BACKGROUNDEpstein-Barr virus (EBV) commonly infects the general population and has been associated with nasopharyngeal carcinoma (NPC), which has a high incidence in certain regions. This study aimed to address how EBV variations contribute to the risk of NPC.

METHODSUsing logistic regression analysis and based on the sequence variations at EBV-encoded RPMS1, a multi-stage association study was conducted to identify EBV variations associated with NPC risk. A protein degradation assay was performed to characterize the functional relevance of the RPMS1 variations.

RESULTSBased on EBV-encoded RPMS1 variations, a single nucleotide polymorphism (SNP) in the EBV genome (locus 155391: G>A, named G155391A) was associated with NPC in 157 cases and 319 healthy controls from an NPC endemic region in South China [P < 0.001, odds ratio (OR) = 4.47, 95% confidence interval (CI) 2.71-7.37]. The results were further validated in three independent cohorts from the NPC endemic region (P < 0.001, OR = 5.20, 95% CI 3.18-8.50 in 168 cases vs. 241 controls, and P < 0.001, OR = 5.27, 95% CI 4.06-6.85 in 726 cases vs. 880 controls) and a non-endemic region (P < 0.001, OR = 7.52, 95% CI 3.69-15.32 in 58 cases vs. 612 controls). The combined analysis in 1109 cases and 2052 controls revealed that the SNP G155391A was strongly associated with NPC (P(combined) < 0.001, OR = 5.27, 95% CI 4.31-6.44). Moreover, the frequency of the SNP G155391A was associated with NPC incidence but was not associated with the incidences of other EBV-related malignancies. Furthermore, the protein degradation assay showed that this SNP decreased the degradation of the oncogenic RPMS1 protein.

CONCLUSIONSOur study identified an EBV variation specifically and significantly associated with a high risk of NPC. These findings provide insights into the pathogenesis of NPC and strategies for prevention.

Adult ; Aged ; Carcinoma ; Case-Control Studies ; China ; epidemiology ; Epstein-Barr Virus Infections ; complications ; epidemiology ; virology ; Female ; Genetic Association Studies ; Genome, Viral ; Herpesvirus 4, Human ; genetics ; isolation & purification ; Humans ; Incidence ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; epidemiology ; virology ; Neoplasm Proteins ; genetics ; Pilot Projects ; Polymorphism, Single Nucleotide ; Risk Assessment ; methods ; Tumor Cells, Cultured ; Viral Proteins ; genetics

Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD). This article presented the inhibitory activity of pentapeptides on the EV71 infection in rhabdomyosarcoma (RD) and suckling mice. The EV71 VP1 capsid protein expression levels and mRNA levels were analyzed by Western blotting and real-time PCR. The antiviral activity of pentapeptides in vivo was evaluated by weight changes and EV71 VP1 protein expression levels in intestines of suckling mice. Results revealed that the pentapeptide P010157 was able to inhibit EV71 replication in RD cells. After being incubated with the P010157 at a concentration of 100 microg x mL(-1) for 48 h, the level of EV71 vp1 mRNA in RD cells decreased by (92.0 +/- 6.3)%. The estimated EC50 was 2.2 microg x mL(-1). P010157 was able to inhibit EV 71-induced cytopathic effect (CPE) in RD cells. The cytotoxic activity of the compound was evaluated against RD cells by MTS assay. The results showed that P010157 had no obvious toxicity. In addition, the treated mice with P010157 did not exhibit weight loss, as was observed in untreated mice. EV71 replication reduced significantly as revealed by Western blotting. These findings suggest that P010157 could prevent EV71 proliferation in vitro and in vivo. P010157 is a novel compound for antiviral therapies against EV71, which merited further investigation.
Animals ; Antiviral Agents ; pharmacology ; Capsid Proteins ; genetics ; metabolism ; Enterovirus A, Human ; physiology ; Enterovirus Infections ; metabolism ; Humans ; Mice ; Oligopeptides ; pharmacology ; RNA, Messenger ; metabolism ; Rhabdomyosarcoma ; metabolism ; pathology ; virology ; Tumor Cells, Cultured ; Virus Replication ; drug effects

MicroRNAs (miRNAs) play an important role in infection and replication of virus in host cells. To identify cellular miRNAs involved in the host response to enterovirus 71 (EV71) infection, we examined miRNAs effects on the replication of EV71 in rhabdomyosarcoma cells. We constructed target gene of miRNAs screening system. 3'untranslated region (UTR) dual luciferase reporter analysis was used to identify putative miRNA targets in the EV71 virus genome. First, 13 segments of EV71 virus genomes were inserted to the pMIR vector and the luciferase expression were assayed to identify the target gene of putative miRNA. The reporter gene expression of the cells transfected with the vector containing 5'-UTR was significantly downregulated. Then we screened the miRNAs that may target to 5'-UTR using online analysis programs. Furthermore, Western blotting and real-time PCR test were performed to investigate the effect of miRNAs on viral replication. The study showed that miR373 and miR542-5p could suppress the replication of EV71 virus through binding to the 5'-UTR gene. Cellular miRNAs could regulate the replication of EV71 virus in host cells, and our paper should report the role of miR373 and miR542-5p in this regulation for the first time. Our findings supported the notion that the cellular miRNAs might be essential in the host-pathogen interactions.
3' Untranslated Regions ; 5' Untranslated Regions ; Down-Regulation ; Enterovirus A, Human ; physiology ; Enterovirus Infections ; virology ; Gene Expression ; Host-Pathogen Interactions ; Humans ; MicroRNAs ; genetics ; Rhabdomyosarcoma ; virology ; Tumor Cells, Cultured ; Virus Replication

In order to explore the relationship between human papilloma virus ( HPV) and upper gastrointestinal cancer(esophageal cancer), An esophageal squamous cell carcinoma(ESCC) tissue was obtained from a 76 year old Chinese female patient from Anyang city, a high-incidence area for esophageal cancer, in China. Transplanted tumor was formed through direct SCID mouse tumorigenicity experiment and cultured monolayer cells were obtained after several passages and screenings Immunofluorescence test, cell growth curve, soft agar assay, chromosome analysis and tissues HE staining were also performed to confirm the epithelial cell origin. Cell DNA STR typing results showed that no three alleles was observed,indicating no contamination of human cells. DNA analysis revealed the presence of HPV type 18 DNA in this cell line. DOLINK test found the E6 protein expression of HPV virus. We concluded that the established cell line is a new esophageal squamous cell-origincarcinoma cell line with HPV DNA positive and expression of viral oncoprotein. It provides new cytologic material for performing etiology studies on the occurrence and development of esophageal cancer.
Aged ; Animals ; Carcinoma, Squamous Cell ; virology ; Cell Proliferation ; China ; Esophageal Neoplasms ; virology ; Female ; Human papillomavirus 18 ; genetics ; isolation & purification ; Humans ; Male ; Mice ; Mice, SCID ; Papillomavirus Infections ; virology ; Tumor Cells, Cultured ; cytology ; virology ; Tumor Virus Infections ; virology

Establishing Epstein-Barr virus(EBV)-specific cytolytic T lymphocytes(EBV-CTLs) from peripheral blood mononuclear cells(PBMCs) for adoptive immunotherapy has been reported in EBV-associated malignancies including Hodgkin's lymphoma and nasopharyngeal carcinoma(NPC). In the current study, we performed ex vivo expansion of tumor-infiltrating lymphocytes(TILs) obtained from NPC biopsy specimens with a rapid expansion protocol using anti-CD3 monoclonal antibody(OKT3), recombinant human interleukin(IL)-2, and irradiated PBMCs from healthy donors to initiate the growth of TILs. Young TIL cultures comprised of more than 90% of CD3+ T cells, a variable percentage of CD3+CD8+ and CD3+CD4+ T cells, and less than 10% of CD3-CD16+ natural killer cells, a similar phenotype of EBV-CTL cultures from PBMCs. Interestingly, TIL cultures secreted high levels of the Th1 cytokines, interferon gamma (IFNγ) and tumor necrosis factor-alpha (TNF-α), and low levels of the Th2 cytokines, IL-4 and IL-10. Moreover, young TILs could recognize autologous EBV-transformed B lymphoblast cell lines, but not autologous EBV-negative blast cells or allogeneic EBV-negative tumor cells. Taken together, these data suggest that ex vivo expansion of TILs from NPC biopsy tissue is an appealing alternative method to establish T cell-based immunotherapy for NPC.
Biopsy ; CD3 Complex ; analysis ; CD4 Antigens ; analysis ; CD8 Antigens ; analysis ; Cells, Cultured ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Interferon-gamma ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-2 ; pharmacology ; Interleukin-4 ; metabolism ; Lymphocytes, Tumor-Infiltrating ; immunology ; virology ; Monocytes ; pathology ; Muromonab-CD3 ; pharmacology ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; virology ; Receptors, IgG ; analysis ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo establish human colorectal tissue model in HIV-1 mucosal infection and by using pseudotyped virus to simulate the biological process of HIV-1 mucosal infection from HIV-1 entering into mucosa to local infection establishment.

METHODSTumor adjacent normal colorectal tissues were obtained with informed consent. After excised the muscularis externa, the mucosa and submucosa were dissected into the same blocks and cultured in 12-well cell culture plates. The cultured tissue structure and morphology were observed from day 0 to day 13 by staining with the hematoxylin eosin (HE), and the tissue activity was detected by 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The established tissues explants were infected by a single cycle replicated pseudotyped virus and propagated for 6 - 7 days, then subjected to the detection of p24 production within supernatant to verify the applicability of the model for the studying of HIV-1 mucosal infection. The applicability of the established explants for safety and reactivity evaluation of mucosa topical drugs was conducted by the using of first generation antiseptic Nonoxynol-9 (N-9) as an example.

RESULTSHE staining showed the structure of colorectal tissue was remained well until 5(th) day and still evident until 13(th) day. The tissue activity of cultured mucosa was above 80% at day 4, and still remained over 50% at day 7 as detected by MTT assay. After infected by pseudo virus, the increased level of p24 was detected from supernatant collected on 1(st), 4(th), 8(th) day, which indicated a local infection was created. In addition, the dose changing of N-9 was reflected sensitively by the activity of this model.

CONCLUSIONEx vivo human colorectal tissue model mimic HIV-1 mucosal infection was established that can be used to replicate the bioprocess of human HIV-1 mucosal infection.

Colon ; pathology ; virology ; HIV Infections ; pathology ; virology ; HIV-1 ; Humans ; Intestinal Mucosa ; pathology ; virology ; Models, Biological ; Rectum ; pathology ; virology ; Tissue Culture Techniques ; methods ; Tumor Cells, Cultured

Adolescent ; Adult ; Aged ; Cells, Cultured ; DNA, Viral ; blood ; Female ; Flow Cytometry ; Hepatitis B Core Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B, Chronic ; metabolism ; virology ; Humans ; Interferon-alpha ; blood ; metabolism ; Interleukin-18 ; blood ; metabolism ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Male ; Middle Aged ; Oligodeoxyribonucleotides ; pharmacology ; Polymerase Chain Reaction ; methods ; Toll-Like Receptor 9 ; agonists ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Young Adult

OBJECTIVETo establish a method for efficient transfer of 1.3-fold HBV/C genome into HepG2 cell line using adenoviral vector system for studying the replication and antigen expression of HBV.

METHODSThe 1.3-copy overlength genome of HBV genotype C was constructed and cloned into the shuttle vector pAdTrack. After confirmation of the constructed HBV genome by sequencing, the resultant plasmid linearized by digestion with Pme I was transformed into competent E.coli Adeasier-1 cells. The recombinants of pAdEasy-HBV/C were linearized by digestion with Pac I and transfected into the packaging cells (293 cells) via liposome. HepG2 cells were then infected with a proper quantity of the recombinant adenoviruses. The HBV DNA level and HBeAg and HBsAg titers were detected in the cell medium.

RESULTSThe 1.3-fold overlength HBV/C genome was efficiently transferred into HepG2 cells via the adenoviral vector system, which resulted in initiation of the virus replication and protein expression in the cells using the viral replication mechanism.

CONCLUSIONThe adenovirus vector system (AdEasy) allows convenient and effective transfer of HBV genome into HepG2 cells, and provides a convenient means for screening therapeutic drugs against HBV.

Adenoviridae ; genetics ; metabolism ; Carcinoma, Hepatocellular ; genetics ; virology ; Genetic Vectors ; genetics ; Genome, Viral ; genetics ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; genetics ; virology ; Transduction, Genetic ; Tumor Cells, Cultured

OBJECTIVETo establish a method for efficient induction and expansion of Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) in vitro and evaluate the possibility of using this strategy for treatment of nasopharyngeal carcinoma (NPC).

METHODSEBV-transformed B lymphoblastoid cells (BLCLs) were used as the antigen stimuli and antigen-presenting cells. EBV-specific CTL was induced by co-culture of the autologous peripheral blood mononuclear cells (PBMCs) and the irradiated BLCLs, and expanded with a cocktail method consisting of OKT-3, irradiated homologous PBMC, and IL-2. The specific activity of the CTL against the NPC cells was measured with MTT assay.

RESULTSEBV-specific CTL was successfully induced and expanded by 600 folds. The killing efficiency of the CTL was 76% for autologous BLCLs, 13% for homologous BLCLs, 51% for autologous NPC cells, and 27% for homologous CNE cell line, and after expansion, the corresponding killing efficiencies were 63%, 25%, 49%, and 33%, respectively. The non-specific killing only slightly increased after the expansion.

CONCLUSIONEBV-specific CTL can be successfully induced and expanded in vitro for specific killing of autologous NPC cells, suggesting the potential of this strategy in the treatment of NPC.

Antigen-Presenting Cells ; cytology ; immunology ; Antigens, Viral ; immunology ; B-Lymphocytes ; cytology ; immunology ; virology ; Cells, Cultured ; Coculture Techniques ; Herpesvirus 4, Human ; immunology ; Humans ; Immunotherapy, Adoptive ; Nasopharyngeal Neoplasms ; immunology ; pathology ; therapy ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; virology ; Tumor Cells, Cultured

No abstract available.
Cell Culture Techniques ; Cell Line, Tumor ; Cells, Cultured ; Cells, Immobilized ; Hepacivirus/isolation & purification/*physiology ; Hepatocytes/physiology/*virology ; Humans ; Models, Biological ; RNA, Viral/analysis ; *Virus Replication