[Genome epidemiology and tropical spastic paraparesis associated with human T-cell lymphotropic virus type 1]. / Epidemiología genómica y paraparesia espástica tropical asociada a la infección por el virus linfotrópico humano de células T tipo 1.

OBJECTIVE: Characterize the genomic environment of the sequences adjacent to human T-cell lymphotropic virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in different regions of Colombia and Japan. METHODS: A total of 71 recombinant clones with human genome sequences adjacent to 5' LTR in patients with HAM/TSP were compared to the Genome Browser and GenBank databases. Sixteen structural and compositional genome variables were identified, and statistical analysis was conducted in the R computer program, version 2.8.1, in a 0.5 Mb window. RESULTS: A total of 43.0% of the proviruses were located in the group C chromosomes; 74% of the sequences were located in the telomeric and subtelomeric regions (P < 0.05). A cluster analysis was used to establish the hierarchical relations between the genome characteristics included in the study. The analysis of principal components identified the components that defined the preferred genome environments for proviral integration in cases of HAM/TSP. CONCLUSIONS: HTLV-1 was integrated more often in chromatin regions rich in CpG islands with a high density of genes and LINE type repetitions, and DNA transposons which, overall, would form the genomic environments targeted for integration. This new scenario will promote substantial changes in the field of public health and in epidemiological management of infectious diseases. It will also foster the development of powerful tools for increasing the efficiency of epidemiological surveillance.

Human T-Lymphotropic Virus (HTLV) Type I in vivo Integration in Oral Keratinocytes.

Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14% (8/14) HAM/TSP patients and 27.28% (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

[Clonal expansion and genomic characterization of the human T-cell lymphotropic virus type I during the integration process in adult T-cell leukemia/lymphoma]. / Expansión clónica y caracterización genómica del proceso de integración del virus linfotrópico humano tipo I en la leucemia/linfoma de células T en adultos.

INTRODUCTION: Although the integration of human T-cell lymphotropic virus type I into the T-cells is not a random process, the mechanistic details are not understood. OBJECTIVES: The characteristics of the flanking host chromatin were evaluated at the integration sites in adult T-cell leukaemia/lymphoma (ATLL) patients infected with the virus. MATERIALS AND METHODS: From seven leukemic Colombian patients positive for the human T-cell lymphotropic virus type I (HTLV-I), lymphocyte DNA samples were extracted and amplified by inverse polymerase chain reaction (IPCR). Clonal expansion and human genome nucleotide composition in an extension of 50 bp was determined. To establish the characteristics of the human genome flanking provirus, 61 IPCR sequences from Colombian and Japanese ATLL patients, were analyzed in silico to obtain insights about the genomic structure, functions and nature of associated chromatin. RESULTS: The clonal expansion of cell clones was predominantly oligoclonal. From 61 IPCR sequences, 155 alignments with homology higher than 95% (e-value < 0.05) were screened. Seventy-five percent of those sequences corresponded to non coding elements that include repetitive and non-repetitive DNA. Fifty percent of the proviral integrations were associated with chromosomes of A and B groups. Viral DNA integration tended to favor exons of genes that replicated early, controlled the cell cycle, or were involved in signal transduction. CONCLUSIONS: The results indicated that HTLV-I integration was preferentially directed towards genomic environments with high C:G content, and toward genes that replicate early, regulate cell cycle or involved with signal transduction.

[Autoimmune syndrome in the tropical spastic paraparesis/myelopathy associated with human T-lymphotropic virus infections]. / Síndrome autoinmune en la paraparesia tropical espástica/mielopatía asociada a la infección por el virus linfotrópico humano tipo I de la costa pacífica colombiana.

INTRODUCTION: Previous reports have given evidence that in tropical spastic paraparesis (TSP)/human T-lymphotrophic virus (HTLV-I)-associated myelopathy (HAM), an autoimmune process occurs as part of its pathogenesis. OBJECTIVE: The roles of autoimmunity and the molecular mimicry was evaluated in TSP/HAM patients. MATERIALS AND METHODS: Plasma samples were characterized from patients in the Pacific coastal region of Colombia. Thirty-seven were identified as TSP/HAM, 10 were diagnosed with adult T-cell leukemia virus, 22 were asymptomatic carriers but seropositive for HTLV-I and 20 were seronegative and served as negative controls. Plasmatic levels of the following were determined: antinuclear antibody (ANA) levels, anticardiolipine-2 (ACL-2), interferon- (IFN-gamma) and interleukin-4 (IL-4). Using Western blot, the crossreactivity of the seropositive and seronegative samples was evaluated against proteins extracted from several central nervous system components of non infected Wistar rats. The HTLV-I seropositive plasmas were crossreacted with a monoclonal tax (LT4 anti-taxp40) from spinal cord neurons of non infected Wistar rats. RESULTS: Of the TSP/HAM patients, 70.2% were reactive against ANA and 83.8% against ACL-2, in contrast with those ATL and asymptomatic seropositives subjects that were not reactive (P<0.001). Moreover, 70.3% had detectable levels of IFN and 43.2% had detectable IL-4. LT4 anti-taxp40 and plasma of TSP/HAM exhibited cross reactivity with a MW 33-35 kDa protein from the rat spinal cord nuclei. CONCLUSION: Support was provided for the existence of an autoimmune syndrome mediated by molecular mimicry; the syndrome was responsible for some of the axonal degeneration observed in TSP/HAM patients.

[Construction of an adenoassociated, viral derived, expression vector to correct the genetic defect in Morquio A disease]. / Construcción de un vector de expresión derivado de virus adenoasociados para corregir in vitro el defecto genético de la enfermedad de Morquio A.

INTRODUCTION: Mucopolysaccharidosis IVA (Morquio A) is caused by a deficiency of N-acetylgalactosamine-6-sulphate-sulphatase, a lysosomal enzyme required for the stepwise degradation of keratan-sulfate and chondroitin-6-sulfate. A deficiency in this enzyme results in an accumulation of glycosaminoglycans in several tissues. Currently, no effective therapies exist and only supportive measures are used to treat some manifestations of the disease. An ideal therapy is one that can be administrated early in life, has low mortality, and leads to long-term expression of the enzyme. Gene therapy emerges as a potential alternative to correct the genetic defect in MPS IVA. OBJECTIVE: Adenoassociated virus-derived expression vectors (AAV) were constructed to correct in vitro the enzyme deficiency in mucopolysaccharidosis IVA. MATERIALS AND METHODS: Adenoasociated virus-derived vectors containing the human GALNS gene and driven by the citomegalivirus immedited-early promoter were constructed using a free-adenoviral protocol. HEK293 cells and human skin Morquio A fibroblasts were transfected with the recombinat vectors. Enzyme activity was measured in cells 24 and 48 hours post-transfection. RESULTS: Free-adenovirus recombinant AAV vectors were obtained with titres up to 2.08x1010 capsids/mL. HEK293 cells and Morquio A fibroblasts transfected with vectors showed GALNS activity up to 3.05 nmoles/mg/h 48 hours post-transfection. CONCLUSION: The AAV mediated the in vitro expression of GALNS enzyme in the transfected cells. These results are the first step towards a gene therapy alternative to Morquio A disease using adenoassociated virus-derived vectors.

Serological and virological evaluation of human T-lymphotropic virus type 1 infection in family groups from Tumaco, Colombia.

INTRODUCTION: To date there has been no statistical evaluation of the profiles of immunoglobulin classes and viral replication as variables in the study of HTLV-1 infection and circulation among families in virus-endemic areas of Colombia. OBJECTIVE: To evaluate the correlation of several immunological and molecular characteristics with the transmission and circulation of HTLV-1 among families in the town of Tumaco. MATERIALS AND METHODS: Plasma levels of HTLV-1 specific immunoglobulin classes IgG, IgM and IgA1, as well as IgG and sIgA in oral fluids, were calculated for 32 members of 10 family groups from Tumaco in which the mother and at least one child were infected with the virus. Levels of the different immunoglobulin classes were correlated with viral RNA circulating in plasma or oral fluids and the proviral burden as detected by RT-PCR. RESULTS: Significant differences were determined between mothers and carrier children for immunoglobulin levels (p=0.037) and proviral burden (p=0.002). The overall estimate of IgG in plasma and sIgA in oral fluids could be correlated with the circulation of free viral RNA in both fluids and high proviral burden, and associated with HAM/TSP mothers. The detection of anti- tax IgG in plasma revealed differences between HAM/TSP mothers and their offspring. CONCLUSION: The study of immunological and molecular variables permitted the analysis of HTLV-1 circulation among families of Tumaco. The strong correlation between levels of IgM specific for the virus and viral RNA circulating in fluids indirectly confirmed the transmission of HTLV-1 among families.

Envelope sequence variation and phylogenetic relations of human T cell lymphotropic virus type 1 from endemic areas of Colombia.

The HTLV-1 envelope gene of 12 TSP/HAM patients from two endemic areas of southwest Colombia (Tumaco and Buenaventura) was amplified by nested PCR, sequenced, and compared with previously reported HTLV-1 envelope sequences from isolates worldwide. In general, the sequence divergences among all Colombian samples ranged from 0.1 to 1.6%. Some amino acid substitutions, referring to the ATK-1 prototype strain in the surface domain gp46 and in p21, were highly prevalent in southwest Colombia, suggesting a geographical clustering of mutations in the envelope gene. The phylogenetic analysis showed that the Colombian isolates belong to the HTLV-1a lineage with minor subgroups. The genetic distance between Colombian and Japanese isolates ranged from 0.1 to 1.8%; in comparison, the genetic distance between Colombian and Caribbean isolates ranged from 0.4 to 2.2%. Our results strongly suggest that the actual quasispecies populations in southwest Colombia have been generated by separate, differently timed introductions of virus.

Global differential expression of genes located in the Down Syndrome Critical Region in normal human brain.

BACKGROUND: The information of gene expression obtained from databases, have made possible the extraction and analysis of data related with several molecular processes involving not only in brain homeostasis but its disruption in some neuropathologies; principally in Down syndrome and the Alzheimer disease. OBJECTIVE: To correlate the levels of transcription of 19 genes located in the Down Syndrome Critical Region (DSCR) with their expression in several substructures of normal human brain. METHODS: There were obtained expression profiles of 19 DSCR genes in 42 brain substructures, from gene expression values available at the database of the human brain of the Brain Atlas of the Allen Institute for Brain Sciences", (http://human.brain-map.org/). The co-expression patterns of DSCR genes in brain were calculated by using multivariate statistical methods. RESULTS: Highest levels of gene expression were registered at caudate nucleus, nucleus accumbens and putamen among central areas of cerebral cortex. Increased expression levels of RCAN1 that encode by a protein involved in signal transduction process of the CNS were recorded for PCP4 that participates in the binding to calmodulin and TTC3; a protein that is associated with differentiation of neurons. That previously identified brain structures play a crucial role in the learning process, in different class of memory and in motor skills. CONCLUSION: The precise regulation of DSCR gene expression is crucial to maintain the brain homeostasis, especially in those areas with high levels of gene expression associated with a remarkable process of learning and cognition.

INTRODUCCIÓN: La información de la expresión de genes consignada en bases de datos, ha permitido extraer y analizar información acerca procesos moleculares implicados tanto en la homeostasis cerebral y su alteración en algunas neuropatologías. OBJETIVOS: Correlacionar los niveles de transcripción de 19 genes localizados en la región crítica del cromosoma 21, asociada a Síndrome de Down (DSCR), con la localización cerebral y su coexpresión en diferentes subestructuras del cerebro humano. MÉTODOS: A partir de valores de expresión génica disponibles en la base de datos del proyecto cerebro humano del Atlas del Cerebro del "Allen Institute for Brain Sciences" (http://human.brain-map.org/), se construyeron perfiles de expresión de 19 genes DSCR en 42 subestructuras cerebrales. Además, utilizando métodos estadísticos multivariados se analizaron los patrones de coexpresión de genes DSCR en el cerebro normal. RESULTADOS: En el núcleo caudado, el núcleo accumbens y el putamen además de las Áreas centrales 2, 3 y 4, se determinaron los valores de expresión más elevados para los genes incluidos RCAN1, que codifica para una proteína involucrada en el proceso de transducción de señales de SNC; PCP4 cuya proteína interviene en la unión a la calmodulina y TTC3 una proteína que interviene en la diferenciación de neuronas. Las subestructuras identificadas con una elevada expresión de estos genes, están asociadas con procesos de aprendizaje, en diferentes tipos de memoria y habilidades motoras. CONCLUSIONES: La regulación de la expresión de los genes DSCR es clave para mantener la homeostasis cerebral, especialmente en aquellas áreas de mayor expresión, las cuales están asociadas con procesos sumamente importantes.

Serological and virological evaluation of human T-lymphotropic virus type 1 infection in family groups from Tumaco, Colombia / Evaluación serológica y virológica de la infección por el virus linfotrópico de células T humanas de tipo 1 en grupos familiares de Tumaco, Colombia

Introduction: To date there has been no statistical evaluation of the profiles of immunoglobulin classes and viral replication as variables in the study of HTLV-1 infection and circulation among families in virus-endemic areas of Colombia. Objective: To evaluate the correlation of several immunological and molecular characteristics with the transmission and circulation of HTLV-1 among families in the town of Tumaco. Materials and methods: Plasma levels of HTLV-1 specific immunoglobulin classes IgG, IgM and IgA1, as well as IgG and sIgA in oral fluids, were calculated for 32 members of 10 family groups from Tumaco in which the mother and at least one child were infected with the virus. Levels of the different immunoglobulin classes were correlated with viral RNA circulating in plasma or oral fluids and the proviral burden as detected by RT-PCR. Results: Significant differences were determined between mothers and carrier children for immunoglobulin levels (p=0.037) and proviral burden (p=0.002). The overall estimate of IgG in plasma and sIgA in oral fluids could be correlated with the circulation of free viral RNA in both fluids and high proviral burden, and associated with HAM/TSP mothers. The detection of anti- tax IgG in plasma revealed differences between HAM/TSP mothers and their offspring. Conclusion: The study of immunological and molecular variables permitted the analysis of HTLV-1 circulation among families of Tumaco. The strong correlation between levels of IgM specific for the virus and viral RNA circulating in fluids indirectly confirmed the transmission of HTLV-1 among families.

Introducción. Todavía no hay una evaluación estadística de los perfiles de las clases de inmuno- globulina s y la replicación viral, como variables para estudiar la infección y la circulació n del HTLV-1 en familias de zonas endémicas en Colombia. Objetivo. Evaluar la correlación de varias características inmunológicas y moleculares, con la transmisión y circulación del virus en familias del municipio de Tumaco. Materiales y métodos. Se calcularon los niveles de IgG, IgM e IgA1 en plasma, e IgG y IgA secretoria en fluido oral, de 32 miembros de 10 grupos familiares de Tumaco, en los que la madre y, al menos, un hijo estaban infectados con el virus. La concentración de las diferentes clases de inmunoglobulinas se pudo correlacionar con la circulación de ARN viral libre en plasma y fluido oral, y la carga proviral, según su detección mediante reacción en cadena de la polimerasa de transcripción inversa. Resultados. Se encontraron diferencias significativas en los niveles de inmunoglobulinas (p=0,037) y en la carga proviral (p=0,002) entre madres e hijos portadores. La estimación total de IgG en plasma e IgA secretoria en fluido oral, se pudo correlacionar con la circulación de ARN viral libre en ambos fluidos y una alta carga proviral, y se asoció con las madres paraparesia espástica tropical o mielopatía asociada con el HTLV-1. La detección en plasma de IgG anti-Tax reveló diferencias entre ellas y sus hijos. Conclusión. El estudio de las variables inmunológicas y moleculares permitió analizar la circulación del HTLV-1 en familias de Tumaco. La fuerte asociación entre los niveles de IgM específica para el virus y el ARN viral circulante en los fluidos y la carga proviral, confirmó indirectamente la transmisión intrafamiliar del virus.

Global differential expression of genes located in the Down Syndrome Critical Region in normal human brain / Expresión diferencial global de genes localizados en la Región Crítica del Síndrome de Down en el cerebro humano normal

Background: The information of gene expression obtained from databases, have made possible the extraction and analysis of data related with several molecular processes involving not only in brain homeostasis but its disruption in some neuropathologies; principally in Down syndrome and the Alzheimer disease. Objective: To correlate the levels of transcription of 19 genes located in the Down Syndrome Critical Region (DSCR) with their expression in several substructures of normal human brain. Methods: There were obtained expression profiles of 19 DSCR genes in 42 brain substructures, from gene expression values available at the database of the human brain of the Brain Atlas of the Allen Institute for Brain Sciences", (http://human.brain-map.org/). The co-expression patterns of DSCR genes in brain were calculated by using multivariate statistical methods. Results: Highest levels of gene expression were registered at caudate nucleus, nucleus accumbens and putamen among central areas of cerebral cortex. Increased expression levels of RCAN1 that encode by a protein involved in signal transduction process of the CNS were recorded for PCP4 that participates in the binding to calmodulin and TTC3; a protein that is associated with differentiation of neurons. That previously identified brain structures play a crucial role in the learning process, in different class of memory and in motor skills. Conclusion: The precise regulation of DSCR gene expression is crucial to maintain the brain homeostasis, especially in those areas with high levels of gene expression associated with a remarkable process of learning and cognition.

Introducción: La información de la expresión de genes consignada en bases de datos, ha permitido extraer y analizar información acerca procesos moleculares implicados tanto en la homeostasis cerebral y su alteración en algunas neuropatologías. Objetivos: Correlacionar los niveles de transcripción de 19 genes localizados en la región crítica del cromosoma 21, asociada a Síndrome de Down (DSCR), con la localización cerebral y su coexpresión en diferentes subestructuras del cerebro humano. Métodos: A partir de valores de expresión génica disponibles en la base de datos del proyecto cerebro humano del Atlas del Cerebro del "Allen Institute for Brain Sciences" (http://human.brain-map.org/), se construyeron perfiles de expresión de 19 genes DSCR en 42 subestructuras cerebrales. Además, utilizando métodos estadísticos multivariados se analizaron los patrones de coexpresión de genes DSCR en el cerebro normal. Resultados: En el núcleo caudado, el núcleo accumbens y el putamen además de las Áreas centrales 2, 3 y 4, se determinaron los valores de expresión más elevados para los genes incluidos RCAN1, que codifica para una proteína involucrada en el proceso de transducción de señales de SNC; PCP4 cuya proteína interviene en la unión a la calmodulina y TTC3 una proteína que interviene en la diferenciación de neuronas. Las subestructuras identificadas con una elevada expresión de estos genes, están asociadas con procesos de aprendizaje, en diferentes tipos de memoria y habilidades motoras. Conclusiones: La regulación de la expresión de los genes DSCR es clave para mantener la homeostasis cerebral, especialmente en aquellas áreas de mayor expresión, las cuales están asociadas con procesos sumamente importantes.

Epidemiología genómica y paraparesia espástica tropical asociada a la infección por el virus linfotrópico humano de células T tipo 1 / Genome epidemiology and tropical spastic paraparesis associated with human T-cell lymphotropic virus type 1

OBJETIVO: Caracterizar el ambiente genómico de las secuencias adyacentes al virus linfotrópico humano de células T tipo 1 (HTLV-1) en pacientes con paraparesia espßstica tropical y mielopatía asociada a la infección con HTLV-1 (PET/MAH) de diferentes regiones de Colombia y del Japón. MÉTODOS: Se enfrentaron 71 clones recombinantes con secuencias del genoma humano adyacentes al 5'-LTR de pacientes con PET/MAH, a las bases de datos del Genome Browser y del Gen-Bank. Se identificaron y analizaron estadísticamente 16 variables genómicas estructurales y composicionales mediante el programa informßtico R, versión 2.8.1, en una ventana de 0,5 Mb. RESULTADOS: El 43,0 por ciento de los provirus se localizaron en los cromosomas del grupo C; 74 por ciento de las secuencias se ubicaron en regiones teloméricas y subteloméricas (P < 0,05). Un anßlisis de conglomerados permitió establecer las relaciones jerßrquicas entre las características genómicas incluidas en el estudio; el anßlisis de componentes principales identificó las componentes que definieron los ambientes genómicos preferidos para la integración proviral en casos de PET/MAH. CONCLUSIONES: El HTLV-1 se integró con mayor frecuencia en regiones de la cromatina ricas en islas de citocina fosfato guanina (CpG), de alta densidad de genes y de repeticiones tipo LINE (elemento disperso largo [long interspersed element]) y transposones de ADN que, en conjunto, conformarían los ambientes genómicos blanco de integración. Este nuevo escenario promoverß cambios sustanciales en el campo de la salud pública y en el manejo epidemiológico de las enfermedades infecciosas, y permitirß desarrollar potentes herramientas para incrementar la eficiencia de la vigilancia epidemiológica.

OBJECTIVE: Characterize the genomic environment of the sequences adjacent to human T-cell lymphotropic virus type 1 (HTLV-1) in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in different regions of Colombia and Japan. METHODS: A total of 71 recombinant clones with human genome sequences adjacent to 5' LTR in patients with HAM/TSP were compared to the Genome Browser and GenBank databases. Sixteen structural and compositional genome variables were identified, and statistical analysis was conducted in the R computer program, version 2.8.1, in a 0.5 Mb window. RESULTS: A total of 43.0 percent of the proviruses were located in the group C chromosomes; 74 percent of the sequences were located in the telomeric and subtelomeric regions (P < 0.05). A cluster analysis was used to establish the hierarchical relations between the genome characteristics included in the study. The analysis of principal components identified the components that defined the preferred genome environments for proviral integration in cases of HAM/TSP. CONCLUSIONS: HTLV-1 was integrated more often in chromatin regions rich in CpG islands with a high density of genes and LINE type repetitions, and DNA transposons which, overall, would form the genomic environments targeted for integration. This new scenario will promote substantial changes in the field of public health and in epidemiological management of infectious diseases. It will also foster the development of powerful tools for increasing the efficiency of epidemiological surveillance.

Human T-Lymphotropic virus (HTLV) type I in vivo integration in oral keratinocytes

Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP) patients and 11 asymptomatic carrier individuals (AC) coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14 percent (8/14) HAM/TSP patients and 27.28 percent (3/11) AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

Caracterización genómica de la integración in vitro del VIH-1 en células mononucleares de sangre periférica, macrófagos y células T de Jurkat / Genomic characterization of HIV-1 in vitro integration in peripheral blood mononuclear cells, macrophages and Jurkat T cells

Introducción: La mayor parte del genoma celular es accesible a la integración retroviral; sin embargo, se propone que este proceso no es aleatorio y es dependiente de cada retrovirus. Objetivos: Identificar y caracterizar las regiones del genoma humano en donde ocurre la integración del virus de la inmunodeficiencia humana de tipo 1 (VIH-1) en células mononucleares de sangre periférica, macrófagos y células T de Jurkat infectadas. Materiales y métodos: Se seleccionaron 300 secuencias de ADN humano obtenidas por el método de ligación mediada por PCR, previamente depositadas en el GenBank. Utilizando el programa BLAST, sólo 264 de ellas se incluyeron en el estudio, pues se pudo obtener información sobre localización cromosómica, genes anotados, secuencias repetidas, número de islas CpG y tiempo medio de replicación, entre otras variables genómicas. Estas secuencias se exportaron a otras bases de datos. Resultados: El 53% (140/264) de las integraciones se registraron en bandas G. El 70,45% de los provirus se localizaron en los genes humanos anotados, mientras que el restante lo hizo en elementos repetidos. En general, la selección del sitio de integración se relacionó con las características locales genómicas y estructurales de la cromatina, entre las que se incluyen secuencias Alu-Sx y L1, densidad génica y de islas CpG, remodelación de la cromatina y tiempo de replicación. Éstas influenciarían la interacción eficiente del complejo de preintegración con los genomas celulares. Conclusión: Se determinó que la integración del VIH-1 en los genomas celulares estudiados estaría condicionada por características diferenciales de la cromatina y por procesos epigenéticos que influirían la selección del sitio blanco de integración.

Introduction: Most of the infected host cell genome is available for retroviral integration; however, it has been proposed that this process does not occur at random and depends upon each type of retrovirus. Objective: The objective is to identify and characterize differences in human genome regions of peripheral blood mononuclear cells, macrophages and Jurkat T cells in which integration of HIV-1 occurs. Material and Methods: Three hundred human DNA genome sequences, previously deposited in the GenBank, were selected at random. Using program BLAST, only 264 of them were included in the study because relevant information about chromosomal position, associated genes, repetitive sequences, number of CpG islands and average replication time was available; these sequences were exported to other data bases for analysis. Results: 53% (140/264) of integrations were located on G bands. 70.45% of provirus was located in human genes and the rest was located in repetitive elements. In general the integration site selection was correlated with genomics and structural characteristics of cell chromatin including Alu-Sx and L1 sequences, gene and CpG island densities, remodeling of chromatin, and replication time. All of them would influence the efficient interaction between the pre-integration complex and target cell genomes. Conclusion: It was determined that HIV-1 integration in target cellular genomes would be conditioned by differential characteristics of associated chromatin and by epigenetic processes that would influence the selection of integration sites.

Expansión clónica y caracterización genómica del proceso de integración del virus linfotrópico humano tipo I en la leucemia/linfoma de células T en adultos / Clonal expansion and genomic characterization of the human T-cell lymphotropic virus type I during the integration process in adult T-cell leukemia/lymphoma

Introducción. Aunque la integración del virus linfotrópico humano tipo I no es al azar, se desconocen muchos de los detalles de este proceso. Objetivo. Evaluar las características de la cromatina celular adyacente a secuencias provirales en pacientes con leucemia/linfoma de células T en adultos asociada al virus. Materiales y métodos. Se extrajo el ADN de biopsias de siete pacientes colombianos con leucemia/linfoma de células T en adultos y positivos para el virus linfotrópico humano tipo I. Éste se amplificó mediante reacción inversa en cadena de la polimerasa, para determinar el grado de expansión clónica y su composición de nucleótidos. A partir de 61 secuencias de ADN humano adyacentes a provirus, provenientes de pacientes leucémicos colombianos y japoneses, se efectuó un análisis in silico para obtener datos sobre su integración, las características de la cromatina y sus funciones asociadas. Resultados. La expansión de clones celulares fue predominantemente oligoclónica. De las 61 secuencias de ADN adyacente a provirus, se seleccionaron 155 alineamientos que cumplieron con los criterios de inclusión (homologías≥95%, e-value≤0,05). De éstos, 74,84% fueron secuencias no codificantes repetidas y no repetidas. El 45,95% de las integraciones provirales se localizó en los cromosomas de los grupos A y B. Se observaron tendencias de integración hacia exones de genes que se replican tempranamente, regulan el ciclo celular y participan en la transducción de señales. Conclusiones. Los resultados permiten postular que la integración del virus linfotrópico humano tipo I se dirigiría hacia un ambiente genómico caracterizado por elevado contenido de C:G, genes de replicación temprana que regularían el ciclo celular y la transducción de señales.

Introduction. Although the integration of human T-cell lymphotropic virus type I into the T-cells is not a random process, the mechanistic details are not understood. Objectives. The characteristics of the flanking host chromatin were evaluated at the integration sites in adult T-cell leukaemia/lymphoma (ATLL) patients infected with the virus. Materials and methods. From seven leukemic Colombian patients positive for the human T-cell lymphotropic virus type I (HTLV-I), lymphocyte DNA samples were extracted and amplified by inverse polymerase chain reaction (IPCR). Clonal expansion and human genome nucleotide composition in an extension of 50 bp was determined. To establish the characteristics of the human genome flanking provirus, 61 IPCR sequences from Colombian and Japanese ATLL patients, were analyzed in silico to obtain insights about the genomic structure, functions and nature of associated chromatin. Results. The clonal expansion of cell clones was predominantly oligoclonal. From 61 IPCR sequences, 155 alignments with homology higher than 95% (e-value <0.05) were screened. Seventy-five percent of those sequences corresponded to non coding elements that include repetitive and non-repetitive DNA. Fifty percent of the proviral integrations were associated with chromosomes of A and B groups. Viral DNA integration tended to favor exons of genes that replicated early, controlled the cell cycle, or were involved in signal transduction. Conclusions. The results indicated that HTLV-I integration was preferentially directed towards genomic environments with high C:G content, and toward genes that replicate early, regulate cell cycle or involved with signal transduction.

Modelación molecular y variación estructural de las integrasas de dos retrovirus humanos: HTLV-I y VIH-1 / Molecular modeling and structural variation of two human retrovirus integrases: HTLV-I and HIV-1

Objetivo: Analizar las características moleculares y de variación de secuencias de las integrasas del HTLV-I y del VIH-1 y sus variantes poblacionales. Metodología: Análisis de secuencias y estructuras obtenidas de diferentes bases de datos; para ello se utilizaron programas computacionales de modelación de estructuras proteicas e identificación de sustituciones polimórficas en secuencias de aminoácidos de integrasas del HTLV-I y VIH-1 previamente reportadas. Materiales y métodos: Tanto la integrasa del HTLV-I como la del VIH-1 son proteínas compuestas por 288 residuos de aminoácidos. Se encontró un parecido de estructuras terciarias entre los dominios catalíticos de las IN de VIH-1, ASV y RSV con la del HTLV- I. A partir de 103 secuencias completas de la integrasa del VIH-1 se registraron, en 46 codones, un total de 53 sustituciones que se localizaron en diferentes posiciones de la proteína nativa; las más frecuentes fueron: N27G (32,1%), A265V (30,1%), L101I (31,1%) y T123A (27,0%). Ninguna de las sustituciones más frecuentemente encontradas generó un cambio en el plegamiento nativo de la correspondiente región. Conclusión: La estructura tridimensional del dominio central catalítico de la integrasa condicionaría su actividad y su relación con moléculas potencialmente inhibidoras. Las sustituciones observadas fueron neutrales sin alterar la estructura nativa. Los resultados obtenidos confirman que la integrasa es un nuevo y promisorio blanco para el desarrollo de terapias antirretrovirales más efectivas en el siglo XXI...

Objective: To analyze the molecular characteristics and aminoacid sequence variations of HTLV-I and of HIV-1 integrases and their population variants. Materials and methods: Data mining and analysis of integrase sequences and protein structure data bases by using appropriate software for modelling and search for polymorphic substitutions in HTLV-I and HIV-1 integrase amino acid sequences previously reported. Results: HTLV-I and HIV-1 integrases are proteins of 288 amino acid residues. Structural modeling of tertiary folding of HTLV-I integrase catalytic central domain’s, showed closed structural characteristic with those of HIV-1, ASV and RSV. From 103 full amino acid sequences of HIV-1 integrase, 53 substitutions located in 46 different codons were recorded. The more frequents correspond to N27G (32,1%), L101I (31,1%), A265V (30,1%) and T123A (27,0%). None of these frequent substitutions introduced changes in the folding of HIV-1 native integrase. Conclusion: The tridimensional structure of central catalytic domain would influence the integrase activity and its relationship with potentially inhibitory molecules. Those observed aminoacid substitutions were neutral and do not alter the native protein structure. Our data confirm those previously published, and enable us to propose that IN is a new and promissory target for develop more effective antiviral therapies in the XXI century...

Sindrome autoinmune en la paraparesia tropical espastica/ mielopatia asociada a la infeccion por el virus linfotropico / Autoimmune syndrome in the tropical spastic paraparesis/myelopathy associated with human T-lymphotropic virus infections

Introduccion. Trabajos previos han aportado evidencias de que en la paraparesia espastica tropical/mielopatia asociada con el virus linfotropico humano tipo I, existe un componente autoinmune asociado a su patogenesis. Objetivo. Evaluar el estado autoinmune y la existencia de mimetismo molecular en pacientes con paraparesia espastica tropical del pacifico colombiano. Materiales y metodos. A partir de muestras de plasma de 37 pacientes con paraparesia espastica tropical/mielopatia asociada al HTLV-I, 10 con leucemia de celulas T del adulto, 22 individuos portadores asintomáticos y 20 seronegativos para el HTLV-I, se determinaron niveles plasmaticos de anticuerpos antinucleares y anticardiolipina-2 y de interferon e interleucina- 4. Se evaluo, por Western blot, la reactividad cruzada de plasmas contra proteinas obtenidas de varias fuentes celulares normales del sistema nervioso. Ademas, se estudio la reactividad cruzada de plasmas de seropositivos y del anticuerpo monoclonal LT4 anti-taxp40 en secciones de medula espinal de ratas Wistar no infectadas. Resultados. El 70,2 por ciento y el 83,8 por ciento de los pacientes con paraparesia espastica tropical fueron reactivos para anticuerpos ANA y ACL-2, respectivamente, en contraste con los de leucemia de celulas T del adulto y los seropositivos asintom¨¢ticos (P<0,001). Ademas, el 70,3 por ciento y el 43,2 por ciento de los pacientes con paraparesia espastica tropical tuvieron niveles detectables de IFN-¦Ã e IL-4, respectivamente. El anticuerpo LT4 anti tax-p40 y los plasmas de paraparesia espastica tropical/mielopatia asociada al HTLV-I mostraron una reaccion cruzada con una proteina de PMr 33-35 kDa, obtenida del nucleo de neuronas de la medula espinal de ratas Wistar no infectadas. Conclusion. Se obtuvieron evidencias que apoyan la existencia de un sindrome autoinmune mediado por mimetismo molecular como parte de la etiopatogenesis de la degeneracion axonal observada en la paraparesia espastica tropical en pacientes colombianos de la costa pacifica.

Construcción de un vector de expresión derivado de virus adenoasociados para corregir in vitro el defecto genético de la enfermedad de Morquio A / Construction of an adenoassociated, viral derived, expression vector to correct the genetic defect in Morquio A disease

Introducción. La mucopolisacaridosis IV A (Morquio A) es una enfermedad de depósito lisosómico causada por la deficiencia en la actividad de la enzima N-acetil-galactosamina- 6-sulfato-sulfatasa que produce la acumulación intralisosómica de queratán y condroitín-6-sulfato. Hasta el momento, su manejo es paliativo, por lo que las investigaciones se han enfocado en establecer una terapia que pueda aplicarse tempranamente y garantice la expresión estable de la enzima. En este sentido, la terapia génica se presenta como una de las potenciales alternativas terapéuticas para corregir el defecto genético en la mucopolisacaridosis IV A. Objetivo. Construir vectores de expresión derivados de virus adenoasociados para corregir in vitro la deficiencia enzimática en la mucopolisacaridosis IV A. Materiales y métodos. Se produjeron vectores derivados de virus adenoasociados que portaban el gen humano de la enzima N-acetil-galactosamina-6-sulfato-sulfatasa dirigido por el promotor temprano del citomegalovirus humano, empleando un sistema libre de adenovirus. Se transfectaron células HEK293 y fibroblastos humanos Morquio A con los virus recombinantes, y se determinó la actividad enzimática en el lisado celular a las 24 y 48 horas después de la transfección. Resultados. Se obtuvieron virus adenoasociados recombinantes, libres de adenovirus, con títulos hasta de 2,08 x 1010 cápsides/ml. Tanto en células HEK293 como en fibroblastos Morquio A transfectados, se obtuvieron actividades enzimáticas hasta de 3,05 nmoles/mg por hora, 48 horas después de la transfección. Conclusión. Los virus recombinantes producidos expresaron in vitro la enzima GALNS en las células transfectadas. Estos resultados constituyen el paso inicial para el desarrollo de una terapia génica para la enfermedad de Morquio A empleando vectores derivados de virus adenoasociados.

La genotipificación y fenotipificación de la resistencia del virus de la inmunodeficiencia humana tipo 1 (VIH-1) a los fármacos antirretrovirales / Genotypification and phenotypification of the viral resistence to anti-retroviral agents from type I immunodeficiency human virus

En diciembre de 2002 existía en el mundo 42 millones de personas infectadas por el virus de la inmunodeficiencia humana tipo 1 (VIH-1). Cómo se ha llegado a esta cifra, es una de las preguntas que tiene como base de respuesta la gran adaptación evolutiva del virus. Las poblaciones del VIH-1 replican como distribuciones complejas de genomas diferentes, pero genéticamente relacionadas, denominadas cuasiespecies. Así, las cepas virales que circulan alrededor del mundo presentan gran heterogeneidad de genotipos distribuidos como agrupaciones genéticas naturales con distribuciones geográficas características y dinámicas de infección y dispersión diferentes. El aumento de la capacidad de acción antiviral, facilitado por el desarrollo de nuevos fármacos, ha incrementado la frecuencia de mutantes virales resistentes. Este nuevo panorama evolutivo dificulta la aplicación de una terapia antirretroviral exitosa. Numerosos estudios retrospectivos realizados a lo largo de los años han demostrado la correlación entre la presencia de mutaciones y el fracaso terapéutico de la infección por el virus. En tal sentido ha sido importante desarrollar pruebas de genotipificación y fenotipificación que puedan discriminar las mutaciones asociadas con la resistencia a un determinado antirretroviral. Aunque no son instrumentos completamente seguros, si se convierten en herramientas poderosas para poder diseñar de manera más confiable tratamientos terapéuticos más efectivos y que retarden al máximo la aparición de cepas resistentes. Además, se pueden emplear en la vigilancia fina de la pandemia del SIDA/VIH uno de los problemas más graves de salud pública del mundo actual

Estudio de la seroprevalencia de la infección por los virus linfotrópicos humanos (HTLV) I y II en poblaciones del departamento de Córdoba, Colombia / Study of seroprevalence of infection by humanT-Lymphotropic virus I/II in Cordoba, Colombia

Introducción: Se determinó la seroprevalencia de los virus linfotrópicos humanos tipos I y II (HTLV-I y II) en varios municipios del Departamento de Córdoba. Materiales y métodos: Se analizaron 962 muestras de suero mediante la prueba de ELISA y de Western blot confirmatorio. Se analizaron genéticamente 5 aislamientos virales por RFLP y secuenciación de la región LTR. Resultados: Se confirmaron 20 aislamientos como HTLV-I y uno como HTLV-II. La seroprevalencia de la infección por el HTLV-I en la muestra obtenida del departamento de Córdoba fue 2.1/100 (20/962), y para el HTLV-II 0.1/100 (1/962). Mediante análisis genéticos se determinó que tres correspondieron al subtipo africano b y dos el cosmopolita a. Conclusión: Este es el primer registro de la infección tanto por HTLV-I como por HTLV-II informado para el departamento de Córdoba en Colombia