). The frequency of Tg-specific T cells (3.0%) was very similar to those of TPO-specific CD8-positive T cells (2.9%). Subgroup analyses revealed a steady increase of the number of epitope-specific CD8-positive T cells from 0.6 ± 1.0% at initial diagnosis up to 9.4 ± 18.3% in patients with long-lasting disease. Analyses of the number of thyroid-infiltrating cells as well as the cytotoxic capacity revealed a similar picture for TPO- and Tg-specific T cells. CONCLUSION: We here report for the first time that both antigens, TPO and Tg, are recognized by CD8-positive T cells and are involved in the thyroid destruction process leading to clinical disease manifestation.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Autoanticorpos/análise , Doença de Hashimoto/imunologia , Imunidade Celular , Iodeto Peroxidase/antagonistas & inibidores , Proteínas de Ligação ao Ferro/antagonistas & inibidores , Tireoglobulina/antagonistas & inibidores , Glândula Tireoide/imunologia , Adulto , Idoso , Especificidade de Anticorpos , Autoanticorpos/química , Autoantígenos/química , Biópsia por Agulha Fina , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Epitopos/análise , Epitopos/química , Feminino , Bócio/imunologia , Bócio/metabolismo , Bócio/patologia , Antígeno HLA-A2/metabolismo , Doença de Hashimoto/metabolismo , Doença de Hashimoto/patologia , Humanos , Iodeto Peroxidase/química , Proteínas de Ligação ao Ferro/química , Masculino , Pessoa de Meia-Idade , Tireoglobulina/química , Glândula Tireoide/patologia , Adulto JovemRESUMO
Natural killer (NK) cells belong to the innate immune system. Besides their role in antitumor immunity, NK cells also regulate the activity of other cells of the immune system, including dendritic cells, macrophages, and T cells, and may, therefore, be involved in autoimmune processes. The aim of the present study was to clarify the role of NK cells within this context. Using two mouse models for type 1 diabetes mellitus, a new subset of NK cells with regulatory function was identified. These cells were generated from conventional NK cells by incubation with IL-18 and are characterized by the expression of the surface markers CD117 (also known as c-Kit, stem cell factor receptor) and programmed death (PD)-ligand 1. In vitro analyses demonstrated a direct lysis activity of IL-18-stimulated NK cells against activated insulin-specific CD8(+) T cells in a PD-1/PD-ligand 1-dependent manner. Flow cytometry analyses revealed a large increase of splenic and lymphatic NK1.1(+)/c-Kit(+) NK cells in nonobese diabetic mice at 8 wk of age, the time point of acceleration of adaptive cytotoxic immunity. Adoptive transfer of unstimulated and IL-18-stimulated NK cells into streptozotocin-treated mice led to a delayed diabetes development and partial disease prevention in the group treated with IL-18-stimulated NK cells. Consistent with these data, mild diabetes was associated with increased numbers of NK1.1(+)/c-Kit(+) NK cells within the islets. Our results demonstrate a direct link between innate and adaptive immunity in autoimmunity with newly identified immunoregulatory NK cells displaying a potential role as immunosuppressors.