RESUMO
Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207(-) cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14(+)CD16(-) blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14(-) haematopoietic precursors possess a much higher differentiation potential than CD14(+) precursor cells.
Assuntos
Células Epidérmicas , Epiderme/embriologia , Células-Tronco Hematopoéticas/citologia , Células de Langerhans/citologia , Modelos Biológicos , Fenótipo , Antígenos CD34/metabolismo , Sangue Fetal/citologia , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Receptores de Lipopolissacarídeos/metabolismo , Estatísticas não Paramétricas , Técnicas de Cultura de TecidosRESUMO
SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular processes such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less studied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. Small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. A decreased SRPK1/SRPK1a ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells as well as in normal human erythroid progenitor cells. Mass spectrometric analysis of SRPK1a-associated proteins identified multiple classes of RNA-binding proteins including RNA helicases, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and mRNA-associated proteins. Several of the SRPK1a-copurifying proteins have been previously identified in ribosomal and pre-ribosomal complexes, thereby suggesting that SRPK1a may play an important role in linking ribosomal assembly and/or function to erythroid differentiation in human leukaemic cells.
Assuntos
Diferenciação Celular , Eritrócitos/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Bases , Primers do DNA , Humanos , Células K562 , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/fisiologiaRESUMO
The Raf kinases are key signal transducers activated by mitogens or oncogenes. The best studied Raf isoform, Raf-1, was identified as an inhibitor of apoptosis by conventional and conditional gene ablation in mice. c-raf-1(-)(/)(-) embryos are growth retarded and anemic, and die at midgestation with anomalies in the placenta and fetal liver. Here, we show that Raf-1-deficient primary erythroblasts cannot be expanded in culture due to their accelerated differentiation into mature erythrocytes. In addition, Raf-1 expression is down-regulated in differentiating wild-type cells, whereas overexpression of activated Raf-1 delays differentiation. As recently described for human erythroid precursors, we find that caspase activation is necessary for the differentiation of murine fetal liver erythroblasts. Differentiation-associated caspase activation is accelerated in erythroid progenitors lacking Raf-1 and delayed by overexpression of the activated kinase. These results reveal an essential function of Raf-1 in erythropoiesis and demonstrate that the ability of Raf-1 to restrict caspase activation is biologically relevant in a context distinct from apoptosis.
Assuntos
Caspases/metabolismo , Diferenciação Celular/fisiologia , Ativação Enzimática/fisiologia , Eritrócitos/citologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Sequência de Aminoácidos , Animais , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-raf/genéticaRESUMO
Stem cell factor (SCF)-induced activation of phosphoinositide-3-kinase (PI3K) is required for transient amplification of the erythroblast compartment. PI3K stimulates the activation of mTOR (target of rapamycin) and subsequent release of the cap-binding translation initiation factor 4E (eIF4E) from the 4E-binding protein 4EBP, which controls the recruitment of structured mRNAs to polysomes. Enhanced expression of eIF4E renders proliferation of erythroblasts independent of PI3K. To investigate which mRNAs are selectively recruited to polysomes, we compared SCF-dependent gene expression between total and polysome-bound mRNA. This identified 111 genes primarily subject to translational regulation. For 8 of 9 genes studied in more detail, the SCF-induced polysome recruitment of transcripts exceeded 5-fold regulation and was PI3K-dependent and eIF4E-sensitive, whereas total mRNA was not affected by signal transduction. One of the targets, Immunoglobulin binding protein 1 (Igbp1), is a regulatory subunit of protein phosphatase 2A (Pp2a) sustaining mTOR signaling. Constitutive expression of Igbp1 impaired erythroid differentiation, maintained 4EBP and p70S6k phosphorylation, and enhanced polysome recruitment of multiple eIF4E-sensitive mRNAs. Thus, PI3K-dependent polysome recruitment of Igbp1 acts as a positive feedback mechanism on translation initiation underscoring the important regulatory role of selective mRNA recruitment to polysomes in the balance between proliferation and maturation of erythroblasts.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Retroalimentação Fisiológica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Fator de Células-Tronco/farmacologia , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Ativação Enzimática/efeitos dos fármacos , Eritroblastos/citologia , Eritroblastos/efeitos dos fármacos , Eritropoetina/farmacologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/enzimologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR , Fator de Crescimento Transformador beta/farmacologiaRESUMO
BACKGROUND: The stem cell marker Octamer-4 (OCT-4) is expressed in human endometrium. Menstrual cycle-dependency of OCT-4 expression has not been investigated to date. METHODS: In a prospective, single center cohort study of 98 women undergoing hysteroscopy during the follicular (n = 49) and the luteal (n = 40) phases of the menstrual cycle, we obtained endometrial samples. Specimens were investigated for OCT-4 expression on the mRNA and protein levels using reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. Expression of OCT-4 was correlated to menstrual cycle phase. RESULTS: Of 89 women sampled, 49 were in the follicular phase and 40 were in the luteal phase. OCT-4 mRNA was detected in all samples. Increased OCT-4 mRNA levels in the follicular and luteal phases was found in 35/49 (71%) and 27/40 (68%) of women, respectively (p = 0.9). Increased expression of OCT-4 protein was identified in 56/89 (63%) samples. Increased expression of OCT-4 protein in the follicular and luteal phases was found in 33/49 (67%) and 23/40 (58%) of women, respectively (p = 0.5). CONCLUSIONS: On the mRNA and protein levels, OCT-4 is not differentially expressed during the menstrual cycle. Endometrial OCT-4 is not involved in or modulated by hormone-induced cyclical changes of the endometrium.
Assuntos
Biomarcadores/metabolismo , Endométrio/metabolismo , Fase Folicular/metabolismo , Regulação da Expressão Gênica , Fase Luteal/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Adulto , Células-Tronco Adultas/metabolismo , Estudos de Coortes , Endométrio/citologia , Feminino , Humanos , Histeroscopia , Imuno-Histoquímica , Fator 3 de Transcrição de Octâmero/genética , Projetos Piloto , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The cooperation of stem cell factor (SCF) and erythropoietin (Epo) is required to induce renewal divisions in erythroid progenitors, whereas differentiation to mature erythrocytes requires the presence of Epo only. Epo and SCF activate common signaling pathways such as the activation of protein kinase B (PKB) and the subsequent phosphorylation and inactivation of Foxo3a. In contrast, only Epo activates Stat5. Both Foxo3a and Stat5 promote erythroid differentiation. To understand the interplay of SCF and Epo in maintaining the balance between renewal and differentiation during erythroid development, we investigated differential Foxo3a target regulation by Epo and SCF. Expression profiling revealed that a subset of Foxo3a targets was not inhibited but was activated by Epo. One of these genes was Cited2. Transcriptional control of Epo/Foxo3a-induced Cited2 was studied and compared with that of the Epo-repressed Foxo3a target Btg1. We show that in response to Epo, the allegedly growth-inhibitory factor Foxo3a associates with the allegedly growth-stimulatory factor Stat5 in the nucleus, which is required for Epo-induced Cited2 expression. In contrast, Btg1 expression is controlled by the cooperation of Foxo3a with cyclic AMP- and Jun kinase-dependent Creb family members. Thus, Foxo3a not only is an effector of PKB but also integrates distinct signals to regulate gene expression in erythropoiesis.
Assuntos
Eritropoese/fisiologia , Eritropoetina/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Fator de Células-Tronco/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/fisiologia , Células Cultivadas , Análise por Conglomerados , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismoRESUMO
Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor-induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Neoplasias/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores de Transcrição/genética , Células 3T3 , Animais , Sequência de Bases , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Clonagem Molecular , Primers do DNA , Ativação Enzimática , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead , Genes Reporter , Células-Tronco Hematopoéticas/citologia , Humanos , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Fatores de Transcrição/fisiologiaRESUMO
BACKGROUND: Acquired benign vocal fold lesions are among the most common causes of voice problems. Since the local impact of estrogen and progesterone receptors in laryngeal tissue is discussed controversially, the presence of sex hormone receptors in benign vocal fold alterations needs to be clarified. GOAL OF THE STUDY: To investigate the expression of estrogen-alpha receptors (ER-alpha), estrogen-beta receptors (ER-beta), progesterone receptors (PR) and androgen receptors (AR) in acquired benign vocal fold alterations. METHODS: Laryngeal epithelial specimens of 14 patients (13 female, 1 male) taken intraoperatively were investigated using immunohistochemistry in order to objectify ER-alpha, ER-beta, PR and AR. Macroscopically and histopathologically diagnosed edemas of Reinke's space (n = 10), vocal fold polyps (n = 3) and vocal fold nodules (n = 1) were enrolled in this study. RESULTS: No specific nuclear immunohistochemical staining could be seen in the biopsies taken. Only unspecific staining patterns could be observed. CONCLUSION: Sex hormone receptors could not be detected in the specimens tested, thus, any direct influence of sex hormones on the development of benign vocal fold lesions is rather unlikely. The results of this study confirm the impact of vocal fold stress and biomechanical abnormalities on their development due to voice overstraining and abuse.
Assuntos
Doenças da Laringe/metabolismo , Receptores de Esteroides/metabolismo , Prega Vocal/metabolismo , Adulto , Idoso , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Doenças da Laringe/cirurgia , Edema Laríngeo/metabolismo , Edema Laríngeo/cirurgia , Masculino , Pessoa de Meia-Idade , Receptores Androgênicos/metabolismo , Receptores de Progesterona/metabolismo , Prega Vocal/cirurgiaRESUMO
Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Kruppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in beta-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eritrócitos/fisiologia , Eritropoese/fisiologia , Regulação da Expressão Gênica , Hemoglobinas/metabolismo , Fenótipo , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos Hormonais/metabolismo , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/genética , Eritrócitos/citologia , Perfilação da Expressão Gênica , Fatores de Transcrição Kruppel-Like , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno/metabolismo , Fatores de Transcrição/genéticaRESUMO
OBJECTIVES: To investigate the association of two common genetic polymorphisms of the gene encoding for endothelial nitric oxide synthase (Nos3), the enzyme catalyzing the production of nitric oxide (NO), with occurrence of the polycystic ovary syndrome (PCOS). METHODS: In a prospective case-control study, we analyzed 2 polymorphisms of the Nos3 gene cluster (Nos 3 exon 7 Glu298Asp and 27-base pair repeat in intron 4 of Nos3) in a series of 210 premenopausal Caucasian women with PCOS and 171 healthy controls using pyrosequencing and PCR, respectively. Women completed a detailed questionnaire and underwent a peripheral venous puncture, ultrasonography, and a standardized oral glucose tolerance test (OGTT). RESULTS: Genotype frequencies were not significantly different among women with PCOS and controls for the exon 7Nos3 and the intron 4Nos3 polymorphism (p=0.3 and 0.2, respectively). CONCLUSIONS: In our series, two common polymorphisms of the Nos3 gene cluster were not associated with occurrence of PCOS.
Assuntos
Óxido Nítrico Sintase Tipo III/genética , Síndrome do Ovário Policístico/enzimologia , Adulto , Estudos de Casos e Controles , DNA/química , DNA/genética , Feminino , Genótipo , Teste de Tolerância a Glucose , Humanos , Síndrome do Ovário Policístico/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Estudos Prospectivos , Estatísticas não ParamétricasRESUMO
Interleukin-1 (IL1) is a multifunctional cytokine and IL1-mediated inflammatory processes have been proposed to influence the processes of ovulation, fertilization and implantation. All these parameters are also affected in women with polycystic ovary syndrome (PCOS). This study investigated the association of common polymorphisms of the interleukin-1 genes (IL1A and IL1B) with the occurrence and clinical characteristics of PCOS. We evaluated one polymorphism of the IL1alpha gene (IL1A C[-889]T) and two of the IL1beta gene (IL1B promoter C[-511]T and IL1B exon 5 position +3953) in 105 Caucasian women with PCOS and 102 healthy Caucasian controls by polymerase chain reaction. For the mutated IL1A allele, allele frequencies in women with PCOS and controls were 60% and 46%, respectively, versus 40% and 54%, respectively, for the wild type allele. Allele frequencies in women with PCOS and controls were 59% (54%) and 61% (41%), respectively, for the mutated IL1B promoter (mutated IL1B exon 5) and 41% (46%) and 39% (59%), respectively, for the wild type alleles. Presence of a polymorphism in the interleukin-1alpha but not the interleukin-1beta gene was found to correlate with the occurrence of PCOS (p=0.04; odds ratio 1.8). The serum level of FSH and subsequent LH/FSH ratio correlated with the polymorphism of IL1A within the PCOS group (p=0.005 and 0.01, respectively). We have shown that a common polymorphism of the interleukin-1alpha but not interleukin-1beta gene is associated with the presence of PCOS and with clinical parameters of women affected by this condition.
Assuntos
Alelos , Frequência do Gene , Interleucina-1alfa/genética , Interleucina-1beta/genética , Síndrome do Ovário Policístico/genética , Polimorfismo de Nucleotídeo Único , Adulto , Éxons/genética , Feminino , Humanos , Interleucina-1alfa/sangue , Interleucina-1beta/sangue , Síndrome do Ovário Policístico/sangueRESUMO
BACKGROUND: The human larynx is assumed to be a steroid receptor target organ. There are only very limited data on the evidence of steroid receptors in the vocal folds, although voice alterations due to hormonal influence and treatment have been found. GOAL OF THE STUDY: To investigate the expression of estrogen alpha, progesterone, and androgen receptors in human vocal folds (vocalis muscle, glands, lamina propria, epithelium). METHODS: Immunohistochemically, vocal fold cadaver specimens of 15 autopsied patients (6 women, 9 men), which were taken approximately 4 to 8 hours postmortem were investigated. Furthermore, one (male) vocal fold biopsy obtained intraoperatively during a laryngectomy was tested. RESULTS: No specific immunohistochemical staining for the different types of steroid hormones investigated could be observed in either the postmortem taken biopsies nor the intraoperatively one. However, several unspecific staining patterns could be observed. CONCLUSION: The results of this study contradict recently published data and question the expression of sex hormone receptors in the vocal folds. Main causes of false interpretations of unspecific staining are discussed.
Assuntos
Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Prega Vocal/metabolismo , Distúrbios da Voz/diagnóstico , Distúrbios da Voz/metabolismo , Adulto , Biópsia , Feminino , Nível de Saúde , Humanos , Imuno-Histoquímica , Masculino , Prega Vocal/patologiaRESUMO
A prerequisite for proper investigation of self-renewal and differentiation of hematopoietic cells is the possibility to obtain large quantities of homogenous primary progenitors under defined conditions, allowing meaningful biochemical and molecular analyses. These cells should show renewal and differentiation characteristics similar to the in vivo situation. The serum-free culture systems delineated in this chapter meet these requirements, employing primary hematopoietic cells derived from murine fetal liver and human umbilical cord blood, which show physiological self-renewal responses to cytokine/hormone combinations, which in vivo are involved in stress hematopoiesis. We describe the expansion and sustained proliferation of multipotent (mouse) and erythroid (mouse and human) progenitors, responding to physiological signals. Moreover, both mouse and human erythroid progenitors can be induced to undergo synchronous terminal differentiation by addition of high levels of erythropoietin. If fetal liver cells from p53-/- mice are used, respective multipotent and erythroid cells undergo immortalization without an obvious Hayflick crisis, but otherwise retain their primary cell characteristics. Finally, both primary and immortal mouse progenitors can be subjected to genetic manipulation via retroviral constructs with high efficiency.
Assuntos
Embrião de Mamíferos/fisiologia , Células Precursoras Eritroides/fisiologia , Sangue Fetal/fisiologia , Fígado/fisiologia , Animais , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Citocinas/fisiologia , Embrião de Mamíferos/citologia , Sangue Fetal/citologia , Humanos , Fígado/citologia , Camundongos , Camundongos KnockoutRESUMO
OBJECTIVE: To investigate the association of a common 5G/4G polymorphism of the plasminogen activator inhibitor-1 gene (PAI1) with occurrence and clinical characteristics of the polycystic ovary syndrome (PCOS). STUDY DESIGN: In a case-control study, we evaluated a series of 106 Caucasian women with PCOS and 102 healthy controls. Women completed a detailed questionnaire and underwent a peripheral venous puncture, ultrasonography, and a standardized oral glucose tolerance test (OGTT). The PAI1 gene promoter polymorphism was evaluated using PCR. RESULTS: Allele and genotype frequencies were not significantly different among women with PCOS and controls (P=0.3 and 0.6, respectively). In women with PCOS, presence of the 5G/4G polymorphism of PAI1 was not associated with changes in serum hormone levels or with clinical characteristics. CONCLUSIONS: The 5G/4G polymorphism of the PAI1 promoter is not associated with occurrence and phenotype of the PCOS.
Assuntos
Inibidor 1 de Ativador de Plasminogênio/genética , Síndrome do Ovário Policístico/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Humanos , Síndrome do Ovário Policístico/etiologia , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas/genética , População Branca/genéticaRESUMO
Estrogen-induced loss of estrogen receptor (ER) alpha expression limits estrogen responsiveness in many target cells. However, whether such a mechanism contributes to changes in vascular endothelial ER alpha and/or ER beta levels is unclear. Using RT-PCR assays, we did not find any regulation of ER alpha or ER beta mRNA expression in human uterine artery endothelial cell (HUAEC) nuclear extracts on stimulation with 17 beta-estradiol for 1 or 2 h. By contrast, Western analysis on HUAEC extracts revealed that 17 beta-estradiol was capable of down-regulating both ER alpha and ER beta protein starting 1 h after treatment, an effect that can be blocked by pretreatment with tamoxifen as well as with the proteasome inhibitor lactacystin. The proteolysis inhibitors insulin, cycloheximide, and puromycin impede ER alpha, but not ER beta, turnover. Ubiquitin, but not its competitive inhibitor methyl-ubiquitin, induces rapid turnover of both ERs in a cell-free system of MCF-7 and HUAEC extracts. We, thus, propose the existence of estrogen-induced ER degradation that serves to control physiological responses in an estrogen target tissue, i.e. human vascular endothelium, by down- regulating ER alpha as well as ER beta through different proteasomal uptake mechanisms.
Assuntos
Acetilcisteína/análogos & derivados , Cisteína Endopeptidases/farmacologia , Endotélio Vascular/metabolismo , Complexos Multienzimáticos/farmacologia , Receptores de Estrogênio/metabolismo , Útero/irrigação sanguínea , Acetilcisteína/farmacologia , Trifosfato de Adenosina/farmacologia , Especificidade de Anticorpos , Artérias , Western Blotting , Núcleo Celular/metabolismo , Sistema Livre de Células , Células Cultivadas , Cicloeximida/farmacologia , Endotélio Vascular/ultraestrutura , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/farmacologia , Cinética , Complexos Multienzimáticos/antagonistas & inibidores , Complexo de Endopeptidases do Proteassoma , Puromicina/farmacologia , RNA Mensageiro/análise , Receptores de Estrogênio/genética , Tamoxifeno/farmacologiaRESUMO
BACKGROUND: Endometriosis is characterized by the presence of endometrium-like tissue outside the uterus. This condition causes painful periods, chronic pelvic pain, subfertility and a profound reduction in quality of life, especially during women's reproductive years. Currently available medical therapies offer comparatively little therapeutic benefit and are often burdened by considerable side effects. However, since clinical evidence shows that pregnancy leads to alleviation of endometriotic symptoms, we have for the first time examined the effect of human chorionic gonadotrophin (HCG) injections on symptoms such as dysmenorrhea and pelvic pain. PATIENTS AND METHODS: Thirty-one patients with histologically verified endometriosis refractory to therapy received 1 to 2 intramuscular injections of 1500 to 5000 IU HCG per week for a period of 3-12 months. A QoL questionnaire and the visual analog pain intensity scale (VAS) were used to evaluate quality of life and pain intensity, respectively, before and after three months of treatment. RESULTS: Three months of HCG therapy led to a highly significant reduction of endometriosis-related pain (p<0.001, Wilcoxon test) and to improvement of disease-related parameters such as sleeplessness (p<0.001), irritability (p<0.001), overall discomfort (p<0.001), depressive moods (p<0.001) and painful defecation (p=0.01). Dyspareunia and dysmenorrhea also clearly improved (both p<0.001), though HCG did not lead to significant reduction of dysuria (p=0.66). Prolonged therapy with HCG for up to 12 months (mean: 4.42 months) did not lead to reduction of the beneficial effect. CONCLUSIONS: HCG injections lead to significant and clinically relevant reduction in pain intensity and to greatly improved quality of life in women with therapy-refractory endometriosis. The remarkable clinical effect of parenteral HCG in our study will have to be confirmed in additional trials but clearly indicates an extremely promising new perspective in the treatment of endometriosis.
Assuntos
Gonadotropina Coriônica/uso terapêutico , Endometriose/tratamento farmacológico , Adulto , Gonadotropina Coriônica/administração & dosagem , Endometriose/complicações , Endometriose/diagnóstico , Endometriose/fisiopatologia , Feminino , Humanos , Injeções Intramusculares , Pessoa de Meia-Idade , Dor/diagnóstico , Dor/etiologia , Medição da Dor , Estudos Prospectivos , Qualidade de Vida , Estatísticas não Paramétricas , Inquéritos e Questionários , Fatores de TempoRESUMO
While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/farmacologia , Tecido Adiposo/citologia , Adulto , Animais , Bovinos , Separação Celular , Forma Celular/efeitos dos fármacos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Microscopia Crioeletrônica , Meios de Cultura Livres de Soro , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/ultraestrutura , Sangue Fetal/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Microscopia de Fluorescência por Excitação MultifotônicaRESUMO
OBJECTIVE: To assess the association between transsexualism and allele and genotype frequencies of the common cytochrome P450 (CYP) 17 -34 T>C single nucleotide polymorphism (SNP). DESIGN: Case-control study. SETTING: Academic research institution. PATIENT(S): 102 male-to-female (MtF) and 49 female-to-male (FtM) transsexuals, 756 male controls, and 915 female controls. INTERVENTION(S): Buccal swabs and multiplex polymerase chain reaction on a microarray system. MAIN OUTCOME MEASURE(S): Analysis of the CYP17 -34 T>C SNP. RESULT(S): CYP17 -34 T>C SNP allele frequencies were statistically significantly different between FtM transsexuals and female controls (CYP17 T: 55/98 [56%] and CYP17 C: 43/98 [44%] versus CYP17 T: 1253/1826 [69%] and CYP17 C: 573/1826 [31%], respectively). In accordance, genotype distributions were also different between FtM transsexuals and female controls using a recessive genotype model (CYP17 T/T+T/C: 39/49 [80%] and C/C 10/49 [20%] vs. CYP17 T/T+T/C: 821/913 [90%] and C/C 92/913 [10%], respectively). The CYP17 -34 T>C allele and genotype distributions were not statistically significantly different between MtF transsexuals and male controls. Of note, the CYP17 -34 T>C allele distribution was gender-specific among controls (CYP17 C: males; 604 of 1512 [40%] vs. females; 573 of 1826 [31%]). The MtF transsexuals had an allele distribution equivalent to male controls, whereas FtM transsexuals did not follow the gender-specific allele distribution of female controls but rather had an allele distribution equivalent to MtF transsexuals and male controls. CONCLUSION(S): These data support CYP17 as a candidate gene of FtM transsexualism and indicate that loss of a female-specific CYP17 T -34C allele distribution pattern is associated with FtM transsexualism.
Assuntos
Hormônios Esteroides Gonadais/metabolismo , Polimorfismo de Nucleotídeo Único , Esteroide 17-alfa-Hidroxilase/genética , Transexualidade/genética , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Esteroide 17-alfa-Hidroxilase/metabolismo , Transexualidade/enzimologia , População Branca/genéticaRESUMO
BACKGROUND: Luteinizing hormone (LH) and human chorionic gonadotropin (HCG) target their receptor in gonadal and nongonadal cells to stimulate steroidogenesis and cell growth. The aim of the present study was to investigate the expression of HCG/LH-R in endometriosis to elucidate a possible impact of LH and HCG on this disease. MATERIALS AND METHODS: Analysis of HCG/LH-R protein expression in 23 paired samples of ectopic and eutopic tissue of cycling women with endometriosis and in endometrial samples from 22 healthy controls was conducted via immunofluorescence. HCG and HCG/LH-R gene expression in endometriotic lesions was confirmed by reverse-transcriptase polymerase chain reaction. RESULTS: In endometriotic implants, epithelial HCG/LH-R was found in 12/23 samples. No significant differences in HCG/LH-R levels were observed when compared with glands of uterine endometrium from the same patients or healthy controls. Messenger RNA transcripts for HCG were detected in all 12 samples, whereas HCG/LH-R mRNAs were observed in 10 of the 12 endometriotic lesions investigated. CONCLUSIONS: Although HCG/LH-R was not found to be selectively upregulated in endometriosis, the mere presence of HCG/LH-R in endometriotic tissue may suggest sensitivity of endometriosis to HCG and LH that target HCG/LH-R.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/biossíntese , Endometriose/metabolismo , Hormônio Luteinizante/biossíntese , Receptores do LH/biossíntese , Adulto , Gonadotropina Coriônica Humana Subunidade beta/genética , Feminino , Humanos , Hormônio Luteinizante/genética , Pessoa de Meia-Idade , Receptores do LH/genéticaRESUMO
In a prospective case-control study we investigated the association of a common polymorphism of the interleukin 1 receptor antagonist gene (IL-1 RA) with the occurrence and the clinical characteristics of polycystic ovary syndrome (PCOS). Allele frequencies did not vary statistically significantly among women with PCOS and healthy controls or within the PCOS group with respect to their clinical characteristics.