Genotipificación, niveles de expresión y estado físico del virus del papiloma humano en pacientes colombianos con cáncer de células escamosas en la cavidad oral / Genotyping, levels of expression and physical status of human papilloma virus in oropharyngeal squamous cell carcinoma among Colombian patients

Introducción. Uno de los factores de riesgo del carcinoma de células escamosas en la cavidad oral es la infección por el virus del papiloma humano (HPV), cuyas prevalencias dependen de la región geográfica. Objetivo. Identificar los tipos del virus del papiloma humano más frecuentes en el cáncer de la cavidad bucal, sus niveles de expresión y el estado físico del genoma viral. Materiales y métodos. Se seleccionaron 46 pacientes que asistían a los servicios de cirugía de cabeza y cuello en Bogotá, Manizales y Bucaramanga. El examen histopatológico de las muestras incluidas en el estudio demostró la presencia de carcinoma de células escamosas en la cavidad oral en todas ellas. Se extrajo el ADN para genotipificar el virus y determinar el estado físico de su genoma, y el ARN para determinar los transcritos virales mediante reacción en cadena de la polimerasa en tiempo real. Resultados. La prevalencia del virus del papiloma humano en los tumores fue de 21,74% (n=10) y el tipo viral más frecuente fue el HPV-16 (nueve casos). La expresión viral del HPV-16 fue baja (una de 11 copias) y el estado físico predominante fue el mixto (ocho casos), con prevalencia de la disrupción en el sitio de unión de E1 y E2 (2525 a 3720 nucleótidos). Conclusión. En los pacientes con carcinoma de cavidad oral incluidos en este trabajo, la frecuencia del virus del papiloma humano fue relativamente baja (21,7 %) y el tipo viral más frecuente fue el HPV-16, el cual se encontró en forma mixta y con baja expresión de E7 , lo cual puede ser indicativo de un mal pronóstico para el paciente.

Introduction: One of the risk factors for squamous cell oropharyngeal carcinoma is infection with the human papilloma virus (HPV), with prevalences that vary depending on the geographical region. Objective: To identify the most frequent HPV viral types in oropharyngeal cancer, the levels of expression and the physical condition of the viral genome. Materials and methods: Forty-six patients were included in the study from among those attending head and neck surgical services in the cities of Bogotá, Manizales and Bucaramanga. In the histopathological report all study samples were characterized as oropharyngeal squamous cell carcinoma. DNA extraction was subsequently performed for HPV genotyping and to determine the physical state of the viral genome, as well as RNA to determine viral transcripts using real-time PCR. Results: HPV prevalence in tumors was 21.74% (n=10) and the most common viral type was HPV-16 (nine cases). Viral expression for HPV-16 was low (one of 11 copies) and the predominant physical state of the virus was mixed (eight cases), with disruption observed at the E1 - E2 binding site (2525 - 3720 nucleotides). Conclusion: The prevalence of HPV associated with oropharyngeal carcinoma among the Colombian study population was 21.7%, which is relatively low. The most frequent viral type was HPV-16, found in a mixed form and with low expression of E7 , possibly indicating a poor prognosis for these patients.

Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K. METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones. RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293. CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins. .

Células epiteliais do timo são possível reservatório viral e transmitem o HTLV-1 para linfócitos T CD4 / Thymic epithelial cells are infected by htlv-1 and transmit the virus to cd4 t lymphocytes

O timo é um órgão linfoide primário, sítio do desenvolvimento de células T, provendofatores críticos e coordenados que induzem e suportam o comprometimento delinhagem, diferenciação e sobrevivência dessas células. A presença das célulasnão-linfóides, principalmente as células epiteliais do timo (TEC) no parênquimatímico promove a migração e diferenciação coordenada dos linfócitos T. Os linfócitosT são o principal alvo do Virus linfotrópico T humano (HTLV-1), agente etiológico daleucemia/linfoma associado ao HTLV-1 (ATL) e de doença que compromete osistema nervoso/muscular (HAM/TSP). É desconhecida a causa que leva a uma ououtra doença. A resposta imune antiviral mediada por células T é ineficiente nessaspatologias. Mesmo que o vírus tenha tropismo pelos linfócitos T, ele é capaz deinfectar outros tipos celulares por contato direto entre células ou por partículas viraislivres. Linfócitos ativados recirculam pelos órgãos linfoides, incluindo o timo, onde ascélulas epiteliais tímicas (TEC) interagem intimamente com as células recirculantes,promovendo uma possível via de transmição do HTLV-1. No nosso trabalho,observamos que as TECs possuem os receptores para a entrada do vírus (GLUT-1e Neuropilina-1). Experimentos in vitro mostraram que as TECs podem serinfectadas pelo HTLV-1 por linhagens de linfócitos derivados de pacientesportadores de ATL e de HAM/TSP. Essas infecções ocorreram tanto por contatodireto entre as células, quanto por sobrenadante contendo partículas virais livresderivadas do sobrenadante dos linfócitos. O vírus pode ser observado após 24horas e 10 dias de cultivo, quando a maioria das células estava infectada. Atravésde microscopia eletrônica de transmissão, foram observadas partículas viraisbrotando de estruturas semelhantes a corpos multivesculares nas TECs...

The thymus is a primary lymphoid organ, site of developing T cells, providing acoordinated set of critical factors to induce and support lineage commitment,differentiation and survival of these cells. The presence of non-lymphoid cellsthrough the thymic parenchyma serves to provide coordinated migration anddifferentiation of T lymphocytes. T lymphocytes are the main target of Human TLymphotropic Virus type 1 (HTLV-1). This virus is the etiologic agent of HTLV-1associated lymphoma and leukemia (ATL) or a disease of muscular/nervous systems(HAM/TSP), but it is unknown what triggers to one or other disease. T-cell mediatedimmune response against viral proteins is not effective in both diseases. Althoughthe virus has T lymphocyte tropism, it can infect other cells by cell contact and cellfreevirus. Activated T lymphocytes circulates around lymphoid organs, including thethymus, where the thymic epithelial cells (TEC) strongly interact with recirculatingcells, so infected lymphocytes could transmit the virus to TEC. Interestingly, in ourwork we observed that TEC expresses molecules related to the HTLV-1 transmission(GLUT-1, Neuropilin 1). In vitro experiments showed that TEC could be infected bycell lineages derived from ATL and HAM/TSP patients. These infections happenedfrom cell contact and by cell-free virus derived from cell supernatants. The virus canbe seen after 24h and after 10 days, when most cells in the culture were infected. Bytransmission electron microscopy, virus particles were observed budding frommultivesicular bodies like structures in TEC. Cytokines and chemokines wereincreased at mRNA level soon after contact with HTLV-1 containing supernatantderived from lymphocytes. In addition, type 1 interferon and interferon stimulatedgenes were decreased in HTLV-1 infected TEC...

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.

Epidemiología y bioinformática en el estudio de la Leucemia Linfoma de Células T del Adulto asociada a la infección con VLHT-1 / Using epidemiology and bioinformatics for studying HTLV-1 infection-associated adult T-cell leukemia

Objetivos Establecer la relación entre el número de provirus VLHT-1 y las características de la cromatina adyacente en casos de Leucemia Linfoma de Células T del Adulto. Metodología Se realizó una revisión sistemática y un metaanálisis de la literatura publica que considero como variables de estudio los provirus por cromosoma y características estructurales y funcionales de la cromatina adyacente a los sitios de integración. La concordancia entre los resultados de la evaluación que emitieron dos expertos fue evaluada con el coeficiente de Spearman Rho. Se evaluó el sesgo de publicación mediante el gráfico de embudo y el estadígrafo Egger. De acuerdo con los resultados de la evaluación de la heterogeneidad se aplicó el modelo de efectos fijos para la combinación de los resultados de las integraciones que ocurrieron en: secuencias codificantes y secuencias codificantes de acuerdo con su función molecular. Resultados La concordancia entre expertos evaluadores fue de 0,7. No se encontró sesgo de publicación. Se determinó homogeneidad entre los estudios seleccionados (p>0,05). El provirus VLHT-1 se integró en secuencias en regiones teloméricas y subteloméricas. La combinación de los resultados mostró una integración sitio dirigida hacia regiones codificantes del genoma humano (p<0,05). Conclusión En su conjunto los resultados permiten concluir que la integración proviral no es al azar en LCCTA; ésta ocurrió en regiones reguladoras o de control; que explicarían algunos de los proceso moleculares involucrado en leukomogénesis.

Objectives Establishing a correlation between the number of HTLV-1 provirus and the characteristics of the genomic environment in ATL cases. Methodology A systematic search was made of publications as well as a meta-analysis of the pertinent literature considering proviruses per chromosome and structural and functional characteristics of flanking chromatin regions as variables. The concordance of experts' study was evaluated by Spearman Rho correlation. Publication bias was analysed by funnel plot and the Egger statisgrapher. A fixed effects model was applied according to heterogeneity evaluation to combine the results of integration occurring in coding sequences as well as coding sequences according to their molecular function. Results The expert concepts' Kappa index was 0.7 and no publication bias was observed. The meta-analysis result was homogeneous (p>0.05). HTLV-1 integration was directed towards several chromosomes' telomeric and subtelomeric regions. The combination of published results in the articles which were analysed supported the hypothesis of integration events being site-directed towards coding regions of the human genome (p<0.05). Moreover, the groups of genes having enzymatic and receptor functions was statistically significant. Conclusion The results led to concluding that HTLV-I integration in the ATLL cases analysed here was not random but was directed towards regulatory regions. Such results could help to explain the role of some processes involved in leukemogenesis.

Resistance to inhibitors of the human immunodeficiency virus type 1 integration

This review will summarize the role of integrase in HIV-1 infection, the mechanism of integrase inhibitors and resistance with an emphasis on raltegravir (RAL), the first integrase inhibitor licensed to treat HIV-1 infection.

Expresión de oncogenes y estado de integración del Papilomavirus Humano (HPV) como posibles marcadores de progresión maligna en pacientes con lesiones cervicales: estudio preliminar en la ciudad de Buenos Aires / Viral oncogene expression and integration status of the human papillomavirus as possible markers of malignant progression in patients with cervical lesions

Introducción: el Papilomavirus Humano (HPV) ha sido identificado como agente causal del cáncer cervical, induciendo lesiones cervicales de bajo grado que, eventualmente, se malignizan debido a distintos factores. La expresión de oncogenes virales y el estado de integración viral son necesarios para el desarrollo de neoplasias, por lo que podrían utilizarse como marcadores de progresión maligna en éste tipo de lesiones. Objetivo: evaluar la expresión de los oncogenes virales E6/E7 de HPV16 y 18 y el estado del genoma viral como posibles marcadores de progresión maligna. Materiales y métodos: se evaluaron muestras de lesiones cervicales (n = 27 controles (Ctrl), n = 18 CIN I, n= 24 CIN II/III y n = 32 Carcinomas invasivos (CC) derivadas del Hospital Gral. de Agudos C. Durand, CABA. Se evaluó la presencia de genoma consenso HPV y específico HPV16 y 18 mediante dPCR. Se determinó la expresión de oncogenes virales E6/E7 de HPV16 y HPV 18 y el estado de integración viral se analizó mediante PCR-APOT. Resultados: se observó un aumento significativo de presencia de genoma viral en correlación con el grado de lesión analizada CIN I: 77,8 %, CIN II/III: 83,3 % y CC: 100 % en comparación con el grupo control (25,9 %, p<0,001). La infección con HPV16 fue significativamente mayor en todos los grupos (vs. HPV18, p<0,001), encontrándose coinfección en varios casos. La expresión de los oncogenes virales de HPV16 fue significativamente mayor en comparación con HPV18 (p<0,0001). El estado físico de los genomas virales mostró una tendencia a la forma integrada en correlación con el grado de lesión analizada, encontrándose mayor presencia de genomas integrados en CC (vs. Episomal y/o episomal e integrado p<0,02) de HPV16. Para HPV18, sólo pudimos observar genomas integrados en CIN II/III (100 %) y CC (50 %). Sólo el 12,5 % de los genomas HPV 18 fueron detectados episomal e integrado. Conclusiones: observamos altas frecuencias de infección con HPV16 en comparación con HPV 18...

Introduction: Human Papillomavirus (HPV) has been identified as causal agent of cervical cancer, inducing cervical low grade lesions that, eventually, maligniza due to different factors. Viral oncogene expression and integration status are necessary for neoplasic development, and they could be used as malignant progression markers in these types of lesions. Aim: Evaluate HPV16 and 18 (E6/E7) viral oncogene expression and genome integration status as possible malignant progression markers. Materials and methods: Cervical samples derived from Hospital GA C. Durand (CABA) were evaluated (n = 27 healthy controls -Ctrl-, n = 18 CINI, n = 24 CIN II/III abd b = 32 Invasive Carcinomas (CC). HPV consensus and HPV16 and 18 specific genomes were evaluated by PCR. Viral oncogene expression (E6/E7) of HPV16 and HPV18 was determined, as well as viral integration status was determined by means of PCR-APOT. Results: A significant increase in viral genome presence was observed in correlation with the degree of the lesion analyzed CIN I: 77,9%, CIN II/III: 83,3% y CC: 100% in comparison to control Group (25,9% p<0,001). The infection with HPV16 was significantly greater in all the groups in comparison with HPV18 (p<0,001); co-infection was detected in several cases. HPV 16 oncogene expression was greater than the one showed by HPV-18 (p<0,0001). The physcal state of the viral genomes showed a tendency to the integrated form in correlation with the degree of analyzed lesion, detecting most of integrated HPV16 genomes in CC group (vs. episomal and/or episomal and integrated, p<0,02), FORM II/III (100%) and CC (50%). Only a minor fraction of HPV18 genomes in CC where found to be, both, episomal and integrated (12,5%). Conclusions: We have detected higher HPV16 infection frequencies in comparison with HPV18 in all analyzed groups. On the other hand, we observed an increase in HPV-16 onocogene expression in comparison to HPV18 ones. HPV16 was found predominantly integrated...

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.

Characterization of the long-terminal repeat single-strand tail-binding site of Moloney-MuLV integrase by crosslinking

Processing of viral DNA by retroviral integrase leaves a dinucleotide single-strand overhang in the unprocessed strand. Previous studies have stressed the importance of the 5' single-stranded (ss) tail in the integration process. To characterize the ss-tail binding site on M-MuLV integrase, we carried out crosslinking studies utilizing a disintegration substrate that mimics the covalent intermediate formed during integration. This substrate carried reactive groups at the 5' ss tail. A bromoacetyl derivative with a side chain of 6 A was crosslinked to the mutant IN 106-404, which lacks the N-terminal domain, yielding a crosslinked complex of 50 kDa. Treatment of IN 106-404 with N-ethylmaleimide (NEM) prevented crosslinking, suggesting that Cys209 was involved in the reaction. The reactivity of Cys209 was confirmed by crosslinking of a more specific derivative carrying maleimide groups that spans 8A approximately. In contrast, WT IN was not reactive, suggesting that the N-terminal domain modifies the reactivity of the Cys209 or the positioning of the crosslinker side chain. A similar oligonucleotide-carrying iodouridine at the 5'ss tail reacted with both IN 106-404 and WT IN upon UV irradiation. This reaction was also prevented by NEM, suggesting that the ss-tail positions near a peptide region that includes Cys209.

Estudo da integração genômica do HTLV-1 e da clonalidade das células leucêmicas: linfoma de células T do adulto (ATL) na Bahia / Study of HTLV-I proviral integration and T cell clonality in adult: T-cell leukemia/lymphoma (ATL) in Bahia

A leucemia/linfoma de células T do adulto (ATL) constitui uma forma grave de leucemial linfoma que ocorre, em geral, na vida adulta e é causada pelo vírus linfotrópico de células T humanas (HTLV-I). O HTL V-I está presente em cerca de 1,8 por cento da população da cidade de Salvador, Bahia, Brasil. No entanto, apenas cerca de 5 por cento dos infectados desenvolvem ATL. O HTLV-I é um retrovírus que integra o seu DNA proviral no DNA genômico da célula hospedeira (principalmente células T CD4+) num local que presume-se randômico e, acredita-se que, para o desenvolvimento da A TL, é necessário ocorrer a expansão clonal das células infectadas. Assim, a detecção da integração proviral e da clonalidade da células leucêmicas são de fundamental importância para o diagnóstico da ATL e definição do tratamento. O presente trabalho buscou identificar os locais de integração do DNA proviral do HTLV -I no DNA da célula hospedeira e o tipo de expansão clonal observada em amostras de PBMC e células de tecido de lesões de pacientes com diagnóstico clínico-patológico de A TL do estado da Bahia. A determinação do sítio de integração do DNA proviral na célula hospedeira foi feita, em 24 pacientes, através das técnicas de IPCR e ILPCR. Estas técnicas permitiram identificar o sítio de integração proviral em pacientes de ATL de diferentes formas clínicas e observou-se que a integração do provírus não ocorreu de forma preferencial em nenhum cromossomo. Em apenas três pacientes houve interrupção de seqüências codificantes e na maioria dos demais pacientes analisados, o provírus integrou-se em regiões próximas aos centrômeros, conhecidas como regiões repetitivas alfóides. O padrão de clonalidade das células leucêmicas foi determinado, em 36 pacientes, através da análise por PCR do rearranjo dos genes que codificam para a cadeia y do TCR. Observou-se assim um padrão monoclonal das células T para todos os pacientes com forma clínica aguda...

Adult T-cell leukemia/lymphoma (ATL) is a severe neoplasm that usually occurs in adults, and that is caused by human T-cell lymphotropic virus (HTLV-I) infection. In Salvador, Bahia, Brazil, a HTLV-I seroprevalence rate of 1.8% was observed in healthy subjects. However, generally, only 5% of infected individuals develop ATL. HTLV-I is a retrovirus that randomly integrates its proviral DNA into the host genome (specially, T CD4+ cells), and clonal expansion of infected cells is believed to result in the onset of ATL. Therefore, detection of monoclonal provirus integration is important to ATL diagnosis and treatment definition. The aim of this study was to investigate the HTLV-I proviral integration sites and T cell clonality in samples of PBMC and cutaneous biopsies of patients with clinical and histopathologic diagnosis ofATL from Bahia. Using inverse PCR (IPCR and ILPCR) we identified provirus integration sites in PBMC and cutaneous biopsies of 24 patients in diverse clinical subtypes of ATL. No chromosome bias was evident among different patients. In three patients occurred interruptionof transcriptional units and in most patients the provirus was integrated in alphoid regions near centrosomes. T-cell clonality was assessed by detection of the rearranged TCR-ã genes. Monoclonal pattern was observed in acute patients. In the other clinical subtypes, smallnumber of patients showed oligoclonal and policlonal patterns. According to these results, we showed that inverse PCR and TCR-ã PCR are efficient for integration site determination and infected cells clonality patterns in ATL patients and could be used in diagnosis and patientfollow up in Bahia.

Integración Viral y Cáncer de Cuello Uterino / HPV integration and cervical cancer

El cáncer de cuello uterino (CCU) constituye un problema generalizado de salud que ocupa el segundo lugar a nivel mundial entre las neoplasias malignas de la mujer. Las infecciones persistentes con virus del papiloma humano (VPH) de alto riesgo son reconocidas actualmente como un factor necesario en el desarrollo de CCU y sus lesiones precursoras. Uno de los procesos que parece estar más involucrado en el origen de las células malignas, es el evento de integración del virus al genoma del huésped, relacionado también con la progresión de lesiones preinvasivas y el mantenimiento del fenotipo transformado. Estos eventos de integración, especialmente de VPH tipo 16 se dan en secuencias específicas del genoma viral, principalmente en su región E1/E2, interrumpiendo la secuencia y permitiendo que exista desregulación de las actividades de transcripción, conduciendo a la sobre-expresión de las oncoproteínas virales E6 y E7 que normalmente inactivan genes supresores de tumores, como p53 y pRb, responsables del control en importantes puntos de chequeo del ciclo celular. Actualmente el proceso de integración viral, es considerado como una alteración genética importante que caracteriza las displasias malignas, con potenciales aplicaciones como marcador de progresión de lesiones precursoras y herramienta de diagnóstico

Caracterização do estado físico do ADN de papilomavírus humanos em lesões cervicais pré-malignas e malignas / Characterization of the physical state of human papillomavirus DNA in cervical lesions premalignant and malignant

A integração do ADN do papilomavírus humano ao genoma celular é um fator essencial para progressão maligna. Este processo usualmente interrompe ou deleta as regiões de sequências de leitura aberta de E1 ou E2, o qual leva à superexpressão das oncoproteínas virais E6/E7 e subsequente transformação maligna...Controle positivos e negativos foram utilizados. Oligonucleotídeos que amplificam a região do ADN humano de 330 bp para a beta-actina foram usados como controle interno. O HPV 16 foi o tipo predominantemente encontrado nas lesões cervicais pré-malignas (87,5%) e nas lesões malignas (77%) e a integração de seu ADN ocorreu em vinte e duas pacientes (71%). Nove (29%) dos casos demonstraram a existência da forma epissomal. O ADN do HPV 18 foi visto em 12,5% das HSIL e em 27% dos carcinomas com integração em 50% dos casos. A diferença entre as formas epissomal e integrada do ADN do HPV 16 foi estatisticamente significativa (p,= 0,05). Apesar das limitações da técnica de PCR em identificar a coexistência das formas epissomal e integrada, nossos resultados sugerem que a integração do ADN do HPV 16 deve ser um útil marcador para progressão maligna.

Mouse mammary tumor virus (MMTV), a retrovirus that exploits the immune system. Genetics of susceptibility to MMTV infection

All animals, including humans, show differential susceptibility to infection with viruses. Study of the genetics of susceptibility or resistance to specific pathogens is most easily studied in inbred mice. We have been using mouse mammary tumor virus (MMTV), a retrovirus that causes mammary tumors in mice, to study virus/host interactions. These studies have focused on understanding the mechanisms that determine genetic susceptibility to MMTV-induced mammary tumors, the regulation of virus gene expression in vivo and how the virus is transmitted between different cell types. We have found that some endogenous MMTVs are only expressed in lymphoid tissue and that a single base pair change in the long terminal repeat of MMTV determines whether the virus is expressed in mammary gland. This expression in lymphoid cells is necessary for the infectious cycle of MMTV, and both T and B cells express and shed MMTV. Infected lymphocytes are required not only for the initial introduction of MMTV to the mammary gland, but also for virus spread at later times. Without this virus spread, mammary tumorigenesis is dramatically reduced. Mammary tumor incidence is also affected by the genetic background of the mouse and at least one gene that affects infection of both lymphocytes and mammary cells has not yet been identified. The results obtained from these studies will greatly increase our understanding of the genetic mechanisms that viruses use to infect their hosts and how genetic resistance to such viruses in the hosts occurs.

Mouse mammary tumor virus (MMTV), a retrovirus that exploits the immune system. Genetics of susceptibility to MMTV infection

All animals, including humans, show differential susceptibility to infection with viruses. Study of the genetics of susceptibility or resistance to specific pathogens is most easily studied in inbred mice. We have been using mouse mammary tumor virus (MMTV), a retrovirus that causes mammary tumors in mice, to study virus/host interactions. These studies have focused on understanding the mechanisms that determine genetic susceptibility to MMTV-induced mammary tumors, the regulation of virus gene expression in vivo and how the virus is transmitted between different cell types. We have found that some endogenous MMTVs are only expressed in lymphoid tissue and that a single base pair change in the long terminal repeat of MMTV determines whether the virus is expressed in mammary gland. This expression in lymphoid cells is necessary for the infectious cycle of MMTV, and both T and B cells express and shed MMTV. Infected lymphocytes are required not only for the initial introduction of MMTV to the mammary gland, but also for virus spread at later times. Without this virus spread, mammary tumorigenesis is dramatically reduced. Mammary tumor incidence is also affected by the genetic background of the mouse and at least one gene that affects infection of both lymphocytes and mammary cells has not yet been identified. The results obtained from these studies will greatly increase our understanding of the genetic mechanisms that viruses use to infect their hosts and how genetic resistance to such viruses in the hosts occurs.

Superantígenos y retrovirus del tumor mamario murino / Superantigens and murine mammary tumor retrovirus

Hosts and their pathogens have co-evolved for millions of years, developing multiple and intimate interactions. Vertebrates have evolved a very complex immune system which pathogens have often been able to circumvent, in some cases even managing to appropriate some of its components for their own purpose. Among the pathogens which do use components of the immune system to survive and propagate, those coding for the expression of superantigens (SAgs) are now under intense scrutiny. Investigations concerning one of these pathogens, the mouse mammary tumor virus (MMTV), led to the understanding of how the expression of such components is a critical step in their life cycle. A number of milk-borne exogenous MMTV infect mice shortly after birth and, when expressed, produce superantigens. Herein, we describe the biological effects of new variants of MMTV. Two of these, BALB14 and BALB2 encoding SAgs with V beta 14+ and V beta 2+ specificities, respectively, were present in BALB/c mice of our colony (BALB/cT); a third variant, termed MMTV LA, originated in (BALB/cTxAKR)F1 mice from recombination between BALB 14 and Mtv-7 endogenous provirus. The recombinant LA virus induces the deletion of V beta 6+ and V beta 8.1+ T cells as a consequence of the acquisition of SAg hypervariable coding region of Mtv-7. The SAg encoded by MMTV LA strongly stimulates cognate T cells in vivo leading to a very effective amplification of lymphoid cells in BALB/c mice, correlating with a high incidence of mammary tumors. These results suggest that the presence of non-productive endogenous proviruses--generally considered to confer a selective advantage to the host by protecting it from infection with exogenous MMTVs encoding cross-reactive SAgs--could also be advantageous for the pathogen by increasing its variability, thus broadening the host range and allowing the expansion of highly tumorigenic variants.

Retrovirus-host interactions. The mouse mammary tumor virus model

Mouse mammary tumor virus (MMTV) is a retrovirus which can induce mammary carcinomas in mice late in life by activation of proto-oncogenes after integration in their vicinity. Surprisingly, it requires a functional immune system to achieve efficient infection of the mammary gland. This requirement became clear when it was discovered that it has developed strategies to exploit the immune response. Instead of escaping immune detection, it induces a vigorous polyclonal T-B interaction which is required to induce a chronic infection. This is achieved by activating and then infecting antigen presenting cells (B cells), expressing a superantigen on their cell surface and triggering unlimited help by the large number of superantigen-specific T cells. The end result of this strong T-B interaction is the proliferation and differentiation of the infected B cells leading to their long term survival.

Bases moleculares de la carcinogénesis viral de papiloma y polioma / The molecular bases of papilomavirus and polyomavirus carcinogenesis

El virus de polioma es capaz de inducir tumores en sus hospederos naturales y transformar células en cultivo. Por otro lado, el virus de papiloma humano se ha relacionado con diversos tipos de neoplasias; de manera particular con lesiones anogenitales humanas. No se conoce con exactitud el mecanismo a través del cual estos virus inducen transformación y tumorigénesis. El presente trabajo muestra algunas de las características de los mecanismos que utilizan los virus mencionados para participar en la transformación y tumorigénesis. Además, se ha encontrado que ciertos aspectos de la infección por el virus de polioma son parecidos a la infección del virus del papiloma (ambos pertenecen a la misma familia Papovaviridae), por lo que se consideran algunas semejanzas y diferencias entre los mismos

Polyomavirus is able to induce tumors in its natural host as well as to transform cells in cultures. On the other hand, human papillomavirus has been involved in several types of neoplasias such as anogenital lesions. Little is known about the mechanisms through which these viruses induce both transformation and tumorigenesis. The present work shows some characteristics of the mechanisms that papillomavirus and polyomavirus use to participate in tumorigenesis. It has also been noticed that the infection caused by polyomavirus resembles that performed by papillomaviruses (which belong to the same Papovaviridae family). Some similarities and differences between these viruses are considered.