RESUMO
BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) is the most common pathogen in orthopaedic surgical site infections (SSIs). However, few studies have investigated the transmission process of orthopaedic MRSA SSI. AIM: To investigate the transmission process of orthopaedic MRSA SSI using epidemiological and molecular analyses and to determine a method to prevent MRSA SSI in nosocomial orthopaedic surgery. METHODS: Active MRSA surveillance, preoperative decolonization and contact precautions for MRSA-positive cases was performed at our institution. Changes in epidemic strains were evaluated and the possibility of transmission from patients in an orthopaedic ward of a Japanese tertiary-care hospital was assessed by genotyping stored MRSA strains. In addition, data on the prevalence of MRSA SSI, MRSA colonization, and use of an alcohol antiseptic agent (mL/patient-days) during 2005-2022 were retrospectively assessed. FINDINGS: SCCmec type II strain in the SSI group decreased over time, associated with fewer outbreaks. Even during a period of high infection rates, no cases of transmission-induced SSI from nasal MRSA carriers were identified. The infection rate correlated negatively with the use of an alcohol antiseptic agent (r = -0.82; P < 0.0001). Two cases among five nasal carriers developed MRSA SSI caused by strains different from those related to nasal colonization. CONCLUSION: The infection control measures for transmission from the hospital reservoirs including strict adherence to hand hygiene and decolonization of carriers is likely to be important for the prevention of orthopaedic MRSA SSI. However, the need for contact precautions for decolonized nasal carriers might be low.
RESUMO
BACKGROUND: The Epstein-Barr virus (EBV) encodes two anti-apoptotic cellular Bcl2 homologs, BALF1 and BHRF1. BHRF1 has an anti-apoptotic activity but is rarely expressed in nasopharyngeal carcinoma (NPC). However, BALF1 is not yet well characterized. OBJECTIVES: The objective of the study was to characterize BALF1 gene. First, the search of its transcriptional expression in EBV-positive B cell lines, EBV-positive Burkitt's lymphoma's cell lines and nasopharyngeal carcinoma's biopsies. Second, the examination of its anti-apoptotic activity in serum dependent assays. STUDY DESIGN: We first analysed the transcriptional expression of BALF1 by reverse transcriptase DNA polymerase chain reaction (RT-PCR) method. For the analysis of its anti-apoptotic activity, we transfected NIH3T3 cells with pBABE-BALF1 expression plasmid and studied serum dependence of these transfectants. RESULTS: BALF1 expression was detected in the latent stage and increased more significantly during the lytic phase in IgG-treated AKATA and TPA-SB-treated P3HR1-TK negative cell lines. As its expression was not affected by the inhibitor of viral DNA synthesis, this gene does not belong to late gene family. When analysed its transcription in Burkitt's lymphoma (BL)-derived cell lines and NPC biopsies, all BL-derived cell lines and more than 80% of NPC biopsies transcribed this gene. The study of serum dependence of BALF1-transfected NIH3T3 cells showed: with 10% of serum, BALF1 transfectants grew significantly more higher cell density than vector alone transfected NIH3T3 cell lines and with 1% of serum, BALF1 transfectants were capable of growing, but with about 40% reduced rate in comparison with those with 10% serum, while vector alone transfected NIH3T3 cells could not almost grow. CONCLUSION: BALF1 gene was transcribed in EBV-associated tumor cells. BALF1 could render cells to serum independent. These results suggest that BALF1 gene could play its role in EBV oncogenesis.
Assuntos
Linfoma de Burkitt/virologia , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virologia , Transcrição Gênica , Proteínas Virais/genética , Células 3T3 , Animais , Biópsia , Linfoma de Burkitt/genética , Divisão Celular , Linhagem Celular Tumoral , DNA Complementar/genética , DNA Viral/genética , Herpesvirus Humano 4/crescimento & desenvolvimento , Humanos , Camundongos , Neoplasias Nasofaríngeas/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin. Potential functions of the BARF1 early gene in EBV oncogenesis emerged from our observations showing expression of BARF1-encoded protein in nasopharyngeal carcinoma biopsies, and induction of either malignant transformation (in rodent fibroblast and human B cell lines) or immortalization (in monkey primary epithelial cells) following BARF1 transfection. We previously reported expression of the BARF1 product as a cytoplasm/membrane-associated protein from 293-tTA cells infected with a BARF1-recombinant adenovirus. Since constitutive expression of BARF1 from this heterologous system became inefficient, we developed a tetracycline-regulatable recombinant vector expressing BARF1 and green fluorescent protein from a dicistronic message. As here reported, stable and efficient expression of BARF1 from this vector in either permissive or non-permissive cell lines, allowed the first sequencing identification and further molecular characterization of BARF1-encoded protein.
Assuntos
Adenoviridae/genética , Regulação Viral da Expressão Gênica , Tetraciclina , Proteínas Virais/biossíntese , Adenoviridae/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Herpesvirus Humano 4/genética , Humanos , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/isolamento & purificaçãoRESUMO
The Epstein-Barr virus (EBV) is associated with two major human epithelial malignancies, where it is likely to play a role in the malignant phenotype: undifferentiated nasopharyngeal carcinoma (100% of cases) and gastric carcinomas (about 10% of cases). We and others have obtained growth transformation of monkey kidney primary epithelial cells by transfection of viral DNA, especially with the BARF1 gene of EBV (Wei et al., 1997). We now report that the same type of primary epithelial cells can be growth-transformed using EBV particles derived from a nasopharyngeal carcinoma tumor line. Not only can these EBV-infected cells grow over 100 passages, escaping senescence, in contrast to their noninfected counterparts, but they can also survive and proliferate at very low cell density. Several subclones were characterized in terms of viral gene expression. All these clones gave a similar pattern, with detection of EBNA1 and BARF1 proteins but absence of LMP1. CD21, which is the main EBV receptor on B lymphocytes, was not expressed on parental monkey kidney epithelial cells nor on EBV-infected cell clones. This model of epithelial cell transformation will be useful for a better investigation of EBV functions critical for oncogenesis of epithelial cells.
Assuntos
Transformação Celular Viral/fisiologia , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virologia , Animais , Testes de Carcinogenicidade , Divisão Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Expressão Gênica , Genes Virais , Genoma Viral , Haplorrinos , Células HeLa , Herpesvirus Humano 4/fisiologia , Humanos , Queratinas/genética , Camundongos , Camundongos Nus , Receptores de Complemento 3d/genética , Células Tumorais Cultivadas , Proteínas da Matriz Viral/genética , Proteínas Virais/genéticaRESUMO
The BARF1 gene encoded by the Epstein-Barr virus induces morphological changes, loss of contact inhibition and anchorage independence in established rodent Balb/c3T3 fibroblast. BARF1 gene was also capable of inducing malignant transformation in a human Louckes B cell line. Our recent study showed that BARF1 gene had an ability to immortalize primary epithelial cells. However we do not know which region(s) of BARF1 protein is(are) responsible for inducing malignant transformation in established rodent cells. Using the deletion mutants, we now localized a malignant transforming region in N-terminal of BARF1 protein. The mutants lacking this region were unable to transform the cells in malignant state. Furthermore, we demonstrated that only the mutants containing this region rendered the cells resistant to apoptosis induced by serum deprivation. Surprisingly, the BARF1 gene was capable of activating anti-apoptotic Bcl-2 expression and this activation was due to the N-terminal transforming region. These data suggest that the cooperation of BARF1 with Bcl-2 is essential for the induction of malignant transformation.
Assuntos
Transformação Celular Neoplásica/patologia , Fibroblastos/patologia , Herpesvirus Humano 4/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Virais/genética , Animais , Apoptose , Transformação Celular Neoplásica/metabolismo , Ensaio de Unidades Formadoras de Colônias , Primers do DNA/química , Fibroblastos/metabolismo , Deleção de Genes , Expressão Gênica , Genes myc/genética , Genes myc/fisiologia , Vetores Genéticos , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Proto-Oncogênicas c-bcl-2/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We reported previously that the EBV BARF1 open reading frame encodes a Mr 31,000-33,000 protein (p31) with potential transforming and oncogenic properties. This gene was found capable of transforming both: (a) the rodent fibroblast lines Balbc/3T3 and NIH3T3 into cells producing aggressive tumors in newborn rats; and (b) the human EBV-negative B-cell line Louckes into cells leading to small tumors, which disappeared 3 weeks after injection. Our recent study showed that BARF1 ORF expression may confer the property of immortalization to primary kidney epithelial cells (M. X. Wei et al., Oncogene, 14: 3073-3081, 1997). Because this suggested that BARF1 could be involved in epithelial malignancy, we investigated its transcriptional and translational expressions in Algerian nasopharyngeal carcinoma (NPC) biopsies by reverse transcription-PCR and immunoblotting using rabbit polyclonal antisera prepared against two synthetic peptides corresponding to distinct, predicted epitopes of the BARF1 protein (NGGVMKEKD, amino acids 172-180, and GKNDKEE, amino acids 203-209). The BARF1 ORF was found to be transcribed and translated in >85% of our NPC biopsies, with high p31 protein level detected in several NPC patient biopsies as well as in NPC-derived xenografts. Our observation of BARF1 expression in a large proportion of NPC epithelial cells suggests that this EBV gene might play an important role in the malignant transformation of human epithelial cells in vivo.
Assuntos
Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Proteínas Virais/biossíntese , Animais , Biópsia , Linhagem Celular , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , Expressão Gênica , Humanos , Imuno-Histoquímica , Linfócitos/metabolismo , Camundongos , Camundongos Nus , Neoplasias Nasofaríngeas/genética , Transplante de Neoplasias , Fases de Leitura Aberta , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transplante Heterólogo , Proteínas Virais/genéticaRESUMO
Emergence of acyclovir (Acy)-resistant herpes simplex virus (HSV) is a major concern in bone marrow transplant recipients. Phenotypic and genetic characterization of thymidine kinase (TK) was done for 7 Acy-susceptible and 11 Acy-resistant HSV-1 isolated from 11 patients. In total, 19 amino acid substitutions were detected that were not related to Acy resistance but to TK gene polymorphism, including 5 mutations that have not been previously reported. The Acy-resistant strain from 1 patient presented no TK gene mutation related to resistance. Five patients (45%) had isolates that harbored point mutations leading to amino acid substitutions that could be associated with Acy resistance. Of the 5 substitutions detected, 3 have not been previously reported (codons 51, 83, and 175). A nucleotide insertion or deletion was detected in resistant isolates from 5 patients (45%); these mutations are located in homopolymer repeats at codon 92 (1 subject) and at codon 146 (4 subjects).
Assuntos
Aciclovir/farmacologia , Antivirais/farmacologia , Transplante de Medula Óssea , Herpesvirus Humano 1/genética , Timidina Quinase/genética , Adolescente , Adulto , Criança , Pré-Escolar , Resistência Microbiana a Medicamentos/genética , Herpes Simples/virologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 1/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Fenótipo , Polimorfismo GenéticoRESUMO
In a previous study of Epstein-Barr virus (EBV) strains in North African nasopharyngeal carcinoma (N PC) biopsies, we have found that the viral strain present was of A/F/W'-I'/Xhol kept/H1-H2 type, while the strain associated with Chinese NPC was the A/"f"/W'I'/Xhol lost/H type. Using the restriction fragment length polymorphism (RFLP) and PCR-RFLP methods, the present study analyzed the H1-H2 variant in different clinical samples from Algeria, including the saliva of healthy EBV-positive individuals and patients with NPC or Hodgkin's disease (HD), as well as HD biopsies and lymphoblastoid cell lines (LCLs) established from the oropharyngeal virus-infected cells. Our results demonstrate that, in contrast to the H1-H2 variant found in NPC biopsies, the H genotype was dominant in HD biopsies. Moreover, H genotype was also dominant in the oropharynx of healthy EBV-positive individuals, of patients with NPC and with HD. Our results clearly indicate that in North Africa the EBV strain present of NPC biopsies is different from that shed in the oropharynx. This may suggest a specific distribution of the H1-H2 variant in the NPC epithelial tumor, whereas the H genotype is dominant in HD biopsies and in the oropharynx. The specific association of both viral strains with these 2 distinct diseases in North Africa may reflect a difference in tumorigenicity.
Assuntos
Herpesvirus Humano 4/genética , Doença de Hodgkin/virologia , Neoplasias Nasofaríngeas/virologia , Orofaringe/virologia , Argélia , Anticorpos Antivirais/análise , Biópsia , Células Cultivadas , Variação Genética , Herpesvirus Humano 4/imunologia , Humanos , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Saliva/virologiaRESUMO
We previously reported that the BARF1 (BamH1-A right frame 1) gene product from Epstein-Barr Virus (EBV) may have oncogenic properties since injection into new-born rats of transfected cell lines resulted in the development of BARF1 expressing tumors, which were aggressive in the case of murine fibroblasts and transient in that of human B lymphocytes. As EBV has been associated with nasopharyngeal carcinoma (NPC) and evidence of BARF1 transcription in this cancer was emerging from our biopsy analyses, we examined the effects of BARF1 transfection into primate primary epithelial cells. The expression of the BARF1 open reading frame in primary monkey kidney epithelial cells led us to the establishment of continuously dividing lines. The BARF1 transfectants showed the major characteristics of immortalized cells: morphological change, short cell doubling time, ability to divide at low cell density and continuous growth over 50 passages. Injection of BARF1 transfectants into nude mice did not induce any tumor. Established subclones were shown to be epithelial cells expressing known keratins as well as the BARF1 coded mRNA and protein. This is the first report indicating that expression of the BARF1 gene product in primary epithelial cells may contribute to the establishment of cell lines.
Assuntos
Linhagem Celular , Transformação Celular Viral/genética , Herpesvirus Humano 4/genética , Rim/patologia , Proteínas Virais/genética , Animais , Testes de Carcinogenicidade , Divisão Celular/genética , Células Cultivadas , Epitélio/patologia , Haplorrinos , Queratinas/genética , Queratinas/metabolismo , Camundongos , Camundongos Nus , Fases de Leitura Aberta , Timidina/metabolismo , Transfecção , Proteínas Virais/metabolismoRESUMO
Nasopharyngeal carcinoma (NPC) is one of important problems of public health in the south of Asian countries and North Africa. The Epstein-Barr virus(EBV) is thought to be a key factor in this cancer as attested by several facts: the constant presence of the EBV genome in all malignant cells, the expression of several viral proteins with known oncogenic activity and the precession of serological modifications. Several proto-oncogenes were also activated in relation with viral gene expression. A possible presence of chromosome deletion in the region where some anti-oncogenes are localized, was also described. From these observations, future objective in NPC research will be to define whether EBV is a sole responsible factor in the development of NPC.
Assuntos
Herpesvirus Humano 4/isolamento & purificação , Neoplasias Nasofaríngeas/virologia , Anticorpos Antivirais/análise , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Humanos , Proteínas Virais/análiseRESUMO
Antibody-dependent cellular cytotoxicity (ADCC) is an important antiviral effector mechanism. ADCC to the protein encoded by the Epstein-Barr virus (EBV) BamHI A rightward open-reading frame-1 (BARF1) was studied by transducing Raji-tk- cells with the BARF1 gene using a retroviral expression vector. The transduced Raji cells expressed BARF1 on the cell surface, as determined by flow cytometry. Sera from chronic and acute infectious mononucleosis and nasopharyngeal carcinoma patients were found to contain antibodies that react with the BARF1 protein. When BARF1-expressing Raji cells were used as targets for ADCC, sera from several nasopharyngeal carcinoma patients demonstrated significant ADCC reactivity, whereas sera from healthy EBV-seronegative and -seropositive persons lacked such reactivity. BARF1-specific ADCC activity could be competitively inhibited with recombinant BARF1 protein. The level of anti-BARF1 antibody activity in sera of patients with EBV-associated diseases suggests that the BARF1 protein may serve as a target on EBV-infected cells for ADCC.
Assuntos
Anticorpos Antivirais/sangue , Citotoxicidade Celular Dependente de Anticorpos , Herpesvirus Humano 4/imunologia , Mononucleose Infecciosa/imunologia , Neoplasias Nasofaríngeas/imunologia , Proteínas Virais/imunologia , Doença Aguda , Western Blotting , Linhagem Celular , Doença Crônica , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Infecções por Herpesviridae/imunologia , Humanos , Neoplasias Nasofaríngeas/virologia , Proteínas Recombinantes de Fusão/imunologia , Transfecção , Células Tumorais Cultivadas , Infecções Tumorais por Vírus/imunologia , Proteínas Virais/genéticaRESUMO
A major 135-kDa DNA binding protein (mDBP) encoded by the BALF2 open reading frame of Epstein-Barr Virus (EBV) is known to be an essential protein for the induction of the lytic cycle. The present investigation was carried out to know whether this protein forms a complex in vivo with other viral DNA binding proteins (DBP) involved in DNA replication: DNA polymerase, EA-D (diffused early antigen), and DNAase. Immunoprecipitation assays followed by mono- and two-dimensional electrophoresis showed that mDBP forms a complex with these three DBP. Other complexes were also found such as EA-D/DNAase, DNA polymerase/DNAase, and DNA polymerase/EA-D. The complexed forms already exist in the early stage of EBV cycle before DNA synthesis is induced in the EBV producer P3HR-1 cell line. The exonuclease activity encoded by DNAase was found to be inhibited when this enzyme complexed with mDBP, while the EBV DNA polymerase retained its activity in the complexed form with mDBP. Our results suggest that these complexes already present before DNA synthesis are necessary for EBV DNA synthesis.
Assuntos
Antígenos Virais/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/metabolismo , Herpesvirus Humano 4/genética , Complexos Multienzimáticos/química , Proteínas Virais/metabolismo , Linhagem Celular , Replicação do DNA , DNA Viral/metabolismo , Eletroforese em Gel Bidimensional , Herpesvirus Humano 4/fisiologia , Humanos , Ligação Proteica , Replicação ViralRESUMO
Epstein-Barr virus (EBV) has been associated with human cancers of lymphocytic or epithelial origin, but viral functions implied in oncogenesis are not yet clear. We previously reported the oncogenic transformation of rodent fibroblast and human B lymphocyte cell lines by the BARF1 coding sequence from EBV. We more recently observed immortalizing effects of this gene on monkey kidney primary epithelial cells. Here we describe an efficient recombinant adenovirus expression system which allowed us to characterize BARF1 translation products, with the help of rabbit polyclonal antibodies raised to the entire protein. The present data demonstrate that BARF1 encodes a 31-33 kDa hydrophobic protein, linked to cell membranes though also recovered in the cytosol, and recognized by human sera from patients with various EBV-related pathologies.
Assuntos
Adenoviridae/genética , Herpesvirus Humano 4/genética , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Recombinação Genética , Proteínas Virais/biossíntese , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/biossíntese , Antígenos Virais/imunologia , Linhagem Celular , Vetores Genéticos , Humanos , Rim , Dados de Sequência Molecular , Biossíntese de Proteínas/imunologia , Coelhos , Proteínas Recombinantes/metabolismo , Proteínas Virais/metabolismoRESUMO
Basic research in Epstein-Barr virus (EBV) molecular genetics has provided means to maintain episomes in human cells, to efficiently deliver episomes with up to 150 kbp of heterologous DNA to human B lymphocytes, and to immortalize human B lymphocytes with EBV recombinants that can maintain up to 120 kbp of heterologous DNA. Episome maintenance requires an EBV nuclear protein, EBNA1, whereas immortalization of cells with EBV recombinants requires EBNA1, EBNA2, EBNA3A, EBNA3C, EBNALP, and LMP1. EBV-derived vectors are useful for experimental genetic reconstitution in B lymphocytes, a cell type frequently used in establishing repositories of human genetic deficiencies. The ability of EBV-infected cells to establish a balanced state of persistence in normal humans raises the possibility that cells infected with EBV recombinants could be useful for genetic reconstitution, in vivo.
Assuntos
Linfócitos B/fisiologia , Antígenos Nucleares do Vírus Epstein-Barr/biossíntese , Vetores Genéticos , Herpesvirus Humano 4/genética , Transfecção/métodos , Linfócitos B/virologia , Linfoma de Burkitt , Linhagem Celular , Transformação Celular Viral , Células Cultivadas , Marcadores Genéticos , Terapia Genética , Humanos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Recombinação Genética , Células Tumorais CultivadasRESUMO
In contrast with most Epstein-Barr virus (EBV)-infected healthy carriers, nasopharyngeal carcinoma patients frequently have increased serum levels of antibodies directed against EBV-DNase. These antibodies are potentially interesting serological markers for the diagnosis and the follow-up of nasopharyngeal carcinoma (NPC). In this context, it is important to determine whether malignant EBV-infected cells are the source of significant amounts of EBV-DNase contributing to antigenic stimulation. Therefore EBV-DNase expression has been investigated in several NPC specimens. A significant expression of this viral enzyme was demonstrated in both fresh biopsies and transplanted tumor lines. The DNase isolated from tumor has a molecular weight varying between 52 and 60 kDa and its activity eluted from a single-stranded DNA affinity column was specifically inhibited by both NPC sera and the rabbit polyclonal antibody against EBV-DNase. The enzyme activity was functional in the presence of 300 mM KCl, with which cellular DNases are completely inhibited. The DNase was mainly localized in epithelial tumor cells of both NPC biopsies and nude mice-derived NPC cells.
Assuntos
Desoxirribonucleases/genética , Herpesvirus Humano 4/enzimologia , Neoplasias Nasofaríngeas/virologia , Proteínas Virais , Desoxirribonucleases/metabolismo , Expressão Gênica , Herpesvirus Humano 4/genética , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Neoplasias Nasofaríngeas/patologia , Testes de Neutralização , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Cases of nasopharyngeal carcinoma (NPC) from North Africa show an unusual bimodal age distribution. As elsewhere, the tumor is closely associated with the presence of Epstein-Barr virus (EBV). The expression of EBV genes and c-onc genes was studied in biopsy specimens from tumors at different clinical stages from 11 young (10 to 30-year-old) and 11 adult (30 to 65-year-old) patients. It was found that the two age groups do not differ in their pattern of gene expression, that there is a tendency for later stage biopsies to express more viral and c-onc transcripts, and that samples expressing larger numbers of EBV genes also tend to express many different c-onc specificities.
Assuntos
Carcinoma/virologia , Expressão Gênica , Herpesvirus Humano 4/genética , Neoplasias Nasofaríngeas/virologia , Proto-Oncogenes , Adolescente , Adulto , África do Norte , Idoso , Envelhecimento , Animais , Carcinoma/patologia , Linhagem Celular , Criança , Dosagem de Genes , Humanos , Pessoa de Meia-Idade , Neoplasias Nasofaríngeas/patologia , Transcrição GênicaRESUMO
To evaluate applications of highly purified recombinant EBV DNAase in the diagnosis and prognosis of NPC, we tested sera from patients with NPC, other EBV-associated diseases and EBV-seropositive and -seronegative healthy subjects by immunoblotting and DNAase inhibitory assay. The results were compared with those obtained by the conventional immunofluorescence assays against the EBV-specified early antigens and capsid antigens. The antigenic specificity of the immunoblotting assay for IgG antibody against the viral enzyme, but not that for the IgA antibody, was correlated with DNAase-inhibitory activity of the sera and their titers of IgG antibodies against the viral early antigens. Purified IgA as well as IgG from NPC sera inhibited enzyme activity with similar efficiency. The use of highly purified viral DNase has increased the sensitivity of detection of the corresponding antibodies by immunoblotting, with the IgG antibody being detected in all but one, and IgA antibody in all but 2, of the 174 NPC sera tested. The IgG antibody was also commonly detected in the other groups of control sera, while the IgA antibody was detected in about 10% of African Burkitt's lymphoma and Algerian Hodgkin's lymphoma patients and less than 3% of the other control subjects. These results suggest that IgA antibody against recombinant EBV DNAase may be useful in the diagnosis of NPC, but the level of this antibody did not appear to be related to clinical stages of this cancer.
Assuntos
Desoxirribonucleases/imunologia , Herpesvirus Humano 4/isolamento & purificação , Imunoglobulina A/sangue , Neoplasias Nasofaríngeas/diagnóstico , Neoplasias Nasofaríngeas/virologia , Animais , Especificidade de Anticorpos , Biomarcadores Tumorais/sangue , Linfoma de Burkitt/sangue , Linfoma de Burkitt/diagnóstico , Linfoma de Burkitt/imunologia , Desoxirribonucleases/biossíntese , Diagnóstico Diferencial , Eletroforese em Gel de Poliacrilamida , Herpesvirus Humano 4/enzimologia , Doença de Hodgkin/sangue , Doença de Hodgkin/diagnóstico , Doença de Hodgkin/imunologia , Humanos , Immunoblotting , Neoplasias Nasofaríngeas/sangue , Neoplasias Nasofaríngeas/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Testes Sorológicos , Spodoptera , TransfecçãoRESUMO
Undifferentiated and poorly differentiated nasopharyngeal carcinoma (NPC) were known to be tightly associated with Epstein-Barr Virus (EBV). Its association with well differentiated NPC was also reported. In the present study, the presence of EBV was investigated by nucleic acid hybridization, Polymerase Chain Reaction (PCR), Immunoblot and in situ hybridization in two well differentiated NPC cell lines (CNE-1 and HK-1) and two other poorly differentiated NPC cell line (CNE-2 and CNE-3). Contrary to previous report indicating the absence of EBV in these cell lines, EBV DNA and proteins were present in all cell lines. The detection of EBV became more easily when the investigation was carried out on the nude mice tumor induced by transplantation of each NPC epithelial cell line. The EBV latent membrane protein (LMP1) was found by in situ hybridization to be integrated partly in the chromosomal DNA of these cell lines. The observations indicate that EBV could persist for a long time in the carcinoma cells established directly from well and poorly differentiated tumor biopsies and from transplantable NPC tumor in nude mice.
Assuntos
DNA Viral/análise , Herpesvirus Humano 4/isolamento & purificação , Southern Blotting , Diferenciação Celular , Herpesvirus Humano 4/genética , Humanos , Immunoblotting , Hibridização In Situ , Neoplasias Nasofaríngeas/patologia , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
In Epstein-Barr virus (EBV)-carrying nonproducer Raji cells, the induction of the viral replicative cycle by chemical treatment is limited to only the early stage and viral DNA synthesis is totally inhibited. We previously showed the absence of two messenger RNAs that are encoded by the BamHI-A fragment of the EBV genome and that correspond to open reading frames BALF2 and BARF1 in chemically induced Raji cells. Since the BALF2 gene encodes a 135-kDa DNA-binding protein which was immunoprecipitated by antibody against ICP8 protein, a key protein in herpes simplex virus replication, we asked whether the lack of productive cycle in Raji cells is due to the absence of expression of the BALF2 gene. We transfected the Raji cell line with the BALF2 gene. After chemical induction, the BALF2-transfected cells expressed not only early antigens but also late antigens. In these cultures, the viral particles were detected by electron microscopy. The expression of late antigens was completely inhibited by arabinofuranosylthymine, which is a specific inhibitor of viral DNA replication. The BALF2 gene might play an essential role in the induction of the EBV-lytic cycle.