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BACKGROUND AND AIMS: The association between white matter abnormalities (WMA) and cognitive decline previously reported in poststroke patients has been mainly documented using visual scales. However, automated segmentation of WMA provides a precise determination of the volume of WMA. Nonetheless, it is rarely used in the stroke population and its potential advantage over visual scales is still unsettled. The objective of this study was to examine whether automated segmentation of WMA provides a better account than the visual Fazekas and Wahlund scales of the decline in executive functions and processing speed in stroke patients. METHODS: The analyses were conducted on the 358 patients of the GRECogVASC cohort with an MRI performed at six months poststroke in the Amiens center. WMA were visually analyzed using the Fazekas (subcortical abnormalities) and Wahlund scales. Segmentation was performed using LST (3.0.3). Following preliminary studies to determine the optimal segmentation threshold, we examined the relationship between cognitive status and WMA volume computed at each threshold using receiver operating characteristic (ROC) curves. Finally, we assessed the ability of both Fazekas and Wahlund visual scores and WMA volume to account for cognitive scores by using a bivariate Pearson correlation analysis, comparing correlation coefficients with the Fisher transformation and repeating correlation analysis after adjustment for the lesion volume. RESULTS: Increasing the threshold led to an underestimation of WMA (P=0.0001) (significant for a threshold ≥0.2) and an improvement in correct rejection of signal changes in the stroke cavity (P=0.02) (significant for a threshold ≤0.5), susceptibility artifacts (P=0.002) (significant for a threshold ≤0.6), and corticospinal degeneration (P=0.03) (significant for a threshold ≤0.5). WMA volume decreased with increasing threshold (P=0.0001). Areas under the curve (AUC) did not differ according to the threshold (processing speed: P=0.85, executive cognitive functions: P=0.7). Correlation coefficients between cognitive scores and WMA were higher for WMA volume than the Fazekas (processing speed: Z=-3.442, P=0.001; executive functions: Z=-2.751, P=0.006) and Wahlund scores (processing speed: Z=-3.615, P=0.0001; executive functions: Z=-2.769, P=0.006). Adjustment for lesion volume did not alter the correlations with WMA volume (processing speed: r=-0.327 [95%CI: -0.416; -0.223], P=0.0001; executive functions: r=-0.262 [95%CI: -0.363; -0.150], P=0.0001). CONCLUSION: This study shows that WMA volume assessed by automated segmentation provides a better account of cognitive disorders than visual analysis. This should favor its wider use to refine imaging determinants of poststroke cognitive disorders.
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BACKGROUND: The association between the pattern of cortical thickness (CT) and executive dysfunction (ED) in mild cognitive impairment (MCI) and subjective cognitive complaints (SCC) is still poorly understood. We aimed to investigate the association between CT and ED in a large French cohort (MEMENTO) of 2323 participants with MCI or SCC. METHODS: All participants with available CT and executive function data (verbal fluency and Trail Making Test [TMT]) were selected (n=1924). Linear regressions were performed to determine relationships between executive performance and the brain parenchymal fraction (BPF) and CT using FreeSurfer. RESULTS: The global executive function score was related to the BPF (sß: 0.091, P<0.001) and CT in the right supramarginal (sß: 0.060, P=0.041) and right isthmus cingulate (sß: 0.062, P=0.011) regions. Literal verbal fluency was related to the BPF (sß: 0.125, P<0.001) and CT in the left parsorbitalis region (sß: 0.045, P=0.045). Semantic verbal fluency was related to the BPF (sß: 0.101, P<0.001) and CT in the right supramarginal region (sß: 0.061, P=0.042). The time difference between the TMT parts B and A was related to the BPF (sß: 0.048, P=0.045) and CT in the right precuneus (sß: 0.073, P=0.019) and right isthmus cingulate region (sß: 0.054, P=0.032). CONCLUSIONS: In a large clinically based cohort of participants presenting with either MCI or SCC (a potential early stage of Alzheimer's disease [AD]), ED was related to the BPF and CT in the left pars orbitalis, right precuneus, right supramarginal, and right isthmus cingulate regions. This pattern of lesions adds knowledge to the conventional anatomy of ED and could contribute to the early diagnosis of AD.
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The presence of vascular neurocognitive impairment (whatever the severity) is always associated with a functional impact and increased risk of dependency and institutionalization. However, vascular cognitive impairment remains underdiagnosed, and the mechanisms underlying post-stroke cognitive disorders are still poorly understood. However, the advent of new criteria and a standardized international neuropsychological battery is expected to lead to improved diagnosis and management, and the development of novel techniques (such as brain imaging and amyloid PET) should improve our understanding of the mechanisms underlying vascular cognitive impairment and help to identify potential targets for therapy.
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Transtornos Cognitivos , Demência Vascular , Neuropsicologia/tendências , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/terapia , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/terapia , Demência Vascular/diagnóstico , Demência Vascular/etiologia , Demência Vascular/terapia , Humanos , Testes Neuropsicológicos , Neuropsicologia/métodos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapiaRESUMO
BACKGROUND: The IFM2009-02 trial studied pomalidomide (4 mg daily, 21/28 versus 28/28) and dexamethasone in very advanced relapsed or refractory multiple myeloma (RRMM). We observed that 40% of patients had a prolonged progression-free survival (PFS) and subsequently overall survival (OS). We sought to analyze the characteristics of these patients and study the effect of long exposure to pomalidomide. DESIGN: We separated the studied population into two groups: 3 months to 1 year (<1 year) and more than 1 year (≥1 year) of treatment with pomalidomide and dexamethasone based on clinical judgment and historical control studies. We then analyzed the characteristics of patients according to duration of treatment. RESULTS: The overall response rate (ORR) for the <1-year group was 43%, the median PFS 4.6 months [95% confidence interval (95% CI) 3.8-6.4] with only 6% at 12 months, and the median OS was 15 months (11.7-20.3) and 40% at 18 months. For the ≥1-year group, the response rate and survival were strikingly different, ORR at 83%, median PFS 20.7 months (14.7-35.4), median OS not reached, and 91% at 18 months. CONCLUSION: Pomalidomide and dexamethasone favored prolonged and safe exposure to treatment in 40% of heavily treated and end-stage RRMM, a paradigm shift in the natural history of RRMM characterized with a succession of shorter disease-free intervals and ultimately shorter survival. Although an optimization of pomalidomide-dexamethasone regimen is warranted in advanced RRMM, we claim that pomalidomide has proven once more to change the natural history of myeloma in this series, which should be confirmed in a larger study.
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Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Talidomida/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Bortezomib/administração & dosagem , Bortezomib/efeitos adversos , Dexametasona/administração & dosagem , Dexametasona/efeitos adversos , Intervalo Livre de Doença , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Recidiva Local de Neoplasia/patologia , Talidomida/administração & dosagem , Talidomida/efeitos adversosRESUMO
The discovery of an almost complete Neanderthal skeleton in a Châtelperronian context at Saint-Césaire 35 years ago changed our perspective on the beginning of the Upper Paleolithic in western Europe. Since then, the Châtelperronian has generally been considered a "transitional" industry rather than an Upper or a Middle Paleolithic industry because of its chronological position, and the association of Neanderthal remains with blades, bone tools and personal ornaments. Several competing hypotheses have been proposed to explain the association between Neanderthals and these types of artefacts including post-depositional mixing, acculturation from anatomically modern human populations, or an independent technological evolution by local Neanderthal populations. Quinçay Cave is the only Châtelperronian site where personal ornaments have been found that does not contain an overlying Upper Paleolithic layer. This means that the post-depositional mixing of later elements into the Châtelperronian may not be used as an explanation for the presence of these materials. We report here on a detailed technological analysis of lithic artefacts from the three Châtelperronian layers at Quinçay Cave. We compare our results with the technology of Mousterian blade industries dating to OIS (oxygen isotope stage) 5, the Mousterian of Acheulian Tradition type B, and the Proto-Aurignacian. We show that the Châtelperronian is sufficiently divergent from the Middle Paleolithic to be classified as a fully Upper Paleolithic industry, with a focus on blade and bladelet production. We also show that the Quinçay Châtelperronian includes retouched bladelets that resemble those found in the Proto-Aurignacian, but were produced in a different manner. We argue that a technological convergence cannot account for these behaviors, since the specific type of retouched bladelet associated with the Châtelperronian was also regularly used by Proto-Aurignacian of neighboring regions. We suggest that the idea of retouched bladelets may have diffused from the northern Proto-Aurignacian to the Quinçay Châtelperronian and that the transmission of the morphology of this desired end-product without the transmission of its manufacturing process may point toward a low degree of social intimacy between these groups. We conclude that the apparent paradox of the Châtelperronian is the result of the complexity of interaction between Neanderthal and anatomically modern human groups in western Europe between 45,000 and 40,000 years ago.
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Evolução Cultural , Homem de Neandertal , Animais , Arqueologia , Cavernas , França , TecnologiaRESUMO
BACKGROUND: The perception of stigmatization of patients with psoriasis is largely due to misconceptions and negative prejudice about this skin disease. 'Uneducated' judgments can give rise to discriminatory behaviours. OBJECTIVE: Evaluate the prevalence, in France, of misconceptions, negative prejudice and discriminatory behaviour towards psoriasis patients. METHODS: Online survey conducted in June 2011, aimed at 1005 persons aged 16-64 years, representative of the French population. The representativeness of the sample was ensured by quota methodology (gender, age, occupation of the interviewed person) after stratification by region and location category. The respondents were asked to respond to a questionnaire on their knowledge of psoriasis, their attitude and main feelings/perceptions towards psoriasis patients. RESULTS: About 62.4% of respondents recognize a lack of information about psoriasis and 19.7% have misconceptions about this disease. About 16.5% believe that psoriasis is contagious, 6.8% believe this skin disease is related to personal hygiene and 3.2% believe that it affects more people with low personal hygiene. About 50.0% of respondents show discriminatory behaviour towards psoriasis patients, reflected by reluctance to maintain friendship ties/a relationship of friendliness (7.6%), to have lunch or dinner with a person with visible manifestations (17.9%), to give a kiss on the cheek in greeting (29.7%), to shake hands (28.8%) and to have sexual relations/intercourse (44.1%). Patients with negative prejudice about the psoriasis frequently have misconceptions towards psoriasis patients. About 52.8% of respondents do not know anyone with psoriasis. Socio-demographic indicators such as gender, education level and rural or urban way of life are not associated with an increased prevalence of misconceptions and/or discriminatory behaviour. CONCLUSION: The lack of knowledge about psoriasis in France is important. There is an urgent need to strengthen information campaigns about psoriasis intended for the General Public.
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Psoríase/psicologia , Estereotipagem , Adolescente , Adulto , França , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: The 2008 World Health Organization (WHO) classification distinguishes three entities among the large granular lymphocytic leukemia (LGL leukemia): T-cell LGL leukemia (T-LGL leukemia), aggressive natural killer (NK) cell leukemia, and chronic NK lymphoproliferative disorders (LPD), the later considered as a provisional entity. Only a few and small cohorts of chronic NK LPD have been published. PATIENTS AND METHODS: We report here clinicobiological features collected retrospectively from 70 cases of chronic NK LPD, and compared with those of T-LGL leukemia. RESULTS: There were no statistical differences between chronic NK LPD and T-LGL leukemia concerning median age [61 years (range 23-82 years)], organomegaly (26%), associated autoimmune diseases (24%), and associated hematological malignancies (11%). Patients with chronic NK LPD were significantly less symptomatic (49% versus 18%, P < 0.001) and the association with rheumatoid arthritis was more rarely observed (7% versus 17%, P = 0.03). The neutropenia (<0.5 × 10(9)/l) was less severe in chronic NK LPD (33% versus 61%, P < 0.001) without difference in the rate of recurrent infections. STAT3 mutation was detected in 12% of the cohort, which is lower than the frequency observed in T-LGL leukemia. Thirty-seven percent of the patients required specific therapy. Good results were obtained with cyclophosphamide. Overall and complete response rates were, respectively, 69% and 56%. Overall survival was 94% at 5 years. CONCLUSION: This study suggests very high similarities between chronic NK LPD and T-LGL leukemias. Since chronic NK LPD is still a provisional entity, our findings should be helpful when considering further revisions of the WHO classification.
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Células Matadoras Naturais/patologia , Leucemia Linfocítica Granular Grande/patologia , Transtornos Linfoproliferativos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Leucemia Linfocítica Granular Grande/classificação , Leucemia Linfocítica Granular Grande/genética , Transtornos Linfoproliferativos/classificação , Transtornos Linfoproliferativos/genética , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fator de Transcrição STAT3/genética , Organização Mundial da SaúdeRESUMO
Neurological diseases are characterized by the complexity of care and by a constant and changing disability. More and more frequently, their impact on the clinical pathway remains unknown. Seven postgraduate rehabilitation students (Master coordination du handicap, université Pierre-et-Marie-Curie, Paris) reconstructed the clinical pathway of 123 patients with various neurological diseases: multiple sclerosis, Alzheimer disease, amyotrophic lateral sclerosis, spinal trauma, Parkinson disease and brain tumors. There was a significant correlation between disease duration and the number of specialists involved in care, the number of prescribed drugs and the number of short-term hospitalizations; there was no correlation with age. This result suggests that with time an increasing number of complications related to the initial neurological disease developed. Hospitalization in rehabilitation units was highly correlated with the degree of disability and also with the help received by the patients during the course of their disease. This result suggests that these hospitalizations were a direct consequence of burn out among relatives. General practitioners (GP) were highly involved only during the initial part of the pathway, and their involvement rapidly declined thereafter, suggesting a probable relation with the specificities and the complexity of care for neurological diseases which induces a progressive transfer of responsibilities from the GP to the hospital. Social care was always incomplete and occurred too late during the course of the disease. The feeling by the patients that their care pathway was chaotic was highly correlated with the quality of the information given to the patient at the time of the announcement of their disease. This study confirms that cares for neurological diseases is highly specific and that expert centers and coordination networks are in a key position to ensure an efficient care pathway.
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Procedimentos Clínicos/organização & administração , Pessoas com Deficiência , Doenças Neurodegenerativas/terapia , Pessoal Técnico de Saúde/estatística & dados numéricos , Progressão da Doença , Hospitalização/estatística & dados numéricos , Humanos , Doenças Neurodegenerativas/epidemiologia , Doenças Neurodegenerativas/psicologia , Prática Profissional/estatística & dados numéricos , Qualidade de Vida , Meio SocialRESUMO
Vascular cognitive impairment (VCI) includes vascular dementia (VaD), vascular mild cognitive impairment (VaMCI) and mixed dementia. In clinical practice, VCI concerns patients referred for clinical stroke or cognitive complaint. To improve the characterization of VCI and to refine its diagnostic criteria, an international group has elaborated a new standardized evaluation battery of clinical, cognitive, behavioral and neuroradiological data which now constitutes the reference battery. The adaption of the battery for French-speaking subjects is reported as well as preliminary results of the on-going validation study of the GRECOG-VASC group [Clinical Trial NCT01339195]. The diagnostic accuracy of various screening tests is reviewed and showed an overall sub-optimal sensitivity (<0.8). Thus, the general recommendation is to perform systematically a comprehensive assessment in stroke patients at risk of VCI. Furthermore,the use of a structured interview has been shown to increase the detection of dementia. In addition to the well known NINDS-AIREN criteria of VaD, criteria of VCI have been recently proposed which are based on the demonstration of a cognitive disorder by neuropsychological testing and either history of clinical stroke or presence of vascular lesion by neuroimaging suggestive of a link between cognitive impairment and vascular disease. A memory deficit is no longer required for the diagnosis of VaD as it is based on the cognitive decline concerning two or more domains that affect activities of daily living. Both VaMCI and VaD are classified as probable or possible. These new criteria have yet to be validated. Considerable uncertainties remain regarding the determinant of VCI, and especially the lesion amount inducing VCI and VaD. The interaction between lesion amount and its location is currently re-examined using recent techniques for the analysis of MRI data. The high frequency of associated Alzheimer pathology is now assessable in vivo using amyloid imaging. The first studies showed that about a third of patients with VaD due to small vessel disease or with poststroke dementia have amyloid PET imaging suggestive of AD. These new techniques will examine the interaction between vascular lesions and promotion of amyloid deposition. Although results of these on-going studies will be available in few years, these data indicate that efforts should be done in clinical practice to reduce underdiagnosis of VCI; VCI should be examined using a specific protocol which will be fully normalized soon for French-speaking patients; the sub-optimal sensitivity of screening tests prompts to use a structured interview to grade Rankin scale and to perform systematically a comprehensive assessment in stroke patients at risk of VCI; poststroke dementia occurring after 3 months poststroke may be preventable by treatment of modifiable vascular risk factors and secondary prevention of stroke recurrence according to recent recommendations.
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Transtornos Cerebrovasculares/diagnóstico , Técnicas de Diagnóstico Neurológico/normas , Testes Neuropsicológicos/normas , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/etiologia , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/etiologia , Demência Vascular/diagnóstico , Demência Vascular/etiologia , Humanos , Acidente Vascular Cerebral/diagnósticoRESUMO
The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.
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Fatores de Ribosilação do ADP/metabolismo , Nucléolo Celular/metabolismo , Proteínas Nucleares , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células 3T3 , Fatores de Ribosilação do ADP/genética , Animais , Nucléolo Celular/ultraestrutura , Senescência Celular , Retroalimentação , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Genes myc , Genes p53 , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-mdm2 , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We describe the clinical, electroencephalography (EEG), and developmental features of a patient with developmental and epileptic encephalopathy due to a homozygous pathogenic variation of mitochondrial glutamate/H+ symporter SLC25A22. Epilepsy began during the first week of life with focal onset seizures. Interictal EEG revealed a suppression-burst pattern with extensive periods of non-activity. The prospective follow-up confirmed developmental encephalopathy as well as ongoing active epilepsy and almost no sign of development at 8 years of age. We confirm in the following paper that SLC25A22 recessive variations may cause a severe developmental and epileptic encephalopathy characterized by a suppression-burst pattern. On the basis of an in-depth literature review, we also provide an overview of this rare genetic cause of neonatal onset epilepsy.
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Encefalopatias/diagnóstico , Deficiências do Desenvolvimento/diagnóstico , Epilepsia/diagnóstico , Proteínas de Transporte da Membrana Mitocondrial/genética , Fenótipo , Encefalopatias/genética , Criança , Pré-Escolar , Deficiências do Desenvolvimento/genética , Eletroencefalografia , Epilepsia/genética , Feminino , Genes Recessivos , Homozigoto , Humanos , Lactente , Recém-Nascido , MutaçãoRESUMO
The c-fms proto-oncogene is a member of a gene family that has been implicated in tumorigenesis. Glycoproteins encoded by c-fms were identified in cat spleen cells by means of an immune-complex kinase assay performed with monoclonal antibodies to v-fms-coded epitopes. The major form of the normal cellular glycoprotein has an apparent molecular weight of 170,000 and, like the product of the viral oncogene, serves as a substrate for an associated tyrosine-specific protein kinase activity in vitro. The results suggest that the transforming glycoprotein specified by v-fms is a truncated form of a c-fms-coded growth factor receptor.
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Glicoproteínas/metabolismo , Proteínas de Neoplasias/metabolismo , Oncogenes , Proteínas Quinases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Gatos , Glicoproteínas/imunologia , Humanos , Camundongos , Peso Molecular , Proteínas de Neoplasias/imunologia , Fosforilação , Proteínas Tirosina Quinases , Proto-Oncogene Mas , RNA/metabolismo , RatosRESUMO
BACKGROUND/AIMS: Despite numerous studies performed in selected populations, major uncertainties regarding the anatomy and patterns of episodic memory deficit still persist. Such objectives require the analysis of consecutive unselected patients with well-delimitated lesions and the use of specific methodology. METHODS: Nonaphasic stroke patients (n = 73) referred for cognitive complaints or deficits were examined using a standardized neuropsychological battery. Verbal episodic memory was assessed by the Grober and Buschke test. RESULTS: Episodic memory deficit was related to lesions of medial temporal, thalamic, frontal, lenticular, and centrum semiovale regions, with a left hemisphere predominance. Although not specific, an immediate recall deficit was frequently observed in thalamic lesions (OR: 5.2, 95% CI: 1.09-24.9). False recognitions (OR: 25.7, 95% CI: 2.7-249) and efficiency of cued recall (OR: 8.6, 95% CI: 1.3-57) were highly specific of frontal damage. When combined with the usual 'frontal lobe' tests, this pattern of memory disorder was an independent predictor (OR: 36, 95% CI: 4.3-302) and increased the sensitivity of diagnosis of frontal damage (sensitivity: 0.92, specificity: 0.89). CONCLUSIONS: This study extends the classical anatomy of episodic verbal memory. In addition, the 'frontal memory pattern' was found to be relatively specific and to be an independent predictor of frontal damage.
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Encéfalo/patologia , Transtornos da Memória/patologia , Transtornos da Memória/fisiopatologia , Acidente Vascular Cerebral/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/fisiopatologia , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Transtornos da Memória/etiologia , Pessoa de Meia-Idade , Testes Neuropsicológicos , Estudos Retrospectivos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/fisiopatologia , Adulto JovemRESUMO
p18(Ink4c) functions as a dedicated inhibitor of cyclin-D-dependent kinases. Loss of Ink4c predisposes mice to tumor development and, in a dose-dependent manner, complements the tumor-promoting effects of various oncogenes. We have now addressed whether Ink4c loss impacts B-cell tumor development in the Emu-Myc transgenic mouse, a model of human Burkitt lymphoma. Loss of one or both alleles did not influence the onset of lymphoma in Emu-Myc transgenics, and did not appreciably affect Myc's proliferative or apoptotic responses in precancerous B cells. Nevertheless, Ink4c loss modulated the effects of Myc-induced transformation by decreasing the frequency of Arf loss, an ordinarily common event in Emu-Myc-induced lymphomas.
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Inibidor de Quinase Dependente de Ciclina p18/fisiologia , Linfoma de Células B/genética , Linfoma de Células B/prevenção & controle , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Apoptose/genética , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p18/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Genes de Cadeia Pesada de Imunoglobulina , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/fisiologia , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
DNA from the human myeloid cell line HL-60 was cotransfected with the cloned thymidine kinase (tk) gene of herpes simplex virus into tk-deficient mouse L cells. tk-positive recipients expressing antigens detected on HL-60 cells were isolated with a fluorescence-activated cell sorter by use of a panel of monoclonal antibodies that detect epitopes on both normal and malignant myeloid cells. Independently sorted populations of transformed mouse cells showed concordant reactivities with four of the monoclonal antibodies in the panel (DU-HL60-4, MY7, MCS.2, and SJ-D1), which suggested that these antibodies reacted to products of a single human gene. A second round of DNA transfection and cell sorting was performed with donor DNA from primary transformants. Two different dominant selection systems were used to isolate secondary mouse L cell and NIH/3T3 cell transformants that coexpressed the same epitopes. Analysis of cellular DNA from secondary mouse cell subclones with a probe specific for human repetitive DNA sequences revealed a minimal human DNA complement containing a characteristic set of restriction fragments common to independently derived subclones. Two glycoproteins, of 130,000 (gp130) and 150,000 (gp150) mol wt, were specifically immunoprecipitated from metabolically labeled lysates of mouse cell transformants and were shown to contain [35S]methionine-labeled tryptic peptides identical to those of analogous glycoproteins expressed in the donor human myeloid cell line. Kinetic and biochemical analyses established that gp130 is a precursor that differs in its carbohydrate moiety from gp150, the mature form of the glycoprotein detected on the cell surface. The isolation of human gene sequences encoding gp150 in a mouse cell genetic background provides the possibility of molecularly cloning the gene and represents a general strategy for isolating human genes encoding differentiation-specific cell surface antigens.
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Antígenos de Superfície/genética , Células-Tronco Hematopoéticas/imunologia , Animais , Linhagem Celular , Clonagem Molecular , DNA Recombinante , Genes , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Camundongos , Peso Molecular , TransfecçãoRESUMO
DNA from a tertiary mouse cell transformant containing amplified human sequences encoding a human myeloid membrane glycoprotein, gp150, was used to construct a bacteriophage lambda library. A single recombinant phage containing 12 kilobases (kb) of human DNA was isolated, and molecular subclones were then used to isolate the complete gp150 gene from a human placental genomic DNA library. The intact gp150 gene, assembled from three recombinant phages, proved to be biologically active when transfected into NIH 3T3 cells. Molecular probes from the gp150 locus annealed with a 4.0-kb polyadenylated RNA transcript derived from human myeloid cell lines and from tertiary mouse cell transformants. The gp150 gene was assigned to human chromosome 15, and was subchromosomally localized to bands q25-26 by in situ hybridization. The chromosomal location of the gp150 gene coincides cytogenetically with the region assigned to the c-fes proto-oncogene, another human gene specifically expressed by myeloid cells.
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Antígenos de Superfície/genética , Mapeamento Cromossômico , Clonagem Molecular , Regulação da Expressão Gênica , Glicoproteínas/genética , Sequência de Bases , Cromossomos Humanos Par 15 , Enzimas de Restrição do DNA/metabolismo , Desoxirribonuclease BamHI , Humanos , Células Híbridas , Hibridização de Ácido Nucleico , Poli A/análise , Proto-Oncogene Mas , RNA/análise , RNA Mensageiro , TransfecçãoRESUMO
The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v-fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development.
Assuntos
Coriocarcinoma/metabolismo , Monócitos/metabolismo , Receptores de Superfície Celular/biossíntese , Neoplasias Uterinas/metabolismo , Animais , Linhagem Celular , Feminino , Glicoproteínas/biossíntese , Humanos , Leucemia Mieloide Aguda/metabolismo , Monócitos/efeitos dos fármacos , Proteínas Oncogênicas Virais/biossíntese , Gravidez , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/biossíntese , Proto-Oncogenes , Coelhos , Receptores de Fator Estimulador de Colônias , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The biosynthesis of macrophage colony-stimulating factor 1 (CSF-1) was examined in mouse NIH-3T3 fibroblasts transfected with a retroviral vector expressing the 554-amino-acid product of a human 4-kilobase (kb) CSF-1 cDNA. Similar to results previously obtained with a 1.6-kb human cDNA that codes for a 256-amino-acid CSF-1 precursor, the results of the present study showed that NIH-3T3 cells expressing the product of the 4-kb clone produced biologically active human CSF-1 and were transformed by an autocrine mechanism when cotransfected with a vector containing a human c-fms (CSF-1 receptor) cDNA. The 4-kb CSF-1 cDNA product was synthesized as an integral transmembrane glycoprotein that was assembled into disulfide-linked dimers and rapidly underwent proteolytic cleavage to generate a soluble growth factor. Although the smaller CSF-1 precursor specified by the 1.6-kb human cDNA was stably expressed as a membrane-bound glycoprotein at the cell surface and was slowly cleaved to release the extracellular growth factor, the cell-associated product of the 4-kb clone was efficiently processed to the secreted form and was not detected on the plasma membrane. Digestion with glycosidic enzymes indicated that soluble CSF-1 encoded by the 4-kb cDNA contained both asparagine(N)-linked and O-linked carbohydrate chains, whereas the product of the 1.6-kb clone had only N-linked oligosaccharides. Removal of the carbohydrate indicated that the polypeptide chain of the secreted 4-kb cDNA product was longer than that of the corresponding form encoded by the smaller clone. These differences in posttranslational processing may reflect diverse physiological roles for the products of the two CSF-1 precursors in vivo.
Assuntos
Fatores Estimuladores de Colônias/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Clonagem Molecular , Fatores Estimuladores de Colônias/genética , DNA/genética , Glicosilação , Humanos , Precursores de Proteínas/genética , Transcrição GênicaRESUMO
The turnover of the colony-stimulating factor 1 receptor (CSF-1R), the c-fms proto-oncogene product, is accelerated by ligand binding or by activators of protein kinase C (PKC), such as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The mechanisms of ligand- and TPA-induced downmodulation were shown to differ by the following criteria. First, in cells in which PKC was downmodulated, CSF-1R reexpressed at the cell surface remained sensitive to ligand but was refractory to TPA-induced degradation. Second, a kinase-defective receptor containing a methionine-for-lysine substitution at amino acid 616 at its ATP-binding site failed to undergo ligand-induced downmodulation but remained responsive to TPA. Following CSF-1 stimulation, no intermediates of receptor degradation could be immunoprecipitated with polyvalent antisera to CSF-1R. In contrast, TPA induced specific proteolytic cleavage of the receptor near its transmembrane segment, resulting in the release of the extracellular ligand-binding domain from the cell and the generation of an intracellular fragment containing the kinase domain. Two-dimensional phosphopeptide mapping demonstrated no new sites of phosphorylation in response to TPA in either the residual intact receptor or the intracellular proteolytic fragment. Therefore, PKC appears not to trigger downmodulation by directly phosphorylating the receptor but, rather, activates a protease which recognizes CSF-1R as a substrate.
Assuntos
Ligantes/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Linhagem Celular , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Humanos , Mapeamento de Peptídeos , Fosforilação , Testes de Precipitina , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Receptor de Fator Estimulador de Colônias de Macrófagos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Ligand-induced tyrosine phosphorylation of the human colony-stimulating factor 1 receptor (CSF-1R) could involve either an intra- or intermolecular mechanism. We therefore examined the ability of a CSF-1R carboxy-terminal truncation mutant to phosphorylate a kinase-defective receptor, CSF-1R[met 616], that contains a methionine-for-lysine substitution at its ATP-binding site. By using an antipeptide serum that specifically reacts with epitopes deleted from the enzymatically competent truncation mutant, cross-phosphorylation of CSF-1R[met 616] on tyrosine was demonstrated, both in immune-complex kinase reactions and in intact cells stimulated with CSF-1. Both in vitro and in vivo, CSF-1R[met 616] was phosphorylated on tryptic peptides identical to those derived from wild-type CSF-1R, suggesting that receptor phosphorylation on tyrosine can proceed via an intermolecular interaction between receptor monomers. When expressed alone, CSF-1R[met 616] did not undergo ligand-induced down modulation, but its phosphorylation in cells coexpressing the kinase-active truncation mutant accelerated its degradation.