RESUMO
In children, pseudohypoparathyroidism (PHP) is a rare but classical cause of basal ganglia calcifications. It is caused by resistance to parathormone (PTH). Hypocalcemia, which may be symptomatic, is its main feature. We report the case of a 13-year-old boy, affected by type Ib PHP revealed by hypocalcemia and seizures, with basal ganglia calcifications on the CT scan. We describe the characteristics of the 2 main types of PHP and emphasize the search for this disease when basal ganglia calcifications are discovered, even fortuitously, on a cerebral CT scan.
Assuntos
Doenças dos Gânglios da Base/etiologia , Calcinose/etiologia , Pseudo-Hipoparatireoidismo/diagnóstico , Adolescente , Doenças dos Gânglios da Base/diagnóstico por imagem , Calcinose/diagnóstico por imagem , Humanos , Hipocalcemia/etiologia , Masculino , Radiografia , Convulsões/etiologiaRESUMO
Monitoring of BCR-ABL transcripts has become established practice in the management of chronic myeloid leukemia. However, nucleic acid amplification techniques are prone to variations which limit the reliability of real-time quantitative PCR (RQ-PCR) for clinical decision making, highlighting the need for standardization of assays and reporting of minimal residual disease (MRD) data. We evaluated a lyophilized preparation of a leukemic cell line (K562) as a potential quality control reagent. This was found to be relatively stable, yielding comparable respective levels of ABL, GUS and BCR-ABL transcripts as determined by RQ-PCR before and after accelerated degradation experiments as well as following 5 years storage at -20 degrees C. Vials of freeze-dried cells were sent at ambient temperature to 22 laboratories on four continents, with RQ-PCR analyses detecting BCR-ABL transcripts at levels comparable to those observed in primary patient samples. Our results suggest that freeze-dried cells can be used as quality control reagents with a range of analytical instrumentations and could enable the development of urgently needed international standards simulating clinically relevant levels of MRD.
Assuntos
Perfilação da Expressão Gênica/métodos , Perfilação da Expressão Gênica/normas , Reação em Cadeia da Polimerase/métodos , Proteínas Tirosina Quinases/genética , RNA Mensageiro/análise , Liofilização , Proteínas de Fusão bcr-abl , Humanos , Indicadores e Reagentes , Células K562 , Reação em Cadeia da Polimerase/normas , Proteínas Tirosina Quinases/análise , Controle de Qualidade , Padrões de ReferênciaRESUMO
This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.
Assuntos
Quimerismo , DNA/sangue , Globinas/genética , Reação em Cadeia da Polimerase , Manejo de Espécimes , Feminino , Dosagem de Genes , Hematócrito , Transplante de Células-Tronco Hematopoéticas , Humanos , Contagem de Leucócitos , Masculino , Papel , Contagem de Plaquetas , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Manejo de Espécimes/métodosRESUMO
Immunocytochemistry (ICC) of thyroid peroxidase (TPO) using the monoclonal antibody MoAb47 has been used as malignancy marker on thyroid fine needle aspiration. However, little is known about the fate of TPO in thyroid carcinoma. We performed a qualitative PCR (Q-PCR) analysis to measure the expression of variants of tpo mRNA in 13 normal tissue samples, 30 benign tumors (BT), 21 follicular carcinomas (FC), 20 classical papillary carcinomas (PCc), 12 follicular variants of papillary carcinomas (PCfv) and nine oncocytic carcinomas (OC). We also studied mutations involving the ras, Braf, ret or pax8 genes. Results of Q-PCR were closely correlated with those of ICC (P < 0.0001; R = 0.59) and showed that overall tpo expression was lower in all carcinomas than in normal and BT (P < 0.05). The ratio tpo2 or tpo3 to tpo1 was inversed in follicular tumors. Genetic mutations were observed in 90% of PCc, 61.9% of FC, 41.7% of PCfv, 0% of OC and 10% in BT. pax8-ppar gamma1 rearrangement was correlated with qualitative changes in tpo mRNA (P < 0.01). These results confirmed the decrease of TPO expression in 97% of thyroid carcinomas regardless of histological type and the overexpression of shorter splice variants in follicular tumors. Both reduction in quantity of TPO and impairment of its maturation process could account for the atypical immunohistochemical reaction of MoAb47 with TPO.
Assuntos
Carcinoma/enzimologia , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Iodeto Peroxidase/genética , Neoplasias da Glândula Tireoide/enzimologia , Adulto , Idoso , Carcinoma/genética , Regulação para Baixo , Feminino , Genes ras/genética , Humanos , Iodeto Peroxidase/análise , Masculino , Pessoa de Meia-Idade , Mutação , Fator de Transcrição PAX8 , Fatores de Transcrição Box Pareados/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-ret/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/genéticaAssuntos
Neoplasias/genética , Neoplasias/prevenção & controle , Proteínas de Fusão Oncogênica/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Europa (Continente) , Proteínas de Fusão bcr-abl/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10(-8)M EGF stimulated AR expression in HMECs to 140-300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5-10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias da Mama/metabolismo , Mama/metabolismo , Glicoproteínas/fisiologia , Substâncias de Crescimento/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adolescente , Adulto , Anfirregulina , Mama/citologia , Mama/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Divisão Celular/fisiologia , Células Cultivadas , Família de Proteínas EGF , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/farmacologia , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Substâncias de Crescimento/farmacologia , Humanos , Imunofenotipagem , Invasividade Neoplásica , Células Tumorais CultivadasRESUMO
Epidermal growth factor (EGF), which plays an important role in normal and tumoral cell growth regulation, displays an ambivalent dose-dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. However, the underlying molecular mechanisms remain obscure. In this study we have examined the regulation of amphiregulin (AR) gene expression by growth inhibitory (10(-9) M) and stimulatory (10(-12) M) EGF concentrations in A431 cells. The time course of AR messenger RNA (mRNA) accumulation was different with 10(-12) and 10(-9) M EGF; AR induction by 10(-9) M EGF peaked between 1 and 1.5 h, then decreased to the basal level within 2 h. Conversely, the induction by 10(-12) M EGF was slightly delayed, but persisted for 4 h. The involvement of tyrosine phosphorylation in AR induction by EGF was suggested by the ability of the tyrosine phosphatase inhibitor sodium orthovanadate to prolong AR expression induced by 10(-12) or 10(-9) M EGF. In the presence of the protein phosphatase 2A inhibitor, okadaic acid, 10(-9) M EGF induced a persistent accumulation of AR mRNA. On the contrary, okadaic acid abrogated the stimulation of AR mRNA level induced by a low EGF concentration, suggesting that both EGF concentrations activated distinct regulatory mechanisms. The signaling components involved in the differential activities of EGF in A431 cells were then examined. We previously reported a relationship between the ambivalent activity of EGF and the p42-mitogen-activated protein (MAP) kinase activity. Thus, 10(-12) M EGF induced a sustained MAP kinase activation, whereas 10(-9) M EGF led to a sharp, but transitory, activation. The MAP kinases are activated by MAP kinase kinases (MEK1 and MEK2). Whereas no significant effect of 10(-12) M EGF could be detected, 10(-9) M EGF was shown to activate MEK1 and, to a lesser extent, MEK2. Also, both MAP kinase activation and AR induction by 10(-9) M, but not by 10(-12) M, EGF were inhibited by the MEK1 inhibitor PD98059. Moreover, the involvement of c-Raf-1 in the signaling pathway induced by EGF was verified. A concentration of 10(-9) M EGF induced stimulation of c-Raf-1 kinase activity, whereas 10(-12) M EGF not only failed to activate c-Raf-1, but led to a moderate decrease in its kinase activity. These results demonstrate that in EGF receptor-overexpressing cells, EGF may differently affect gene expression and cell proliferation through distinct mechanisms of regulation.