RESUMO
A novel class of Ru(II)-based polypyridyl complexes with an auxiliary salicylaldehyde ligand [Ru(phen)2(X-Sal)]BF4 {X: H (1), 5-Cl (2), 5-Br (3), 3,5-Cl2 (4), 3,5-Br2 (5), 3-Br,5-Cl (6), 3,5-I2 (7), 5-NO2 (8), 5-Me (9), 4-Me (10), 4-OMe (11), and 4-DEA (12), has been synthesized and characterized by elemental analysis, FT-IR, and 1H/13C NMR spectroscopy. The molecular structure of 4, 6, 9, 10, and 11 was determined by single-crystal X-ray diffraction analysis which revealed structural similarities. DFT and TD-DFT calculations showed that they also possess similar electronic structures. Absorption/emission spectra were recorded for 2, 3, 10, and 11. All Ru-complexes, unlike the pure ligands and the complex lacking the salicylaldehyde component, displayed outstanding antiproliferative activity in the screening test (10 µM) against CCRF-CEM leukemia cells underlining the crucial role of the presence of the auxiliary ligand for the biological activity. The two most active derivatives, namely 7 and 10, were selected for continuous assays showing IC50 values in the submicromolar and micromolar range against drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells, respectively. These two compounds were investigated in silico for their potential binding to duplex DNA well-matched and mismatched base pairs, since they showed remarkable selectivity indexes (2.2 and 19.5 respectively) on PBMC cells.
Assuntos
Aldeídos , Antineoplásicos , Complexos de Coordenação , Leucemia , Rutênio , Humanos , Ligantes , Leucócitos Mononucleares/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Rutênio/farmacologia , Rutênio/química , Complexos de Coordenação/química , Antineoplásicos/farmacologia , Antineoplásicos/químicaRESUMO
Fungal unspecific peroxygenases (UPOs) are arising as versatile biocatalysts for C-H oxyfunctionalization reactions. In recent years, several directed evolution studies have been conducted to design improved UPO variants. An essential part of this protein engineering strategy is the design of reliable colorimetric high-throughput screening (HTS) assays for mutant library exploration. Here, we present a palette of 12 colorimetric HTS assays along with their step-by-step protocols, which have been validated for directed UPO evolution campaigns. This array of colorimetric assays will pave the way for the discovery and design of new UPO variants.
Assuntos
Colorimetria , Ensaios de Triagem em Larga Escala , Oxigenases de Função Mista/metabolismo , Engenharia de Proteínas/métodosRESUMO
Here we explored the role of interleukin-1ß (IL-1ß) repressor cytokine, IL-1 receptor antagonist (IL-1rn), in both healthy and abnormal hematopoiesis. Low IL-1RN is frequent in acute myeloid leukemia (AML) patients and represents a prognostic marker of reduced survival. Treatments with IL-1RN and the IL-1ß monoclonal antibody canakinumab reduce the expansion of leukemic cells, including CD34+ progenitors, in AML xenografts. In vivo deletion of IL-1rn induces hematopoietic stem cell (HSC) differentiation into the myeloid lineage and hampers B cell development via transcriptional activation of myeloid differentiation pathways dependent on NFκB. Low IL-1rn is present in an experimental model of pre-leukemic myelopoiesis, and IL-1rn deletion promotes myeloproliferation, which relies on the bone marrow hematopoietic and stromal compartments. Conversely, IL-1rn protects against pre-leukemic myelopoiesis. Our data reveal that HSC differentiation is controlled by balanced IL-1ß/IL-1rn levels under steady-state, and that loss of repression of IL-1ß signaling may underlie pre-leukemic lesion and AML progression.
Assuntos
Leucemia Mieloide Aguda , Receptores de Interleucina-1 , Humanos , Receptores de Interleucina-1/genética , Medula Óssea , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proliferação de Células , Antígenos CD34RESUMO
Resumen. El Insomnio infantil definido como la dificultad mantenida, a pesar de la oportunidad y en función etaria, para iniciar o mantener el sueño o su calidad que provoca alteraciones funcionales en el niño y/o familia. Puede repercutir significativamente en la conducta, aprendizaje y metabolismo del niño y en su familia afectando su calidad de vida. La actigrafía nos permite a través de un dispositivo identificar periodos de vigilia y sueño. El objetivo de este trabajo es caracterizar a través de la actigrafía el patrón sueño-vigilia y evaluar la exposición a luz azul en niños menores de 2 años que consultan por insomnio. Se realizó un estudio observacional, descriptivo, transversal de lactantes derivados por insomnio al Centro de Sueño de Red de Salud UC Christus, durante: Marzo 2017-2018, con actigrafía. Fueron 20 actigrafías de 7 días. Edades entre 5 y 22 meses con diagnóstico de insomnio que no respondió al manejo inicial. 8 hombres, 12 mujeres. Horarios promedios: Acostarse: 20:31 hrs. Levantarse: 7:43 hrs. Horarios variables para acostarse: 15/20. 10/20 siestas después de las 16 hrs. Todos presentaron tiempo total de sueño disminuido, con aumentos del tiempo despierto una vez iniciado el sueño. Latencias del sueño aumentadas: 6/20. Eficiencia del sueño disminuidas en 4/20. Despertares nocturnos: promedio: 10.58. Expuestos a luz azul: 14/20. Horas exposición media: 2,4 hr/evento. Concluimos de este estudio que las principales dificultades fueron los despertares nocturnos con largos tiempo de vigilia, con disminución del tiempo total de sueño para la edad, y la actigrafía fue una herramienta de apoyo para objetivar conductas que dificultan la adquisición de un buen patrón de sueño.Palabras Clave: Insomnio infantil, sueño en lactantes, actigrafía, luz azul, despertares.
Abstract. Child insomnia is defined as sustained difficulty, despite the opportunity and according to age group, to initiate or maintain sleep, or a sleep quality causing that causes alterations in the child or family. It can significantly impact behavior, learning and metabolism of the child and his or her family, affecting their quality of life. The actigraphy through a device allows us to identify periods of wakefulness and sleep. The purpose of this work is to characterize sleep-wake patterns through actigraphy and to determine exposure to blue light in children younger than 2 years old, consulting for insomnia. An observational, descriptive, cross-sectional study of infants consulting for insomnia at the Sleep Center of UC Christus health network, in the period from March 2017 to 2018, with actigraphy. Twenty week long actigraphies were performed. The ages of the patients varied between 5 and 22 months, all had a diagnosis of insomnia that did not respond to initial management. Eight patients were male and 12 female. Hourly averages: bedtime: 8:31 PM. Waking up: 7:43 AM. Time variable for bedtime: 15/20. 10/20 NAPs after 16 hrs. All showed decreased sleep, with increases of total awake time once sleep began. Increased sleep latency: 6/20. Sleep efficiency decreased in 4/20. Nighttime Awakenings: average: 10.58. Exposed to blue light: 14/20. Average exposure in hours: 2.4 hr/event. We conclude from this study that the main difficulties were the nighttime awakenings with long time vigil, with decrease of the total sleep time for the age, and the actigraphy was a support tool to record behaviors that hinder a normal sleep pattern acquisition.Key words: Child insomnia, sleep in infants, actigraphy, blue light, awakenings
Assuntos
Humanos , Masculino , Feminino , Lactente , Actigrafia/métodos , Distúrbios do Início e da Manutenção do Sono/diagnóstico , Luz AzulRESUMO
Computed tomography (CT) is not the imaging technique of choice to assess inguinoscrotal pathology, as magnetic resonance or ultrasonography have superior soft tissue contrast resolution and do not involve gonadal exposure to ionizing radiation. However, testicular and inguinoscrotal pathology may be found both as an extension of intra-abdominal processes or incidentally on CT scans requested for other reasons. CT also plays a role in the evaluation of testicular injury when associated to pelvic trauma and in perineal infections with scrotal extension. A pictorial review of testicular and inguinoscrotal involvement in vascular, neoplastic, traumatic, infectious, or inflammatory diseases and in complications of abdominal surgeries is presented. Additionally, the CT appearance of several congenital anomalies and benign processes is depicted.
Assuntos
Neoplasias dos Genitais Masculinos/diagnóstico por imagem , Genitália Masculina/diagnóstico por imagem , Canal Inguinal/diagnóstico por imagem , Doenças Testiculares/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Doenças Vasculares/diagnóstico por imagem , Genitália Masculina/lesões , Humanos , Infecções/diagnóstico por imagem , Inflamação/diagnóstico por imagem , Masculino , Escroto/diagnóstico por imagem , Testículo/diagnóstico por imagem , Testículo/lesõesRESUMO
BACKGROUND: Variants in HIV-coreceptor C-C chemokine receptor type 5 (CCR5) and Human leukocyte antigen (HLA) genes are the most important host genetic factors associated with HIV infection and disease progression. Our aim was to analyze the association of these genetic factors in the presence of clinical symptoms during Primary HIV Infection (PHI) and disease progression within the first year. METHODS: Seventy subjects diagnosed during PHI were studied (55 symptomatic and 15 asymptomatic). Viral load (VL) and CD4 T-cell count were evaluated. HIV progression was defined by presence of B or C events and/or CD4 T-cell counts <350 cell/mm3. CCR5 haplotypes were characterized by polymerase chain reaction and SDM-PCR-RFLP. HLA-I characterization was performed by Sequencing. RESULTS: Symptoms during PHI were significantly associated with lower frequency of CCR5-CF1 (1.8% vs. 26.7%, p = 0.006). Rapid progression was significantly associated with higher frequency of CCR5-CF2 (16.7% vs. 0%, p = 0.024) and HLA-A*11 (16.7% vs. 1.2%, p = 0.003) and lower frequency of HLA-C*3 (2.8% vs. 17.5%, p = 0.035). Higher baseline VL was significantly associated with presence of HLA-A*11, HLA-A*24, and absence of HLA-A*31 and HLA-B*57. Higher 6-month VL was significantly associated with presence of CCR5-HHE, HLA-A*24, HLA-B*53, and absence of HLA-A*31 and CCR5-CF1. Lower baseline CD4 T-cell count was significantly associated with presence of HLA-A*24/*33, HLA-B*53, CCR5-CF2 and absence of HLA-A*01/*23 and CCR5-HHA. Lower 6-month CD4 T-cell count was associated with presence of HLA-A*24 and HLA-B*53, and absence of HLA-A*01 and HLA-B*07/*39. Moreover, lower 12-month CD4 T-cell count was significantly associated with presence of HLA-A*33, HLA-B*14, HLA-C*08, CCR5-CF2, and absence of HLA-B*07 and HLA-C*07. CONCLUSION: Several host factors were significantly associated with disease progression in PHI subjects. Most results agree with previous studies performed in other groups. However, some genetic factor associations are being described for the first time, highlighting the importance of genetic studies at a local level.
Assuntos
Soropositividade para HIV/genética , Antígenos HLA/genética , Fatores Celulares Derivados do Hospedeiro/metabolismo , Receptores CCR5/genética , Argentina , Western Blotting , Contagem de Linfócito CD4 , Progressão da Doença , Haplótipos/genética , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Carga ViralRESUMO
The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (nâ=â9, 32.1%), C (nâ=â1, 3.6%), and CRFs (nâ=â18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous "CRF" B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events.
Assuntos
HIV-1/genética , HIV-1/patogenicidade , Recombinação Genética/genética , Argentina/epidemiologia , Genoma Viral/genética , Infecções por HIV/epidemiologia , HIV-1/classificação , Humanos , Filogenia , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
The internal transcribed spacer (ITS) regions of rDNA genes of 49 Histoplasma capsulatum (48 from clinical samples and one from soil) isolates were examined. Nucleotide sequence heterogeneity within this region was useful for phylogenetic classification of H. capsulatum and species identification. Thus, in 45 of 49 isolates we observed higher percentages of identity in the nucleotide sequences of ITS regions when the isolates studied herein were compared with those reported in our country in the South America B clade. Phylogenetic analyses of rDNA sequences corresponding to the 537 bp of the ITS region obtained from H. capsulatum isolates assigned South America type B clade (45 isolates), North America type 1 and Asia clade (2 isolates each one). H. capsulatum strains isolated from soil and from patients living in Argentina (45 of 49) clustered together with the H. capsulatum isolates of the South America B clade. The high level of genetic similarity among our isolates suggests that almost one genetic population is present in the microenvironment. Isolates described as H. capsulatum var. capsulatum or var. farciminosum (2 isolates) did not form a monophyletic group and were found in the Asia clade. Subsequent studies are needed to properly identify these isolates.
Assuntos
DNA Espaçador Ribossômico/genética , Variação Genética , Histoplasma/genética , Histoplasma/isolamento & purificação , Histoplasmose/microbiologia , Histoplasmose/veterinária , Animais , Argentina/epidemiologia , Sequência de Bases , DNA Fúngico/genética , Histoplasma/classificação , Histoplasmose/epidemiologia , Doenças dos Cavalos/microbiologia , Cavalos , Humanos , Dados de Sequência Molecular , FilogeniaRESUMO
Lactobacillus is normally present in animals and humans colonizing several epithelia, mainly those belonging to the upper gastrointestinal tract. Most of the information about the distribution of Lactobacillus in mice has been obtained by bacterial culture and characterization, and only few reports have described the direct presence of these bacteria in tissues, especially in the gastric mucosa. In this study, we have characterized and evaluated the location and detailed relationship between Lactobacillus and epithelia using a combination of histological, molecular, immunocytochemical and ultrastructural methods. Normal Balb/c mice were sacrificed to study esophagus and stomach. Partial 16S rRNA gene sequencing, Gram, and P.A. Schiff staining allowed us to demonstrate that Lactobacillus murinus isolated from each animal colonize not only the epithelium of the forestomach but also that belonging to the distal esophagus. The pattern of colonization was linear over the keratinized epithelium, and also in a vertical way of focal bacterial aggregates. This was confirmed by transmission electron microscopy, and the nature of bacteria was further assessed by immunocytochemistry. Our results indicate that L. murinus can colonize the stomach and the esophagus epithelia in a biofilm-like manner, possibly acting as a defense barrier against colonization by other bacteria.
Assuntos
Esôfago/microbiologia , Lactobacillus/crescimento & desenvolvimento , Estômago/microbiologia , Animais , Biofilmes , Feminino , Vida Livre de Germes , Imuno-Histoquímica , Lactobacillus/isolamento & purificação , Lactobacillus/fisiologia , Lactobacillus/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
El propósito de esta presentación es dar a conocer los hallazgos clínicos, micológicos e histopatológicos de una aspergilosis pulmonar crónica por Aspergillus nomius, en una mujer de 52 años de edad que sufre de una feohifomicosis diseminada por Exophiala spinifera, con lesiones cutáneas, ganglionares y óseas de 22 años de evolución. La aspergilosis pulmonar se presentó como una neumopatía crónica, de 3 años de duración, que evolucionó hacia la abscedación. Esta infección fúngica se produjo durante el tratamiento con 800 mg/diarios de posaconazol y respondió favorablemente a la administraciónconjunta de caspofungina por vía intravenosa. Finalmente, la enferma fue intervenida quirúrgicamente y se le extirparon los lóbulos medio e inferior del pulmón derecho. En el estudio histopatológico de la pieza quirúrgica se comprobó que Aspergillus nomius invadía los vasos sanguíneos y que se formaba un granuloma epitelioide con células gigantes en torno a las hifas endovasculares. El agente causal deeste caso se aisló de múltiples muestras de expectoración y lavados broncoalveolares, así como de lapieza quirúrgica. Su ubicación taxonómica se hizo en base a estudios de biología molecular. No pudo establecerse una causa clara de inmunodeficiencia en este caso.
Assuntos
Humanos , Feminino , Pessoa de Meia-Idade , Antifúngicos/administração & dosagem , Aspergillus , Aspergilose Broncopulmonar AlérgicaRESUMO
Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp) belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.
Assuntos
Catepsina L/metabolismo , Endopeptidases/metabolismo , Histonas/metabolismo , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina L/análise , Catepsina L/genética , DNA Complementar/genética , Endopeptidases/análise , Endopeptidases/genética , Fertilização , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Dados de Sequência Molecular , Filogenia , Ouriços-do-Mar/citologia , Ouriços-do-Mar/genética , Alinhamento de Sequência , Espermatozoides/metabolismoRESUMO
HIV-1 epidemics in South America are believed to have originated in part from the subtype B epidemic initiated in the Caribbean/North America region. However, circulation of BF recombinants in similar proportions was extensively reported. Information currently shows that many BF recombinants share a recombination structure similar to that found in the CRF12_BF. In the present study, analyzing a set of 405 HIV sequences, we identified the most likely origin of the BF epidemic in an early event of recombination. We found that the subtype B epidemics in South America analyzed in the present study were initiated by a founder event that occurred in the early 1970s, a few years after the introduction of these strains in the Americas. Regarding the F/BF recombinant epidemics, by analyzing a subtype F genomic segment within the viral gene gag present in the majority of the BF recombinants, we found evidence of a geographic divergence very soon after the introduction of subtype F strains in South America. Moreover, through analysis of a subtype B segment present in all the CRF12_BF-like recombination structure, we estimated the circulation of the subtype B strain that gave rise to that recombinant structure around the same time period estimated for the introduction of subtype F strains. The HIV epidemics in South America were initiated in part through a founder event driven by subtype B strains coming from the previously established epidemic in the north of the continent. A second introduction driven by subtype F strains is likely to have encountered the incipient subtype B epidemic that soon after their arrival recombined with them, originating the BF epidemic in the region. These results may explain why in South America the majority of F sequences are found as BF recombinants.
Assuntos
Evolução Molecular , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Recombinação Genética , Análise por Conglomerados , Genoma Viral , Genótipo , HIV-1/isolamento & purificação , Humanos , Epidemiologia Molecular , Filogenia , Análise de Sequência de DNA , América do Sul/epidemiologia , Fatores de TempoRESUMO
Studies of HIV-1 replication kinetics and fitness require an accurate determination of the level of infectious HIV-1 present in virus stocks. The standard technique for measuring the level of replication-competent infectious virus in culture supernatants or patient samples is the tissue culture dose for 50% infectivity (TCID(50)), which provides an accurate assessment of the level of infectious HIV-1. However, it is a time-consuming technique which typically takes two or more weeks to complete and requires PHA-stimulated PBMC from HIV-1 seronegative donors or an appropriate cell line. Thus rapid, cell-free surrogate measures for TCID(50) are desirable. Here, we introduce the virtual TCID(50) technique: a new cell-free method estimating a surrogate of infectious titer by comparing the reverse transcriptase activity in virus stock to that of reference viruses with a known TCID(50) value. We have demonstrated that the virtual TCID(50) obtained through this technique is comparable to the actual infectious TCID(50). This method greatly simplifies the process of accurate HIV-1 titration and is particularly beneficial for studies which require titration of large number of HIV-1 isolates.
Assuntos
Transcriptase Reversa do HIV/metabolismo , HIV-1/patogenicidade , Virologia/métodos , Células Cultivadas , HIV-1/enzimologia , Humanos , Leucócitos Mononucleares , Padrões de ReferênciaRESUMO
BACKGROUND: Cytotoxic T-Lymphocyte (CTL) response drives the evolution of HIV-1 at a host-level by selecting HLA-restricted escape mutations. Dissecting the dynamics of these escape mutations at a population-level would help to understand how HLA-mediated selection drives the evolution of HIV-1. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a study of the dynamics of HIV-1 CTL-escape mutations by analyzing through statistical approaches and phylogenetic methods the viral gene gag sequenced in plasma samples collected between the years 1987 and 2006 from 302 drug-naïve HIV-positive patients. By applying logistic regression models and after performing correction for multiple test, we identified 22 potential CTL-escape mutations (p-value<0.05; q-value<0.2); 10 of these associations were confirmed in samples biologically independent by a Bayesian Markov Chain Monte-Carlo method. Analyzing their prevalence back in time we found that escape mutations that are the consensus residue in samples collected after 2003 have actually significantly increased in time in one of either B or F subtype until becoming the most frequent residue, while dominating the other viral subtype. Their estimated prevalence in the viral subtype they did not dominate was lower than 30% for the majority of samples collected at the end of the 80's. In addition, when screening the entire viral region, we found that the 75% of positions significantly changing in time (p<0.05) were located within known CTL epitopes. CONCLUSIONS: Across HIV Gag protein, the rise of polymorphisms from independent origin during the last twenty years of epidemic in our setting was related to an association with an HLA allele. The fact that these mutations accumulated in one of either B or F subtypes have also dominated the other subtype shows how this selection might be causing a convergence of viral subtypes to variants which are more likely to evade the immune response of the population where they circulate.
Assuntos
HIV-1/genética , Antígenos HLA/imunologia , Mutação , Seleção Genética , Antígenos Virais/genética , Evolução Biológica , Epitopos/genética , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , HIV-1/imunologia , Antígenos HLA-B/imunologia , Humanos , Imunidade , Modelos Estatísticos , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND: HIV-1 is characterized by its rapid genetic evolution and high diversity as a consequence of its error-prone reverse transcriptase and genetic recombination. This latter mechanism is responsible for the creation of circulating recombinant forms (CRFs) found in nature. Previous studies from our lab group have shown that the epidemic in Argentina is characterized by one highly prevalent circulating recombinant form, CRF12_BF, and many related BF recombinant forms. Since transcriptional transactivation of the HIV-1 long terminal repeat (LTR) promoter element requires the essential viral Tat protein, since these genetic structures underwent recombination in variants widely spread in South America, the aim of this work was to study transcriptional activity associated with the recombinant LTR and Tat elements. RESULTS: Differential transcriptional activity was measured for the BF recombinant LTR/Tat complex that is present in widely spread viral variants was demonstrated. This analysis demonstrated a higher activity for the BF complex when compared to its B subtype counterpart. CONCLUSION: This study indicates structural and functional consequences of recombination events within the LTR promoter and Tat transactivator protein of a naturally occurring HIV-1 recombinant form.
Assuntos
Variação Genética , Repetição Terminal Longa de HIV/genética , HIV-1/genética , Ativação Transcricional , Síndrome da Imunodeficiência Adquirida/virologia , Adulto , Linhagem Celular , Criança , Clonagem Molecular , DNA Viral/genética , Produtos do Gene tat/genética , Genes Reporter , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
UNLABELLED: A total of 220 individuals were included in this study, 112 HIV-seronegative healthy individuals and 108 HIV-1-infected patients involving: 18 AIDS patients with Toxoplasmic encephalitis (AIDS-TE), 49 AIDS patients without TE, and 41 asymptomatic patients, were genotyping for DR and DQ loci by molecular biology techniques. Fisher's Exact test was used for statistical analysis. HLA-DQB*0402 and DRB1*08 alleles were associated with a high risk to develop opportunistic infections with neurological involvement, mainly Toxoplasma encephalitis in relationship with subjects healthy (OR = 20.43; Pc = 7.0 x 10(-6) and OR = 11; Pc = 2.6 x 10(-4), respectively); in relationship with AIDS no TE (OR = 6.98; Pc = 0.028 and OR = 4.85; P = 0.012, Pc = 0.14) and with patients in asymptomatic stage (OR = 61.50, Pc = 8.4 x 10(-6) and OR = 19.38; Pc = 3.9 x 10(-4)), respectively. CONCLUSIONS: It was concluded that the presence of HLA-DQB*0402 and DRB1*08 alleles in HIV-1-positive patients could be considered risk factors for developing neurological opportunistic infections, mainly Toxoplasmic encephalitis.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/genética , Encefalite/genética , Antígenos HLA-DQ/genética , Antígenos HLA-DR/genética , Toxoplasmose Cerebral/genética , Infecções Oportunistas Relacionadas com a AIDS/complicações , Argentina/epidemiologia , Estudos de Casos e Controles , Encefalite/complicações , Frequência do Gene , Predisposição Genética para Doença , HIV-1 , Antígenos HLA-DQ/classificação , Cadeias beta de HLA-DQ , Antígenos HLA-DR/classificação , Humanos , Fenótipo , Toxoplasmose Cerebral/complicaçõesRESUMO
Different complex structures of the pol gene have been identified in 284 HIV-1 B/F recombinant sequences obtained from a group of 587 patients under treatment failure from Argentina. To analyze the mosaic structures of these viral sequences and to determine their phylogenetic relationship, the 284 partial pol gene sequences of BF recombinant viruses were amplified by RT-PCR and sequenced. Intersubtype breakpoints were analyzed by bootscanning. Phylogenetic relationships were determined by means of neighbor-joining trees. The analysis of the sequences showed multiple phylogenetic topologies clustering within intersubtype BF reference sequences. At least three different mosaic patterns were found compared to previously described BF-type viruses with unequal distribution in the studied population. The analysis also showed that HIV-1 BF recombinant viruses with diverse mosaic structures are phylogenetically related in their F segments and in selected B fragments with the F1 subtype and with BF recombinant viruses from Brazil, respectively, suggesting a common recombinant ancestor. No association was observed between the prevalence of each mosaic pattern and the frequency of major drug-resistance mutations in PR and RT.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Genes pol/genética , Infecções por HIV/tratamento farmacológico , HIV-1/classificação , Recombinação Genética , Inibidores da Transcriptase Reversa/uso terapêutico , Fármacos Anti-HIV/farmacologia , Argentina , Farmacorresistência Viral , Quimioterapia Combinada , Infecções por HIV/virologia , Protease de HIV/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutação , Filogenia , Inibidores da Transcriptase Reversa/farmacologia , Análise de Sequência de DNA , Falha de TratamentoRESUMO
To monitor HIV-1 diversity in Argentina, a phylogenetic-based analysis of HIV-1 partial pol sequences obtained for resistance testing in 587 treatment failure patients was performed in Buenos Aires city between 2001 and 2003. HIV-1 RNA was isolated from plasma samples and partial pol fragments amplified by RT-PCR. Sequences were obtained by automated sequencing. Phylogenetic analysis was performed and recombination patterns characterized. A total of 299 sequences grouped into clade B (50.94%) and 284 were B/F recombinants (48.38%). Four sequences were grouped into clades A, C, and F (0.68%). The clade C sample, 96105, was found to be a BC recombinant and samples 103396 and 104575 showed the same mosaic pattern with Kisii5009 from Kenya and 97KR004 from Korea, previously described as A2D recombinants. With the presence of two full-length genomes, one from Kenya and one from Korea, and now two partial genomes from Argentina, this recombinant is designated CRF16_A2D. Its presence on three continents shows that CRF16_A2D has a global distribution.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Análise por Conglomerados , Genes pol , Infecções por HIV/epidemiologia , HIV-1/classificação , HIV-1/genética , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Recombinação GenéticaRESUMO
To investigate the immunopathogenic mechanisms of type I autoimmune hepatitis in children, we analyzed by quantitative or semiquantitative reverse transcription-polymerase chain reaction the expression of cytokines interferon (IFN)-gamma, interleukin (IL)-12p40, IL-18, IL-4, IL-10, and IL-12R beta 2. In addition, liver and peripheral blood was collected to investigate the expression of the natural killer T (NKT) cell marker V alpha 24. The presence of NKT cells in hepatic lesions were also identified by immunohistochemistry. The analysis was performed on liver biopsies from 25 children with type I autoimmune hepatitis. As disease controls, we included six children with hepatitis C virus-related chronic hepatitis and nine control livers. The expression of IFN-gamma and IL-12p40 was not detected in controls but was clearly upregulated in pathologic biopsies. In addition, these samples showed an increased expression of IL-18 (p = 0.0003), IL-4 (p = 0.0055), and IL-12R beta 2 (p = 0.007). Western blot analysis confirmed the expression of IL-12p40 and IL-18. However, for IL-18, we detected only the immature biologically inactive polypeptide. The V alpha 24 transcripts were found increased in the liver (p = 0.0007) where V alpha 24(+) cells were also localized, but decreased in peripheral blood mononuclear cells (p = 0.041). In addition to a type I immune response, NKT cells might play a substantial role in the pathogenesis of type I autoimmune hepatitis in children.