RESUMO
CBA macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. Employing a transcriptomic approach, we previously showed the up-regulation of the genes involved in the classical pathway of macrophage activation in resistant mice. However, microarray analyses do not evaluate changes in gene expression that occur after translation. To circumvent this analytical limitation, we employed a proteomics approach to increase our understanding of the modulations that occur during infection and identify novel targets for the control of Leishmania infection. To identify proteins whose expression changes in CBA macrophages infected with L. major or L. amazonensis, protein extracts were obtained and digested and the peptides were characterized using multi-dimensional liquid chromatography coupled with tandem mass spectrometry analyses. A total of 162 proteins were selected as potentially modulated. Using biological network analyses, these proteins were classified as primarily involved in cellular metabolism and grouped into cellular development biological networks. This study is the first to use a proteomics approach to describe the protein modulations involved in cellular metabolism during the initial events of Leishmania-macrophage interaction. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of infection.
Assuntos
Interações Hospedeiro-Patógeno , Leishmania major/imunologia , Leishmania mexicana/imunologia , Macrófagos/química , Macrófagos/parasitologia , Proteoma/análise , Animais , Cromatografia Líquida , Feminino , Leishmania major/patogenicidade , Leishmania mexicana/patogenicidade , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Espectrometria de Massas em TandemRESUMO
CBA/J mice are resistant to Leishmania major infection but are permissive to L. amazonensis infection. In addition, CBA/J macrophages control L. major but not L. amazonensis infection in vitro. Phagocytosis by macrophages is known to determine the outcome of Leishmania infection. Pattern recognition receptors (PRR) adorning antigen presenting cell surfaces are known to coordinate the link between innate and adaptive immunity. The macrophage receptor with collagenous structure (MARCO) is a PRR that is preferably expressed by macrophages and is capable of binding Gram-positive and Gram-negative bacteria. No research on the role of MARCO in Leishmania-macrophage interactions has been reported. Here, we demonstrate, for the first time, that MARCO expression by CBA/J macrophages is increased in response to both in vitro and in vivo L. major infections, but not to L. amazonensis infection. In addition, a specific anti-MARCO monoclonal antibody reduced L. major infection of macrophages by 30%-40% in vitro. The draining lymph nodes of anti-MARCO-treated mice displayed a reduced presence of immunolabelled parasite and parasite antigens, as well as a reduced inflammatory response. These results support the hypothesis that MARCO has a role in macrophage infection by L. major in vitro as well as in vivo.
Assuntos
Leishmania major/imunologia , Leishmaniose/imunologia , Leishmaniose/metabolismo , Macrófagos Peritoneais/imunologia , Receptores Imunológicos/biossíntese , Animais , Anticorpos Antiprotozoários/imunologia , Anticorpos Antiprotozoários/metabolismo , Imunidade Inata , Leishmania major/metabolismo , Leishmania mexicana/imunologia , Leishmania mexicana/metabolismo , Leishmaniose/parasitologia , Leishmaniose/patologia , Linfonodos/imunologia , Linfonodos/patologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos CBA , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Depuradores/biossíntese , Receptores Depuradores/genética , Receptores Depuradores/imunologia , Ativação Transcricional , Regulação para CimaRESUMO
In this study we employed randomly amplified polymorphic DNA patterns to assess the genetic relatedness among 14 Brazilian Trypanosoma evansi stocks from domestic and wild hosts, which are known to differ in biological characteristics. These akinetoplastic stocks were compared with one another, to three Old World (Ethiopia, China and Philippines) dyskinetoplastic stocks of T. evansi, and also with Trypanosoma equiperdum, Trypanosoma brucei brucei, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. Randomly amplified polymorphic DNA analysis showed limited heterogeneity in T. evansi stocks from different hosts and geographical regions of the world, or in other species of the subgenus Trypanozoon. However, minor variations generated random amplification of polymorphic DNA analysis disclosed a pattern consisting of a unique synapomorphic DNA fragment (termed Te664) for the T. evansi cluster that was not detected in any other trypanosome species investigated. Pulsed field gel electrophoresis analysis demonstrated that the Te664 fragment is a repetitive sequence, dispersed in intermediate and minichromosomes of T. evansi. Based on this sequence, we developed a conventional PCR assay for the detection of T. evansi using crude preparations of blood collected either on glass slides or on filter paper as template DNA. Our results showed that this assay may be useful as a diagnostic tool for field-epidemiological studies of T. evansi.
Assuntos
DNA de Protozoário/genética , Trypanosoma/genética , Animais , Sequência de Bases , Southern Blotting , Brasil , Búfalos , Carnívoros , Bovinos , DNA de Protozoário/química , Cães , Eletroforese em Gel de Campo Pulsado , Variação Genética , Cavalos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Técnica de Amplificação ao Acaso de DNA Polimórfico , Roedores , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Suínos , Trypanosoma/química , Trypanosoma/isolamento & purificaçãoRESUMO
The sequence of the spliced-leader gene repeat of a Brazilian Trypanosoma vivax stock from cattle showed high similarity to sequences of West African T. vivax in both intron and intergenic sequences. This is the first evidence based on DNA sequences of close-relatedness between Brazilian and West African T. vivax stocks. A T. vivax-specific diagnostic PCR assay based on spliced-leader gene intergenic sequences was able to amplify DNA from T. vivax stocks from South America (Brazil, Bolivia, and Colombia) and West Africa. Species-specificity of this method was confirmed by results obtained by testing 15 other trypanosomes, including other species and subspecies that can also infect cattle. The PCR assay developed presented high sensitivity, detecting the DNA content of only one parasite and also revealing T. vivax infection in asymptomatic animals without detectable parasitemia by microhematocrit or in Giemsa-stained blood smears. Use of crude preparations from field-blood samples collected on both filter paper and glass slides as DNA template suggested that this method could be useful for the diagnosis of T. vivax in large epidemiological studies.
Assuntos
DNA Intergênico/genética , DNA de Protozoário/genética , RNA Líder para Processamento/genética , Trypanosoma vivax/genética , Animais , Sequência de Bases , Brasil , Bovinos , Sondas de DNA/normas , DNA Intergênico/química , DNA de Protozoário/química , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Alinhamento de Sequência , Ovinos , Especificidade da Espécie , Trypanosoma vivax/classificaçãoRESUMO
A number of studies have pointed out the potential importance of the household in the transmission of schistosomiasis. The clustering of domestic activities associated with water collection, storage, and usage can result in the sharing of transmission sites and infective water contact behaviours. In this study, we employed a variance component method to estimate effects due to individual risk factors and shared residence on the variance in faecal egg counts during Schistosoma mansoni infection. A suite of covariates, which included demographic, socioeconomic, water supply, and water contact behaviour terms, contributed 15% to the variance in faecal egg counts. Shared residence alone accounted for 28% of the variance in faecal egg excretion. When both the suite of covariates and shared residence were considered in the same model, shared residence still contributed 22% to the variance in infection intensity. These results point to the importance of shared residence as a means of capturing the complex interrelationship between shared demographic, socioeconomic, physical environmental, and behavioural factors that influence transmission of schistosomiasis at the household level.
Assuntos
Características da Família , Comportamentos Relacionados com a Saúde , Saúde da População Rural , Esquistossomose mansoni/epidemiologia , Esquistossomose mansoni/transmissão , Água , Adolescente , Adulto , Análise de Variância , Brasil/epidemiologia , Criança , Pré-Escolar , Análise por Conglomerados , Doenças Endêmicas , Fezes/parasitologia , Feminino , Humanos , Higiene , Lactente , Masculino , Pessoa de Meia-Idade , Contagem de Ovos de Parasitas , Prevalência , Fatores de Risco , Esquistossomose mansoni/parasitologia , Fatores Socioeconômicos , Inquéritos e QuestionáriosRESUMO
Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.
Assuntos
Vetores Genéticos/genética , Reação em Cadeia da Polimerase/métodos , Transfecção/métodos , Trypanosoma cruzi/genética , Animais , Meios de Cultura , Primers do DNA , Expressão Gênica , Genes Reporter , Canamicina Quinase/genética , Canamicina Quinase/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.
Assuntos
Genes de Protozoários/genética , Insetos/parasitologia , Plantas/parasitologia , RNA Líder para Processamento/genética , Trypanosomatina/genética , Animais , Sequência de Bases , DNA Recombinante/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Especificidade da EspécieRESUMO
The prevalence of GB virus C (GBV-C) in candidate Brazilian blood donors with normal and elevated alanine aminotransferase levels was found to be 5.2% (5 of 95) and 6.5% (5 of 76), respectively. Among Brazilian patients, GBV-C was found in 9.5% (13 of 137) of cases of hepatitis not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV (non-A-E hepatitis) and in 18.2% (8 of 44) of individuals infected with HCV. Molecular characterization of GBV-C by partial sequencing of the NS3 region showed clustering between members of a single family, implying intrafamilial transmission. In conclusion, these results together suggest that contagion mechanisms which facilitate intrafamilial transmission of GBV-C may partially explain the high prevalence of viremic carriers worldwide.
Assuntos
Flaviviridae/isolamento & purificação , Hepatite Viral Humana/transmissão , Sequência de Bases , Doadores de Sangue , Brasil , Família , Flaviviridae/classificação , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Prevalência , Proteínas não Estruturais Virais/genéticaRESUMO
In this paper we describe a method for the detection of Phytomonas spp. from plants and phytophagous insects using the PCR technique by targeting a genus-specific sequence of the spliced leader (SL) gene. PCR amplification of DNA from 48 plant and insect isolates previously classified as Phytomonas by morphological, biochemical, and molecular criteria resulted in all cases in a 100-bp fragment that hybridized with the Phytomonas-specific spliced leader-derived probe SL3'. Moreover, this Phytomonas-specific PCR could also detect Phytomonas spp. in crude preparations of naturally infected plants and insects. This method shows no reaction with any other trypanosomatid genera or with plant and insect host DNA, revealing it to be able to detect Phytomonas spp. from fruit, latex, or phloem of various host plants as well as from salivary glands and digestive tubes of several species of insect hosts. Results demonstrated that SLPCR is a simple, fast, specific, and sensitive method that can be applied to the diagnosis of Phytomonas among cultured trypanosomatids and directly in plants and putative vector insects. Therefore, the method was shown to be a very specific and sensitive tool for diagnosis of Phytomonas without the need for isolation, culture, and DNA extraction of flagellates, a feature that is very convenient for practical and epidemiological purposes.
Assuntos
Insetos/parasitologia , Plantas/parasitologia , RNA Líder para Processamento/genética , Trypanosomatina/isolamento & purificação , Animais , DNA de Protozoário/análise , Reação em Cadeia da Polimerase , RNA de Protozoário/genética , Sensibilidade e Especificidade , Moldes Genéticos , Trypanosomatina/genéticaRESUMO
The 1988 Upstate New York Live Birth Certificate was the first to record Hispanic ethnicity and country of origin. This registry was used to compare low birthweight and preterm delivery among non-Hispanic white, non-Hispanic black, and Hispanic infants. Risk of low birthweight and preterm delivery was assessed for Hispanics by country of origin. Unconditional backward elimination logistic regression analysis, controlling for confounders was used to assess risk of low birthweight and preterm delivery of Hispanic subgroups compared to non-Hispanic white and non-Hispanic black mothers. The data showed that non-Hispanic black mothers are at greatest risk of low birthweight and preterm delivery. Hispanics as a group have rates similar to non-Hispanic white mothers, although risk of preterm delivery and low birthweight differs among Hispanic ethnic subgroups. This study supports the need to assess Hispanic subgroups separately rather than as a single entity.
Assuntos
Hispânico ou Latino , Recém-Nascido de Baixo Peso , Recém-Nascido Prematuro , Negro ou Afro-Americano , População Negra , América Central/etnologia , Cuba/etnologia , Humanos , Recém-Nascido , México/etnologia , New York/epidemiologia , Porto Rico/etnologia , Análise de Regressão , América do Sul/etnologia , População BrancaRESUMO
TTV is a recently discovered DNA virus, isolated from a patient with post-transfusion hepatitis of unknown etiology by Japanese researchers. In the present study, we evaluated the presence of TTV among chronic liver diseases patients in São Paulo and Pará states, representing two geographically distinct Brazilian regions. TTV DNA was found in 21/105 (20%) and 9/20 (45%) cases from São Paulo and Pará States, respectively. DNA sequence data confirmed the presence of TTV genotypes 1a and 2a, as well as other genotypes not yet described. In conclusion, TTV is present in chronic liver diseases cases from Southeast and North Brazil. However, further studies involving healthy populations are necessary before establishing any causal relationship among TTV and human hepatitis.
Assuntos
Vírus de DNA , Hepatite Viral Humana/transmissão , Hepatite Viral Humana/virologia , Hepatopatias/virologia , Reação Transfusional , Brasil , Doença Crônica , Vírus de DNA/patogenicidade , Genótipo , Hepatite Viral Humana/genética , HumanosRESUMO
We probed DNA from all trypanosomatid genera by slot blot hybridization with an oligonucleotide (SL3') complementary to a sequence of the Phytomonas spliced-leader or mini-exon RNA. The 19-nucleotide probe target site was previously shown to be highly conserved among a limited number of Phytomonas isolates, but diverges in other kinetoplastid genera. Our examination of 84 isolates of various genera of trypanosomatids showed hybridization of this probe exclusively with isolates from plants or insects which could, by morphological, biochemical, and molecular criteria, be considered to belong to the genus Phytomonas. In contrast, no hybridization was observed with flagellates of the genera Blastocrithidia, Crithidia, Endotrypanum, Herpetomonas, Leptomonas, Leishmania, and Trypanosoma. The method detected DNA quantities as low as 50 ng using either radioactive or nonradioactive probes, and was effective with as few as 10(4) intact flagellates. Together, these results suggest that this probe will serve as a convenient marker for taxonomic and epidemiological studies requiring reliable identification of Phytomonas spp. in plants or in putative insect vectors.
Assuntos
DNA de Protozoário/genética , Sondas de Oligonucleotídeos , Sinais Direcionadores de Proteínas/genética , Trypanosomatina/genética , Animais , Insetos , Hibridização de Ácido Nucleico , RNA de Protozoário/genética , Sensibilidade e Especificidade , Especificidade da Espécie , Trypanosomatina/isolamento & purificaçãoRESUMO
The neurodevelopmental outcome of hypoplastic left heart syndrome in infants remains unclear. All 11 survivors of staged surgical repair of hypoplastic left heart syndrome received standardized neurodevelopmental assessments at one regional children's hospital. Seven children (64%) had major developmental disabilities. Quality-of-life outcomes must be considered when management options for children with hypoplastic left heart syndrome are evaluated.
Assuntos
Deficiências do Desenvolvimento/etiologia , Síndrome do Coração Esquerdo Hipoplásico/complicações , Deficiência Intelectual/etiologia , Qualidade de Vida , Paralisia Cerebral/complicações , Feminino , Seguimentos , Técnica de Fontan , Humanos , Síndrome do Coração Esquerdo Hipoplásico/cirurgia , Recém-Nascido , Masculino , Destreza Motora , Resultado do TratamentoRESUMO
A case series design was used to identify cases of cystic periventricular leukomalacia (N = 31) identified by neurosonography at one regional tertiary intensive care nursery. Patients were preterm infants born at < or = 32 weeks of gestation who had cysts involving predominantly the middle-posterior or posterior periventricular regions. Neurodevelopmental evaluations were made for 26 (96%) of 27 survivors. All infants assessed had cerebral palsy (i.e., 54% quadriplegia, 42% diplegia, and 4% hemiplegia). Most cognitive delays and all sensory impairments occurred in children with quadriplegia. Periventricular cysts were most extensive on parasagittal, anteroposterior views. The parasagittal, anteroposterior extent of periventricular cysts was most accurate in predicting the type and severity of motor and cognitive disabilities. Quadriplegia was associated with larger and more extensive cysts.
Assuntos
Paralisia Cerebral/etiologia , Leucomalácia Periventricular/complicações , Paralisia Cerebral/classificação , Feminino , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Leucomalácia Periventricular/diagnóstico por imagem , Leucomalácia Periventricular/patologia , Masculino , UltrassonografiaRESUMO
The purpose of this cohort study was to determine the incidence of and risk factors for major neurodevelopmental impairments among survivors of extreme prematurity. The study cohort comprised 100 infants born between 24 and 28 weeks of gestational age at one tertiary center from 1983 to 1984. Twenty-five infants (25%) died; 75 (75%) survived until follow-up (mean, 60 months). Standardized neurodevelopmental and psychometric assessments were performed in blind fashion on 68 of the 75 surviving children (91% follow-up). Informal assessments (parent, teacher, and physician reports) were obtained instead for seven (9%) children who had relocated outside of the area. Overall, 19 children (25%) had one or more major impairments: mental retardation, 9; cerebral palsy, 4; multiple impairments, 5; and blindness, 1. Despite a high prevalence of impairments, 95% of children (n = 71) were functionally independent [corrected]. Special educational resources were definitely necessary for seven (9%) and possibly needed for 36 (48%) additional children. Univariate analyses revealed four significant risk factors for cerebral palsy: hydrocephalus (relative risk = 12.2), grades III and IV intraventricular hemorrhage (relative risk = 5.8), 5-minute Apgar score lower than 7 (relative risk = 5.7), and bronchopulmonary dysplasia (relative risk = 5.5). Hydrocephalus was the only significant risk factor observed for mental retardation (relative risk = 5.4). Risk factors predicting a need for special education resources included sepsis (relative risk = 24.9), low socioeconomic status (relative risk = 16.3), and nonwhite race (relative risk = 3.0). Thus our data suggest that biomedical factors appear to confer the greatest risk of major impairments; sociodemographic factors appear to have a significant impact on educational risk in extremely premature infants who do not die. Continued follow-up with biomedical and developmental-social interventions appears warranted to decrease the risk of educational underachievement in this population.