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1.
Genome Med ; 5(2): 15, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23419152

RESUMO

BACKGROUND: Human papillomavirus-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) represents a distinct clinical and epidemiological condition compared with HPV-negative (HPV-) HNSCC. To test the possible involvement of epigenetic modulation by HPV in HNSCC, we conducted a genome-wide DNA-methylation analysis. METHODS: Using laser-capture microdissection of 42 formalin-fixed paraffin wax-embedded (FFPE) HNSCCs, we generated DNA-methylation profiles of 18 HPV+ and 14 HPV- samples, using Infinium 450 k BeadArray technology. Methylation data were validated in two sets of independent HPV+/HPV- HNSCC samples (fresh-frozen samples and cell lines) using two independent methods (Infinium 450 k and whole-genome methylated DNA immunoprecipitation sequencing (MeDIP-seq)). For the functional analysis, an HPV- HNSCC cell line was transduced with lentiviral constructs containing the two HPV oncogenes (E6 and E7), and effects on methylation were assayed using the Infinium 450 k technology. RESULTS AND DISCUSSION: Unsupervised clustering over the methylation variable positions (MVPs) with greatest variation showed that samples segregated in accordance with HPV status, but also that HPV+ tumors are heterogeneous. MVPs were significantly enriched at transcriptional start sites, leading to the identification of a candidate CpG island methylator phenotype in a sub-group of the HPV+ tumors. Supervised analysis identified a strong preponderance (87%) of MVPs towards hypermethylation in HPV+ HNSCC. Meta-analysis of our HNSCC and publicly available methylation data in cervical and lung cancers confirmed the observed DNA-methylation signature to be HPV-specific and tissue-independent. Grouping of MVPs into functionally more significant differentially methylated regions identified 43 hypermethylated promoter DMRs, including for three cadherins of the Polycomb group target genes. Integration with independent expression data showed strong negative correlation, especially for the cadherin gene-family members. Combinatorial ectopic expression of the two HPV oncogenes (E6 and E7) in an HPV- HNSCC cell line partially phenocopied the hypermethylation signature seen in HPV+ HNSCC tumors, and established E6 as the main viral effector gene. CONCLUSIONS: Our data establish that archival FFPE tissue is very suitable for this type of methylome analysis, and suggest that HPV modulates the HNSCC epigenome through hypermethylation of Polycomb repressive complex 2 target genes such as cadherins, which are implicated in tumor progression and metastasis.

2.
Nat Cell Biol ; 12(5): 513-9, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20418869

RESUMO

MicroRNAs are small, non-coding RNAs that negatively regulate gene expression. It has been proposed that microRNAs could function in the regulation of innate immunity, but this has not been demonstrated for viral infection. Here we test this hypothesis using the human pathogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) and one of its putative natural cellular targets, primary lymphatic endothelial cells (LECs). We show that an early antiviral microRNA response (6 h post-infection) includes expression of microRNAs that enhance viral gene expression. In particular, the CREB-induced miR-132 microRNA is highly upregulated after infection and has a negative effect on the expression of interferon-stimulated genes, facilitating viral replication. We show a similar function for miR-132 during infection of monocytes with herpes simplex virus-1 (HSV-1) and human cytomegalovirus (HCMV). miR-132 regulates innate antiviral immunity by inhibiting expression of the p300 transcriptional co-activator. p300 is downregulated early after KSHV infection, and inhibition of miR-132 induction restores p300 expression. Furthermore, p300 regulates miR-132 levels, revealing a dynamic equilibrium between miR-132 and p300. By targeting p300, rather than a transcription factor or signalling protein, miR-132 has a broad role in the regulation of antiviral immunity.


Assuntos
Proteína p300 Associada a E1A/genética , Células Endoteliais/imunologia , Regulação da Expressão Gênica/imunologia , Herpesviridae/imunologia , Imunidade Inata , MicroRNAs/fisiologia , Células Cultivadas , Citomegalovirus/imunologia , Proteína p300 Associada a E1A/antagonistas & inibidores , Células Endoteliais/virologia , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 8/imunologia , Humanos , Regulação para Cima
3.
Genes Dev ; 24(2): 195-205, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20080955

RESUMO

Kaposi sarcoma herpesvirus (KSHV) induces transcriptional reprogramming of endothelial cells. In particular, KSHV-infected lymphatic endothelial cells (LECs) show an up-regulation of genes associated with blood vessel endothelial cells (BECs). Consequently, KSHV-infected tumor cells in Kaposi sarcoma are poorly differentiated endothelial cells, expressing markers of both LECs and BECs. MicroRNAs (miRNAs) are short noncoding RNA molecules that act post-transcriptionally to negatively regulate gene expression. Here we validate expression of the KSHV-encoded miRNAs in Kaposi sarcoma lesions and demonstrate that these miRNAs contribute to viral-induced reprogramming by silencing the cellular transcription factor MAF (musculoaponeurotic fibrosarcoma oncogene homolog). MAF is expressed in LECs but not in BECs. We identify a novel role for MAF as a transcriptional repressor, preventing expression of BEC-specific genes, thereby maintaining the differentiation status of LECs. These findings demonstrate that viral miRNAs could influence the differentiation status of infected cells, and thereby contribute to KSHV-induced oncogenesis.


Assuntos
Reprogramação Celular , Células Endoteliais/citologia , Células Endoteliais/patologia , Herpesvirus Humano 8/metabolismo , MicroRNAs/metabolismo , Proteína Oncogênica v-maf/metabolismo , Sarcoma de Kaposi/fisiopatologia , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Regulação para Baixo , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Regulação Viral da Expressão Gênica , Inativação Gênica , Células HeLa , Infecções por Herpesviridae/fisiopatologia , Herpesvirus Humano 8/genética , Humanos
4.
PLoS Pathog ; 5(10): e1000616, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19816565

RESUMO

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Endotélio Vascular/fisiologia , Herpesvirus Humano 8/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Sistema Linfático/fisiologia , Proteínas de Membrana/fisiologia , Receptores Notch/fisiologia , Sarcoma de Kaposi/virologia , Proteínas Adaptadoras de Transdução de Sinal , Endotélio Vascular/citologia , Endotélio Vascular/virologia , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Proteína Jagged-1 , Sistema Linfático/citologia , Sistema Linfático/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/genética , Receptor Notch4 , Receptores Notch/genética , Sarcoma de Kaposi/genética , Proteínas Serrate-Jagged , Transdução de Sinais , Regulação para Cima
5.
PLoS One ; 4(6): e5890, 2009 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-19536280

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is causally related to Kaposi's sarcoma (KS), the most common malignancy in untreated individuals with HIV/AIDS. The adaptive T-cell immune response against KSHV has not been fully characterized. To achieve a better understanding of the antigenic repertoire of the CD8 and CD4 T-cell responses against KSHV, we constructed a library of lentiviral expression vectors each coding for one of 31 individual KSHV open reading frames (ORFs). We used these to transduce monocyte-derived dendritic cells (moDCs) isolated from 14 KSHV-seropositive (12 HIV-positive) and 7 KSHV-seronegative (4 HIV-positive) individuals. moDCs were transduced with up to 3 KSHV ORFs simultaneously (ORFs grouped according to their expression during the viral life cycle). Transduced moDCs naturally process the KSHV genes and present the resulting antigens in the context of MHC class I and II. Transduced moDCs were cultured with purified autologous T cells and the CD8 and CD4 T-cell proliferative responses to each KSHV ORF (or group) was assessed using a CFSE dye-based assay. Two pools of early lytic KSHV genes ([ORF8/ORF49/ORF61] and [ORF59/ORF65/K4.1]) were frequently-recognized targets of both CD8 and CD4 T cells from KSHV seropositive individuals. One pool of late lytic KSHV genes ([ORF28/ORF36/ORF37]) was a frequently-recognized CD8 target and another pool of late genes ([ORF33/K1/K8.1]) was a frequently-recognized CD4 target. We report that both the CD8 and CD4 T-cell responses against KSHV are skewed towards genes expressed in the early and late phases of the viral lytic cycle, and identify some previously unknown targets of these responses. This knowledge will be important to future immunological investigations into KSHV and may eventually lead to the development of better immunotherapies for KSHV-related diseases.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Adulto , Idoso , Antígenos Virais/metabolismo , Proliferação de Células , Estudos de Coortes , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/complicações , Infecções por Herpesviridae/complicações , Humanos , Imunoterapia/métodos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Fases de Leitura Aberta
6.
Cell Host Microbe ; 4(5): 470-83, 2008 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-18996347

RESUMO

The involvement of Toll-like receptor 4 (TLR4) in immunity against human herpesviruses has not been previously demonstrated. We show that infection of endothelial cells with Kaposi sarcoma herpesvirus (KSHV), a human oncogenic virus, leads to rapid suppression of TLR4 expression. This is a mechanism of immune escape as TLR4 mediates innate immunity against KSHV. In vitro, cells lacking TLR4 are more susceptible to KSHV infection, whereas activation of TLR4 protects cells from infection. In vivo, HIV-1-infected individuals carrying a mutant TLR4 allele appear more likely to have multicentric Castleman's disease, a lymphoproliferation associated with enhanced KSHV replication. ERK activation by KSHV structural proteins and the KSHV-encoded vGPCR plays a key role in the TLR4 downregulation, whereas the KSHV vIRF1 also contributes to this effect. Our findings reveal a role for TLR4 in innate immunity against herpesviruses and suggest the potential use of TLR4 agonists for the treatment of KSHV-related neoplasms.


Assuntos
Herpesvirus Humano 8/imunologia , Sarcoma de Kaposi/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células Cultivadas , Regulação para Baixo , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Expressão Gênica , Infecções por HIV/complicações , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/fisiologia , Herpesvirus Humano 8/fisiologia , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transdução de Sinais , Receptor 4 Toll-Like/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral
7.
PLoS One ; 3(6): e2505, 2008 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-18560562

RESUMO

BACKGROUND: Burkitt lymphoma, a childhood cancer common in parts of sub-Saharan Africa, has been associated with Epstein Barr Virus (EBV) and malaria, but its association with human immunodeficiency virus (HIV) is not clear. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a case-control study of Burkitt lymphoma among children (aged < or = 15 years) admitted to the pediatric oncology unit in Blantyre, Malawi between July 2005 and July 2006. Cases were 148 children diagnosed with Burkitt lymphoma and controls were 104 children admitted with non-malignant conditions or cancers other than hematological malignancies and Kaposi sarcoma. Interviews were conducted and serological samples tested for antibodies against HIV, EBV and malaria. Odds ratios for Burkitt lymphoma were estimated using unconditional logistic regression adjusting for sex, age, and residential district. Cases had a mean age of 7.1 years and 60% were male. Cases were more likely than controls to be HIV positive (Odds ratio (OR)) = 12.4, 95% Confidence Interval (CI) 1.3 to 116.2, p = 0.03). ORs for Burkitt lymphoma increased with increasing antibody titers against EBV (p = 0.001) and malaria (p = 0.01). Among HIV negative participants, cases were thirteen times more likely than controls to have raised levels of both EBV and malaria antibodies (OR = 13.2; 95% CI 3.8 to 46.6; p = 0.001). Reported use of mosquito nets was associated with a lower risk of Burkitt lymphoma (OR = 0.2, 95% CI, 0.03 to 0.9, p = 0.04). CONCLUSIONS: Our findings support prior evidence that EBV and malaria act jointly in the pathogenesis of Burkitt lymphoma, suggesting that malaria prevention may decrease the risk of Burkitt lymphoma. HIV may also play a role in the etiology of this childhood tumor.


Assuntos
Linfoma de Burkitt/complicações , Infecções por Vírus Epstein-Barr/complicações , Infecções por HIV/complicações , Malária/complicações , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Malaui , Masculino
8.
Cancer Res ; 67(9): 4042-51, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17483315

RESUMO

Kaposi's sarcoma (KS) is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) and consists of proliferating spindle cells, which are related to lymphatic endothelial cells (LEC). Angiopoietin-2 (Ang2) is a secreted proangiogenic and lymphangiogenic molecule. Here, we show the expression of Ang2 protein in KS and confirm that KSHV infection up-regulates Ang2 in LEC. We show that a paracrine mechanism contributes to this up-regulation. A lentiviral library of individual KSHV-encoding genes, comprising the majority of known latent genes and a selection of lytic viral genes, was constructed to investigate the underlying mechanism of this up-regulation. Two lytic genes, viral interleukin-6 (vIL6) and viral G-protein-coupled receptor (vGPCR), up-regulated Ang2 expression in LEC. Both vIL6 and vGPCR are expressed in KSHV-infected LEC and caused up-regulation of Ang2 in a paracrine manner. KSHV, vIL6, and vGPCR up-regulated Ang2 through the mitogen-activated protein kinase (MAPK) pathway. Gene expression microarray analysis identified several other angiogenic molecules affected by KSHV, including the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) axis, which is also affected by vIL6 and vGPCR in LEC, and matrix metalloproteinases, which could act in concert with Ang2 to contribute to KS development. These findings support the paracrine and autocrine roles of the lytic KSHV-encoded proteins, vIL6 and vGPCR, in KS pathogenesis and identify Ang2 as a potential therapeutic target for this neoplasm.


Assuntos
Angiopoietina-2/biossíntese , Herpesvirus Humano 8/genética , Interleucina-6/genética , Receptores Acoplados a Proteínas G/genética , Sarcoma de Kaposi/virologia , Angiopoietina-2/genética , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Regulação Neoplásica da Expressão Gênica , Biblioteca Gênica , Humanos , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/virologia , Receptores Acoplados a Proteínas G/metabolismo , Sarcoma de Kaposi/irrigação sanguínea , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Regulação para Cima
9.
Curr Biol ; 13(11): 979-84, 2003 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-12781138

RESUMO

The degree of population replacement in the British Isles associated with cultural changes has been extensively debated. Recent work has demonstrated that comparisons of genetic variation in the British Isles and on the European Continent can illuminate specific demographic processes in the history of the British Isles. For example, Wilson et al. used the similarity of Basque and Celtic Y chromosomes to argue for genetic continuity from the Upper Palaeolithic to the present in the paternal history of these populations (see also ). Differences in the Y chromosome composition of these groups also suggested genetic signatures of Norwegian influence in the Orkney Islands north of the Scottish mainland, an important center of Viking activities between 800 and 1300 A.D. More recently, Weale et al. argued for substantial Anglo-Saxon male migration into central England based on the analysis of eight British sample sets collected on an east-west transect across England and Wales. To provide a more complete assessment of the paternal genetic history of the British Isles, we have compared the Y chromosome composition of multiple geographically distant British sample sets with collections from Norway (two sites), Denmark, and Germany and with collections from central Ireland, representing, respectively, the putative invading and the indigenous populations. By analyzing 1772 Y chromosomes from 25 predominantly small urban locations, we found that different parts of the British Isles have sharply different paternal histories; the degree of population replacement and genetic continuity shows systematic variation across the sampled areas.


Assuntos
Cromossomos Humanos Y/genética , Evolução Molecular , Variação Genética , Haplótipos/genética , Genética Populacional , Humanos , Irlanda , Masculino , Repetições de Microssatélites , Reino Unido
10.
Am J Hum Genet ; 72(4): 1058-64, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12629598

RESUMO

We have analyzed and compared mitochondrial DNA variation of populations from the Near East and Africa and found a very high frequency of African lineages present in the Yemen Hadramawt: more than a third were of clear sub-Saharan origin. Other Arab populations carried approximately 10% lineages of sub-Saharan origin, whereas non-Arab Near Eastern populations, by contrast, carried few or no such lineages, suggesting that gene flow has been preferentially into Arab populations. Several lines of evidence suggest that most of this gene flow probably occurred within the past approximately 2,500 years. In contrast, there is little evidence for male-mediated gene flow from sub-Saharan Africa in Y-chromosome haplotypes in Arab populations, including the Hadramawt. Taken together, these results are consistent with substantial migration from eastern Africa into Arabia, at least in part as a result of the Arab slave trade, and mainly female assimilation into the Arabian population as a result of miscegenation and manumission.


Assuntos
Árabes/genética , Mapeamento Cromossômico , Etnicidade/genética , Caracteres Sexuais , África Subsaariana , Feminino , Geografia , Haplótipos , Humanos , Oriente Médio/etnologia
11.
Am J Hum Genet ; 70(6): 1411-20, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11992249

RESUMO

We have analyzed the maternally inherited mitochondrial DNA from each of nine geographically separated Jewish groups, eight non-Jewish host populations, and an Israeli Arab/Palestinian population, and we have compared the differences found in Jews and non-Jews with those found using Y-chromosome data that were obtained, in most cases, from the same population samples. The results suggest that most Jewish communities were founded by relatively few women, that the founding process was independent in different geographic areas, and that subsequent genetic input from surrounding populations was limited on the female side. In sharp contrast to this, the paternally inherited Y chromosome shows diversity similar to that of neighboring populations and shows no evidence of founder effects. These sex-specific differences demonstrate an important role for culture in shaping patterns of genetic variation and are likely to have significant epidemiological implications for studies involving these populations. We illustrate this by presenting data from a panel of X-chromosome microsatellites, which indicates that, in the case of the Georgian Jews, the female-specific founder event appears to have resulted in elevated levels of linkage disequilibrium.


Assuntos
DNA Mitocondrial/genética , Efeito Fundador , Judeus/genética , Cromossomo X/genética , Cromossomo Y/genética , Árabes/genética , Inglaterra , Feminino , Variação Genética/genética , Geografia , Humanos , Desequilíbrio de Ligação , Masculino , Repetições de Microssatélites/genética , Mães , Linhagem
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