RESUMEN
Typing carbapenem-resistant Klebsiella pneumoniae (CR-KPN) is crucial in controlling their dissemination and solving outbreaks. In this context, we searched for an effective, faster, and cheaper alternative technique to type KPN by analyzing the fosAKP sequence. We analyzed the nucleotide sequences of chromosomal fosAKP gene in 350 KPN genomes (70 per sequence type [ST] or clonal complex [CC]). Assembly genomes were randomly downloaded from NCBI and annotated using RAST in PATRIC platform. The isolate STs were verified using multilocus sequence typing 2.0 by the Center for Genomic Epidemiology. Chromosomally encoded fosAKP was confirmed in MLplasmid, and the sequence alignments were performed in Clustal Omega. The amino acid sequences were analyzed using SNAP2 and SMART platforms. Out of the 70 genomes analyzed for each ST/CC, we observed 100% fosA sequence identity for CC258/11, ST15, ST307, and ST101. For ST16, only two fosA sequences were different from each other. We observed differences in amino acid sequences at positions 25 and 79 (ST16) and 86 (ST16, ST101). The C-terminal (amino acid 138, 139, 140) was different for each cluster. None of these polymorphisms is related to the protein active site. Moreover, L25Q (ST16) polymorphism was predicted to probably affect the protein function. We observed that chromosomal fosAKP sequences from KPN are highly conserved in ST15, ST307, ST16, ST101, and CC258/11, suggesting fosAKP sequencing as an alternative, easier, faster, and less expensive technique in identifying epidemiological STs for KPN, and discriminating them from CC258/11.
Asunto(s)
Infecciones por Klebsiella , Klebsiella pneumoniae , Humanos , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/epidemiología , beta-Lactamasas/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Antibacterianos/farmacología , Tipificación de Secuencias Multilocus , Células Clonales/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Acinetobacter baumannii is a successful nosocomial pathogen due to its genomic plasticity. Homologous recombination allows genetic exchange and allelic variation among different clonal lineages and is one of the mechanisms associated with horizontal gene transfer (HGT) of resistance determinants. The main mechanism of colistin resistance in A. baumannii is mediated through mutations in the pmrCAB operon. Here, we describe two A. baumannii clinical isolates belonging to International Clone 7 (IC7) that have undergone recombination in the pmrCAB operon and evaluate the contribution of mobile genetic elements (MGE) to this phenomenon. Isolates 67569 and 72554 were colistin susceptible and resistant, respectively, and were submitted for short- and long-read genome sequencing using Illumina MiSeq and MinION platforms. Hybrid assemblies were built with Unicycler, and the assembled genomes were compared to reference genomes using NUCmer, Cortex, and SplitsTree. Genomes were annotated using Prokka, and MGEs were identified with ISfinder and repeat match. Both isolates presented a 21.5-kb recombining region encompassing pmrCAB. In isolate 67659, this region originated from IC5, while in isolate 72554 multiple recombination events might have happened, with the 5-kb recombining region encompassing pmrCAB associated with an isolate representing IC4. We could not identify MGEs involved in the mobilization of pmrCAB in these isolates. In summary, A. baumannii belonging to IC7 can present additional sequence divergence due to homologous recombination across clonal lineages. Such variation does not seem to be driven by antibiotic pressure but could contribute to HGT mediating colistin resistance. IMPORTANCE Colistin resistance rates among Acinetobacter baumannii clinical isolates have increased over the last 20 years. Despite reports of the spread of plasmid-mediated colistin resistance among Enterobacterales, the presence of mcr-type genes in Acinetobacter spp. remains rare, and reduced colistin susceptibility is mainly associated with the acquisition of nonsynonymous mutations in pmrCAB. We have recently demonstrated that distinct pmrCAB sequences are associated with different A. baumannii International Clones (IC). In this study, we identified the presence of homologous recombination as an additional cause of genetic variation in this operon, which, to the best of our knowledge, was not mediated by mobile genetic elements. Even though this phenomenon was observed in both colistin-susceptible and -resistant isolates, it has the potential to contribute to the spread of resistance-conferring alleles, leading to reduced susceptibility to this last-resort antimicrobial agent.
Asunto(s)
Acinetobacter baumannii/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Colistina/farmacología , Genoma Bacteriano , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Brasil , Células Clonales/metabolismo , Farmacorresistencia Bacteriana/genética , Transferencia de Gen Horizontal , Humanos , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/genética , Neoplasias de la Vesícula Biliar/genética , Indígenas Sudamericanos/genética , Animales , Antineoplásicos/farmacología , Líquido Ascítico/metabolismo , Carcinogénesis/genética , Pruebas de Carcinogenicidad , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Chile , Cisplatino/farmacología , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Dermatoglifia del ADN , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Células Epiteliales/metabolismo , Neoplasias de la Vesícula Biliar/metabolismo , Perfilación de la Expresión Génica , Genes erbB-2/genética , Humanos , Queratina-19/genética , Queratina-7/genética , Masculino , Ratones Endogámicos NOD , Persona de Mediana Edad , Receptor ErbB-2/genética , Análisis de Secuencia de ARN , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , GemcitabinaRESUMEN
BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.
Asunto(s)
Humanos , Animales , Masculino , Persona de Mediana Edad , Antígenos de Carbohidratos Asociados a Tumores/genética , Indígenas Sudamericanos/genética , Neoplasias de la Vesícula Biliar/genética , Líquido Ascítico/metabolismo , Células Tumorales Cultivadas , Pruebas de Carcinogenicidad , Chile , Dermatoglifia del ADN , Proteína p53 Supresora de Tumor/genética , Cisplatino/farmacología , Ratones Endogámicos NOD , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Análisis de Secuencia de ARN , Receptor ErbB-2/genética , Genes erbB-2/genética , Perfilación de la Expresión Génica , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Células Epiteliales/metabolismo , Queratina-19/genética , Queratina-7/genética , Carcinogénesis/genética , Neoplasias de la Vesícula Biliar/metabolismo , Antineoplásicos/farmacologíaRESUMEN
The success of the development of new sugarcane varieties is associated with the ability to correctly select the genitor. The aim of this study was to evaluate the genetic diversity between 113 clones and sugarcane varieties using the Ward-modified location model procedure with added information about the coefficient of parentage and endogamy. In this study, data was used from 100 experiments that evaluated clones; the experimental phase was conducted in 70 places between the years 2002 and 2009 on the outlining in random blocks. According to the diversity analysis, 3 groups formed: G1, G2, and G3, which were composed of 58, 8, and 47 genotypes, respectively. The clones of groups G1 and G3 were the most outstanding. Thus, biparental crossbreeding involving clones and varieties of these 2 groups can efficiently obtain transgressive genotypes. Knowledge of the heterotypic groups indicated by the Ward-modified location model method, along with the parentage information, will make it a lot easier to define the desirable and undesirable crossbreeds for public and private breeding programs that develop sugarcane varieties.
Asunto(s)
Cruzamiento/métodos , Variación Genética , Modelos Genéticos , Saccharum/genética , Brasil , Células Clonales/metabolismo , Genotipo , Fenotipo , Reproducibilidad de los Resultados , Saccharum/clasificación , Saccharum/crecimiento & desarrolloRESUMEN
BACKGROUND: Endometriosis is a multifactorial gynecological disease characterized by the presence of functional endometrium-like tissue in ectopic sites. Several studies have focused on elucidating the immunological, endocrine, environmental and genetic factors involved in endometriosis. However, its pathogenesis is still unclear. METHODS: High-resolution comparative genomic hybridization was applied to screen for genomic imbalances in laser microdissected stromal and epithelial cells from 20 endometriotic lesions and three samples of eutopic endometrium derived from eight patients. The expression of seven stemness-related markers (CD9, CD13, CD24, CD34, CD133, CD117/c-Kit and Oct-4) in endometrial tissue samples was evaluated by immunohistochemistry. RESULTS: Samples of eutopic endometrium showed normal genomic profiles. In ectopic tissues, an average of 68 genomic imbalances was detected per sample. DNA losses were more frequently detected and involved mainly 3p, 5q, 7p, 9p, 11q, 16q, 18q and 19q. Many of the genomic imbalances detected were common to endometriotic stroma and epithelia and also among different endometriotic sites from the same patient. These findings suggested a clonal origin of the endometriotic cells and the putative involvement of stem cells. Positive immunostaining for CD9, CD34, c-Kit and Oct-4 markers was detected in isolated epithelial and/or stromal cells in eutopic and ectopic endometrium in the majority of cases. CONCLUSIONS: The presence of shared genomic alterations in stromal and epithelial cells from different anatomical sites of the same patient and the expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells.
Asunto(s)
Células Madre Adultas/metabolismo , Antígenos CD/metabolismo , Aberraciones Cromosómicas , Endometriosis/metabolismo , Endometrio/metabolismo , Regulación de la Expresión Génica , Adulto , Células Madre Adultas/patología , Antígenos CD/genética , Biomarcadores/metabolismo , Deleción Cromosómica , Células Clonales/metabolismo , Células Clonales/patología , Hibridación Genómica Comparativa , Endometriosis/diagnóstico , Endometriosis/patología , Endometriosis/cirugía , Endometrio/patología , Endometrio/cirugía , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Enfermedades Intestinales/diagnóstico , Enfermedades Intestinales/metabolismo , Enfermedades Intestinales/patología , Enfermedades Intestinales/cirugía , Captura por Microdisección con Láser , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Populações que residem em áreas endêmicas da doença de Chagas são submetidas a múltiplas infecções pelo Trypanosoma cruzi e podem estar infectadas com cepas ou clones com diferentes graus de virulência e susceptibilidade à quimioterapia. Este é um fator importante para o desenvolvimento e morbidade da doença. As cepas de Trypanosoma cruzi representam complexas populações multiclonais, homogêneas ou heterogêneas, com predominância de um clone principal. São biologicamente classificados em Biodemas (Tipos I, II e III) que apresentam diferentes graus de resistência à quimioterapia. Cepas do Tipo I são muito suscetíveis ao tratamento; cepas do Tipo II possuem uma média susceptibilidade (cepa 21SF); cepas de Tipo III são muito resistentes (cepa Colombiana). No presente estudo, é avaliado o resultado do tratamento de camundongos triplamente infectados com clones da cepa 21SF, em comparação com os infectados com a cepa parental. 50 camundongos foram infectados com a cepa 21SF (infecção única). O inóculo foi de 5 x 104 formas tripomastigotas sanguícolas. 80 camundongos foram infectados, sucessivamente, com 3 clones da cepa 21SF (C6, C7 e C8), inóculo: 1x104 formas tripomastigotas (infecção tripla). A infecção simples com cada clone também foi feita. Os camundongos de ambos os grupos foram divididos em dois subgrupos: tratados com Benzonidazol BZ (100 mg/kg/dia - 60 doses) e controles não tratados. Após 60 dias do final do tratamento, os camundongos sobreviventes foram eutanasiados, por exsanguinação, pós-anestesia; o sangue foi coletado para o exame sorológico de imunofluorescência indireta; testes de cura foram realizados (parasitemia, xenodiagnósticos, hemocultura) e seções do coração e músculo esquelético foram coletadas, fixadas e processadas para o estudo histopatológico em cortes corados com Hematoxilina & Eosina. A PCR foi também usada como uma técnica diagnóstica complementar. Os testes parasitológicos mostraram uma positividade variando de 54,4% nos camundongos infectados com a cepa parental e tratados; 33,4 a 66,7% nos animais com infecção única pelos diversos clones e tratados e 26,7% nos camundongos com infecção tríplice, tratados. Os títulos sorológicos (TIFI) variaram de 1:20 a 1:280 nos infectados com cepa parental tratados com BZ e de 1:640 a 1:1280 para controles não tratados. Os títulos sorológicos na infecção única com cada clone variaram de 1:10 a 1:1280 em camundongos tratados e de 1:160 a 1:1280 nos controles não tratados. A PCR revelou positividade em todos os animais infectados, tratados. O resultado final foi obtido pela combinação dos testes parasitológicos com os títulos sorológicos revelando positividade de 6,6% nos camundongos infectados com a cepa parental e tratados; 0 a 18,2% nos animais com infecção única pelos diversos clones e tratados e 12% nos camundongos com infecção tríplice, tratados. Estudo histopatológico: Os camundongos infectados com a cepa parental apresentaram lesões que variavam de leves a moderadas, na maioria dos casos, predominante no miocárdio (animais tratados e controles não tratados) camundongos submetidos à infecção única por cada clone apresentaram lesões semelhantes aos demonstrados pela infecção com cepa parental. Os camundongos com infecção tripla apresentaram uma exacerbação de lesões, evoluindo para a miocardite crônica. Nestes casos, havia intensas lesões no músculo esquelético; animais tratados apresentaram uma nítida redução das lesões no miocárdio e no músculo esquelético. Os resultados da quimioterapia com Benzonidazol em animais triplamente infectados, considerando os testes parasitológicos e sorológicos, revelaram baixos índices de cura e agravamento das lesões tissulares nos camundongos submetidos a múltiplas infecções com clones obtidos da cepa 21SF.
Asunto(s)
Animales , Ratones , Ratones Endogámicos/genética , Células Clonales/metabolismo , Muridae , Quimioterapia/métodos , Trypanosoma cruzi/parasitologíaRESUMEN
Paciente de 43 años diagnosticado con leucemia mieloide crónica en 1998, que fue tratado de forma inicial con interferón alfa. En la terapia posterior adquirió múltiples cambios citogenéticos clonales en células del cromosoma Filadelfia negativas, por lo que se describe el efecto de los inhibidores de la tirosina quinasa de segunda generación sobre esos clones celulares.
Asunto(s)
Humanos , Masculino , Adulto , Células Clonales/fisiología , Células Clonales/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/complicaciones , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Proteínas Tirosina Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/farmacología , Proteínas Tirosina Quinasas/uso terapéuticoRESUMEN
Apesar dos esforços realizados nos últimos quatro anos, as taxas de mortalidade dos bezerros clonados da raça que chegam a termo são ainda altas, cerca de 50%. Demonstrou-se a ocorrência de graves distúrbios cardiorrespiratórios caracterizados por hiperfonese, presença de sopros cardíacos na 1ª e 2ª bulha associadosa dispneias, respiração rude e estertores. Em consequência ao nãofechamento do Forâmen de Botal e do Ducto Arterioso, há mistura de sangue arterial e venoso comprometendo a capacidade de oxigenação do sangue dos bezerros clonados. Observou-se ainda a ocorrência de macrossomia, hipoglicemia, hipotermia, anomaliasdas estruturas umbilicais, anemia e alopecia.
Despite all efforts during the last four years to improve cloned newborn care, the mortality rate of calf after term is still high, around 50%. Clinical symptoms observed in these cloned calf were related to severe cardiopulmonary disorders like hyperphonese, diastolic and systolic cardiac murmurs associated to dyspnea and crackling lung sounds. Due to the patency of the Foramen Ovale and Ductus Arteriosus, in which causes the mix of arterial and venous blood, the blood oxygenation in these cloned calf is compromised. In addition, cloned calf could also present increasedbirth weight, hypoglycemia, hypothermia, umbilical cord abnormalities, anemia, and alopecia.
Asunto(s)
Animales , Bovinos , Clonación de Organismos/veterinaria , Células Clonales/metabolismo , Células Híbridas , Cardiopatías/veterinaria , Puente Cardiopulmonar/veterinariaRESUMEN
Apesar dos esforços realizados nos últimos quatro anos, as taxas de mortalidade dos bezerros clonados da raça que chegam a termo são ainda altas, cerca de 50%. Demonstrou-se a ocorrência de graves distúrbios cardiorrespiratórios caracterizados por hiperfonese, presença de sopros cardíacos na 1ª e 2ª bulha associadosa dispneias, respiração rude e estertores. Em consequência ao nãofechamento do Forâmen de Botal e do Ducto Arterioso, há mistura de sangue arterial e venoso comprometendo a capacidade de oxigenação do sangue dos bezerros clonados. Observou-se ainda a ocorrência de macrossomia, hipoglicemia, hipotermia, anomaliasdas estruturas umbilicais, anemia e alopecia.(AU)
Despite all efforts during the last four years to improve cloned newborn care, the mortality rate of calf after term is still high, around 50%. Clinical symptoms observed in these cloned calf were related to severe cardiopulmonary disorders like hyperphonese, diastolic and systolic cardiac murmurs associated to dyspnea and crackling lung sounds. Due to the patency of the Foramen Ovale and Ductus Arteriosus, in which causes the mix of arterial and venous blood, the blood oxygenation in these cloned calf is compromised. In addition, cloned calf could also present increasedbirth weight, hypoglycemia, hypothermia, umbilical cord abnormalities, anemia, and alopecia.(AU)
Asunto(s)
Animales , Bovinos , Células Clonales/metabolismo , Células Híbridas , Clonación de Organismos/veterinaria , Puente Cardiopulmonar/veterinaria , Cardiopatías/veterinariaRESUMEN
The initial steps towards the generation of cell diversity in the central nervous system of the fruitfly Drosophila melanogaster take place during early phases of embryonic development when a stereotypic population of neural progenitor cells (neuroblasts and midline precursors) is formed in a precise spatial and temporal pattern, and subsequently expresses a particular sequence of genes. The clarification of the positional, temporal and molecular features of the individual progenitor cells in the nerve cord and brain as well as of their specific types of neuronal and/or glial progeny cells forms an essential basis to understand the mechanisms controlling their development. The present study contributes to this effort by tracing the expression of period and timeless, two genes that encode transcription factors with a key role in the molecular mechanism of the biological clock. Using a combination of genetic markers and immunocytochemistry with antibodies specific for period and timeless we define the number, location, origin and lineage of period cells in the nerve cord throughout embryogenesis. We also provide the first description of the expression of timeless in the embryonic central nervous system. We found a major transformation in the number and types of cells that express period and timeless takes place between embryonic and larval life.
Asunto(s)
Linaje de la Célula/genética , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Proteínas Circadianas Period/genética , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Movimiento Celular/fisiología , Sistema Nervioso Central/citología , Células Clonales/metabolismo , Células Clonales/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Larva/genética , Larva/metabolismo , Estadios del Ciclo de Vida/genética , Modelos Biológicos , Neuronas/metabolismo , Neuronas/fisiología , Proteínas Circadianas Period/metabolismoRESUMEN
We have previously demonstrated that the addition of apotransferrin (aTf) to oligodendroglial cell (OLGc) primary cultures accelerates their maturation. Cells treated with aTf developed a multipolar morphology and displayed increased expression of mature OLGc markers. In this work, we studied the effect of Tf overexpression in two OLGc lines, N19 and N20.1. The former cells exhibit characteristics of OLGc precursors (O2A), while N20.1 cells express markers of more mature OLGcs. Using the complete cDNA of the human Tf gene, we obtained clones overexpressing Tf in both cell lines. These clones were evaluated for the expression of OLGc differentiation markers. In agreement with our previous results, we found that in the cells overexpressing Tf, there was an increased O(4), GC, and MBP immunoreactivity. To study the myelinogenic potential of these cells, we co-cultured N19 and N20.1 Tf-transfected cells together with cortical neurons. There was a dramatic increase in the morphological differentiation of the OLGcs accompanied by enhanced GC and MBP expression. The OLGcs appeared to establish contact with neurites and extend their processes along them. Only two MBP isoforms were detected in Tf-overexpressing clones, while all the isoforms were present in the co-cultures, suggesting that there was a modulation of MBP expression by neurons. Concomitantly, we found an increase in several proteins involved in axon-glia interaction, such as MAG, N-CAM, and F3/Contactin. This co-culture system represents a potentially powerful tool to study neuron-glia interactions that occur during myelinogenesis and the role of Tf in this process.
Asunto(s)
Diferenciación Celular/genética , Oligodendroglía/metabolismo , Transferrina/genética , Transferrina/metabolismo , Animales , Biomarcadores/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Comunicación Celular/genética , Línea Celular , Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/metabolismo , Células Clonales/citología , Células Clonales/metabolismo , Técnicas de Cocultivo/métodos , Contactinas , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Ratones , Proteína Básica de Mielina/metabolismo , Vaina de Mielina/metabolismo , Vaina de Mielina/ultraestructura , Glicoproteína Asociada a Mielina/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Oligodendroglía/citología , Ratas , Células Madre/citología , Células Madre/metabolismo , Transfección/métodosRESUMEN
The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte invasion assays to investigate invasion into normal, enzyme-treated, and clinical-mutant RBCs. Analysis of these isolates revealed four distinct invasion profiles. Using En(a-) cells to get an unequivocal estimate of the use of glycophorin A (GPA) as a receptor, we found that the 175-kDa erythrocyte-binding antigen (EBA-175)/GPA pathway was used by a minority of the parasite isolates studied. Although polymorphism of region II domains at specific amino acid positions in both EBA-140 and EBA-181 was found in these field isolates, this did not correlate with invasion profiles and thus receptor selectivity. These studies have further confirmed the existence of a significant diversity of invasion pathways in nature and suggest that additional parasite ligands will have to be targeted to devise global vaccines that will work in the field.
Asunto(s)
Malaria Falciparum/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Polimorfismo Genético/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Brasil , Proteínas Portadoras/química , Proteínas Portadoras/genética , Células Clonales/metabolismo , Eritrocitos/metabolismo , Eritrocitos/parasitología , Genotipo , Humanos , Proteínas de la Membrana , Datos de Secuencia Molecular , Fenotipo , Plasmodium falciparum/aislamiento & purificación , Plasmodium falciparum/patogenicidad , Estructura Terciaria de Proteína/genética , Proteínas Protozoarias/químicaRESUMEN
To investigate collagen turnover and proliferation in dermal fibroblasts from patients with systemic sclerosis (SSc) and their relationship with disease duration and cellular subpopulations, SSc patients were grouped by disease duration (less than 2.5 years or more than 7 years). Control and SSc fibroblasts were obtained from skin biopsies. Collagen biosynthesis was determined by [14C]-proline uptake. Type I/III collagens, gelatinolytic activity, and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were evaluated by electrophoresis, zymography, and enzyme-linked immunosorbent assay, respectively. Total collagen synthesis and the levels of alpha1(I), alpha2(I), and alpha1(III) chains, as well as TIMP-1 and proliferation were increased in fibroblasts only from patients with early-stage SSc. Gelatinolytic activity did not vary among the groups. This metabolic condition favors a higher local fibroblast population and is characterized by a heterogeneous clonal response in which the majority exhibited higher levels of collagen and TIMP-1 synthesis as well as an increase in their proliferation patterns involving hyper-reactive fibroblast subsets.
Asunto(s)
Colágeno/metabolismo , Fibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/fisiopatología , Piel/metabolismo , Piel/fisiopatología , Adulto , Edad de Inicio , Radioisótopos de Carbono , División Celular/fisiología , Células Cultivadas , Células Clonales/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Progresión de la Enfermedad , Regulación hacia Abajo/fisiología , Femenino , Gelatinasas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Prolina/metabolismo , Piel/patología , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
The aim of this study was to investigate the effect of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) on NADPH oxidase activity and gp91-phox gene expression in HL-60 clone 15 cells as they differentiate along the eosinophilic lineage. The results were compared to the eosoniphilic inducers interleukin-5 (IL-5) and butyric acid. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) or IL-5 (200 pM) caused a significant increase in the expression of the eosinophil peroxidase (EPO) and the major basic protein (MBP) genes. Similar results were observed when the cells were cultured with 0.5 mM butyric acid for 5 days. IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml) also caused a significant increase in superoxide release by HL-60 clone 15 cells after 2 days compared with control or with butyric acid-induced cells. After 5 days, these cytokines and butyric acid induced an even stronger release of superoxide. HL-60 clone 15 cells cultured with IFN-gamma and TNF-alpha for 2 days showed a significant increase in gp91-phox gene expression. We conclude that IFN-gamma and TNF-alpha are sufficient to induce the differentiation of HL-60 clone 15 cells to the eosinophilic lineage and to upregulate gp91-phox gene expression and activity of the NADPH oxidase system.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Eosinófilos/efectos de los fármacos , Interferón gamma/farmacología , NADPH Oxidasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Biomarcadores/análisis , Ácido Butírico/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Linaje de la Célula , Células Clonales/efectos de los fármacos , Células Clonales/enzimología , Células Clonales/metabolismo , Activación Enzimática/efectos de los fármacos , Eosinófilos/citología , Eosinófilos/enzimología , Eosinófilos/metabolismo , Eritropoyetina/genética , Eritropoyetina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Interleucina-5/farmacología , Proteínas de Unión a Maltosa , Glicoproteínas de Membrana/genética , NADPH Oxidasa 2 , NADPH Oxidasas/genética , Peroxidasa/genética , Peroxidasa/metabolismoRESUMEN
T cell clones were derived from peripheral blood mononuclear cells of Schistosoma haematobium infected and uninfected individuals living in an endemic area. The clones were stimulated with S. haematobium worm and egg antigens and purified protein derivative. Attempts were made to classify the T cell clones according to production of the cytokines IL-4, IL-5 and IFN-gamma. All the T cell clones derived were observed to produce cytokines used as markers for the classification of Th1/Th2 subsets. However, the 'signature' cytokines marking each subset were produced at different levels. The classification depended on the dominating cytokine type, which was having either Th0/1 or Th0/2 subsets. The results indicated that no distinct cytokine profiles for polarisation of Th1/Th2 subsets were detected in these S. haematobium infected humans. The balance in the profiles of cytokines marking each subset were related to infection and re-infection status after treatment with praziquantel. In the present study, as judged by the changes in infection status with time, the T cell responses appeared to be less stable and more dynamic, suggesting that small quantitative changes in the balance of the cytokines response could result in either susceptibility or resistant to S. haematobium infection
Asunto(s)
Humanos , Animales , Niño , Citocinas/biosíntesis , Schistosoma haematobium/inmunología , Esquistosomiasis Urinaria/inmunología , Linfocitos T Colaboradores-Inductores/clasificación , Antihelmínticos/uso terapéutico , Antígenos Helmínticos , Línea Celular , Células Clonales/clasificación , Células Clonales/metabolismo , Citocinas/análisis , Citocinas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática , Estudios de Seguimiento , Recuento de Huevos de Parásitos , Praziquantel/uso terapéutico , Esquistosomiasis Urinaria/tratamiento farmacológico , Subgrupos de Linfocitos T/clasificación , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Células TH1/clasificación , Células TH1/metabolismo , Células Th2/clasificación , Células Th2/metabolismo , VolumetríaRESUMEN
The production of the lymphokines leukocyte migration inhibition factor (LIF) and migration stimulation factor (MStF) at the level of CD4+ and CD8+ human lymphocyte subsets was investigated. In a first series of experiments, anti-CD4 and anti-CD8 monoclonal antibodies capable of inhibiting the activation by concanavalin-A (Con-A) of the respective T-cell subset were used. It was observed that when CD8+ cell activation was blocked, LIF was always produced after Con-A activation. When CD4+ cell activation was blocked, MStF was produced in five out of nine experiments (no activity in the other four). The addition of N-acetyl-D-glucosamine to block LIF in supernatants of anti-CD8 treated cells was unable to show evidence of masked MStF activity. In a second series of experiments, T-cell clones were established from continuous growing T-lymphocyte cell lines developed from cultures of Con-A activated normal human leukocyte cultures. The phenotype of 22 clones was determined and their ability to produce LIF or MStF investigated. Four clones produced MStF after Con-A activation and all of them were CD3+, CD4-, CD8+. Three clones produced LIF after Con-A activation and all of them were CD3+, CD4+, CD8-. We conclude that LIF is produced by CD4+ cells and MStF by CD8+ cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/farmacología , Factores Quimiotácticos/biosíntesis , Factores Inhibidores de la Migración de Leucocitos/biosíntesis , Linfocinas/biosíntesis , Macrófagos , Linfocitos T/metabolismo , Anticuerpos Monoclonales/farmacología , Células Clonales/efectos de los fármacos , Células Clonales/inmunología , Células Clonales/metabolismo , Concanavalina A/farmacología , Humanos , Activación de Linfocitos , Linfocitos T/clasificación , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Chinese hamster cells (V79) resistant to high concentrations of Cd2+ in the medium were obtained by using the procedure of Beach & Palmiter [(1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2110-2114], which in mouse led to amplification of metallothionein (MT) genes and to an enrichment in cellular MT. The Cd-resistant V79 clones isolated were significantly more resistant than parental cells to oxidative stress by extracellular H2O2 or a mixture of H2O2 and superoxide anion (O2-) generated by xanthine oxidase plus acetaldehyde. On a per-cell basis, there was no difference between the two cells in their total H2O2-decomposing or O2-(-)dismutating activity. The most likely explanation is that an enrichment in MT content in the Cd-resistant cells was responsible for this effect, because of the antioxidant properties already described for this protein.
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Cadmio/farmacología , Supervivencia Celular/efectos de los fármacos , Oxígeno/farmacología , Animales , Línea Celular , Células Clonales/efectos de los fármacos , Células Clonales/metabolismo , Cricetinae , Cricetulus , Resistencia a Medicamentos/genética , Peróxido de Hidrógeno/farmacología , Superóxido Dismutasa/metabolismo , Superóxidos/farmacologíaRESUMEN
KJ23a+ T cell clones, which bear the determinant encoded by the V beta 17a T cell receptor gene segment, frequently recognize IE molecules of various murine H-2 haplotypes. In the presence of IE molecules, thymic maturation of KJ23a+ clones is infrequent. We investigated the basis of this phenomenon by blocking expression of IE molecules with monoclonal anti-IE antibodies in organ cultures of fetal thymus and in neonates from the C57BR/cdJ strain (H-2k, V beta 17a homozygous). Our data support the contention that this process results from deletion of clones with anti-IE reactivity, as functional blocking of the IE molecule results in maturation of IE-reactive clones and increased numbers of KJ23a+ mature cells. In addition, we noted that blocking of functional IE expression in this haplotype permitted development of both CD4+/KJ23a+ and CD8+/KJ23a+ T cells. The CD4+ clones isolated from anti-IE-treated animals were frequently reactive against IEk; we could demonstrate no alloreactivity against B cell or B lymphoma stimulators in the CD4- clones. We conclude that clonal deletion events during thymic development may be initiated by T cell precursor interactions with MHC molecules against which the mature clones display no measurable reactivity. Specifically, clones destined to be MHC Class I-reactive may be deleted during development by interactions with MHC Class II molecules.
Asunto(s)
Antígenos H-2/genética , Tolerancia Inmunológica , Timo/inmunología , Animales , Animales Recién Nacidos/inmunología , Anticuerpos Monoclonales/fisiología , Diferenciación Celular , Células Clonales/inmunología , Células Clonales/metabolismo , Células Clonales/fisiología , Antígenos H-2/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Terapia de Inmunosupresión , Recuento de Leucocitos , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/fisiología , Timo/citología , Timo/fisiologíaRESUMEN
The soft agar technique was employed to investigate factors involved in the regulation of granulopoiesis in ten children with aplastic anemia. Children with AA had greatly reduced numbers of granulocytic colony-forming cells in their bone marrow and in their peripheral blood when compared to "control" children. Colony-stimulating activity was decreased in five of the ten children tested. Serum from eight children with AA did not inhibit colony formation when added to normal adult bone marrow cells in culture. The defect in AA residues in the stem cell, with involvement of the CSA-producing cells in some cases. The serum of these patients does not contain an inhibitor of granulopoiesis.