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Investigates the effect of the Epstein-Barr virus latent membrane protein (LMP) expression on epithelial cells. Methods; Results; Discussion.

Abstract Background The response of serum cholesterol to diet may be affected by the apolipoprotein E (APOE) ε2/ε3/ε4 polymorphism, which also is a significant predictor of variation in the risk of coronary heart disease (CHD) and CHD death. Here, we test the hypothesis that the APOE polymorphism may modulate the cholesterol-raising effect of coffee. Objective We determined the effect of a coffee abstention period and a daily intake of 600 mL coffee on serum cholesterol and triglycerides with respect to the APOE polymorphism. Design 121 healthy, non-smoking men (22%) and women (78%) aged 29–65 years, took part in a study with four intervention periods: 1 and 3) a coffee free period of three weeks, 2 and 4) 600 mL coffee/day for four weeks. Results APOE ε2 positive individuals had significantly lower total cholesterol concentration at baseline (4.68 mmol/L and 5.28 mmol/L, respectively, p = 0.01), but the cholesterol-raising effect of coffee was not influenced significantly by APOE allele carrier status. Conclusions The APOE ε 2 allele is associated with lower serum cholesterol concentration. However, the APOE polymorphism does not seem to influence the cholesterol-raising effect of coffee.

The Epstein-Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein-Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer-promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.

The Epstein-Barr virus (EBV)-encoded LMP1 oncogene has a role in transformation, proliferation, and metastasis of several EBV-associated tumors. Furthermore, LMP1 is critically involved in transformation and growth of EBV-immortalized B cells in vitro. The oncogenic properties of LMP1 are attributed to its ability to upregulate anti-apoptotic proteins and growth signals. The transcriptional regulation of LMP1 is dependent on the context of cellular and viral proteins present in the cell. Here, we investigated the effect of several signaling pathways on the regulation of LMP1 expression. Inhibition of p38 signaling, using p38-specific inhibitors SB203580 and SB202190, downregulated LMP1 in estrogen-induced EREB2.5 cells. Similarly, p38 inhibition decreased trichostatin A-induced LMP1 expression in P3HR1 cells. Exogenous expression of p38 in lymphoblastoid cell lines (LCLs) led to an increase in LMP1 promoter activity in reporter assays, and this activation was mediated by the previously identified CRE site in the promoter. Inhibition of p38 by SB203580 and p38-specific small interfering RNA (siRNA) also led to a modest decrease in endogenous LMP1 expression in LCLs. Chromatin immunoprecipitation indicated decreased binding of CREB-ATF1 to the CRE site in the LMP1 promoter after inhibition of the p38 pathway in EREB2.5 cells. Taken together, our results suggest that an increase in p38 activation upregulates LMP1 expression. Since p38 is activated in response to stimuli such as stress or possibly primary infection, a transient upregulation of LMP1 in response to p38 may allow the cells to escape apoptosis. Since the p38 pathway itself is activated by LMP1, our results also suggest the presence of an autoregulatory loop in LMP1 upregulation.

The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-kappaB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-kappaB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-kappaB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-kappaB factors in both B cells and epithelial cells. Exogenous expression of NF-kappaB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-kappaB activation and LMP1 expression.

The Epstein-Barr virus (EBV)-encoded tumour-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We have previously identified a cyclic AMP-responsive element (CRE) in the B95-8 LMP1 promoter that is essential for transcription activation. Sequencing of LMP1 promoter in the P3HR1-derived EREB2.5 cell line revealed 25 single base pair substitutions in comparison to the B95-8 virus, one of them localized in the CRE element. Sequence variations in this element have been identified in several EBV isolates of both African and Asian origins. The effect of the P3HR1 CRE site variation on binding of factors to the LMP1 promoter sequence (LRS) and promoter activation was investigated with electrophoretic mobility-shift assays and reporter gene transfection assays. ATF1 and CREB1 transcription factors bound with reduced efficiency to the P3HR1 variant and below the detection level to the other tested variants. Accordingly, reporter plasmids carrying the P3HR1 CRE sequence in a B95-8 LRS context displayed 50 % lower activity in all tested cell lines. The impaired ability to activate transcription caused by the C to A substitution in CRE was not apparent when the mutated site was placed in a P3HR1 LRS context and the reporter transfected into Jijoye cells, most likely as a consequence of the other base pair substitutions in P3HR1 LRS. Overall, our results suggest that the mutations in the LRS CRE site have been conserved to adjust LMP1 expression to levels that favour cell survival in certain cellular and environmental contexts.

MODULAR ANALYTICS (Roche Diagnostics) (MODULAR ANALYTICS, Elecsys and Cobas Integra are trademarks of a member of the Roche Group) represents a new approach to automation for the clinical chemistry laboratory. It consists of a control unit, a core unit with a bidirectional multitrack rack transportation system, and three distinct kinds of analytical modules: an ISE module, a P800 module (44 photometric tests, throughput of up to 800 tests/h), and a D2400 module (16 photometric tests, throughput up to 2400 tests/h). MODULAR ANALYTICS allows customised configurations for various laboratory workloads. The performance and practicability of MODULAR ANALYTICS were evaluated in an international multicentre study at 16 sites. Studies included precision, accuracy, analytical range, carry-over, and workflow assessment. More than 700 000 results were obtained during the course of the study. Median between-day CVs were typically less than 3% for clinical chemistries and less than 6% for homogeneous immunoassays. Median recoveries for nearly all standardised reference materials were within 5% of assigned values. Method comparisons versus current existing routine instrumentation were clinically acceptable in all cases. During the workflow studies, the work from three to four single workstations was transferred to MODULAR ANALYTICS, which offered over 100 possible methods, with reduction in sample splitting, handling errors, and turnaround time. Typical sample processing time on MODULAR ANALYTICS was less than 30 minutes, an improvement from the current laboratory systems. By combining multiple analytic units in flexible ways, MODULAR ANALYTICS met diverse laboratory needs and offered improvement in workflow over current laboratory situations. It increased overall efficiency while maintaining (or improving) quality.

Background: The response of serum cholesterol to diet may be affected by the apolipoprotein E (APOE) epsilon2/epsilon3/epsilon4 polymorphism, which also is a significant predictor of variation in the risk of coronary heart disease (CHD) and CHD death. Here, we test the hypothesis that the APOE polymorphism may modulate the cholesterol-raising effect of coffee., Objective: We determined the effect of a coffee abstention period and a daily intake of 600 mL coffee on serum cholesterol and triglycerides with respect to the APOE polymorphism., Design: 121 healthy, non-smoking men (22%) and women (78%) aged 29-65 years, took part in a study with four intervention periods: 1 and 3) a coffee free period of three weeks, 2 and 4) 600 mL coffee/day for four weeks., Results: APOE epsilon2 positive individuals had significantly lower total cholesterol concentration at baseline (4.68 mmol/L and 5.28 mmol/L, respectively, p = 0.01), but the cholesterol-raising effect of coffee was not influenced significantly by APOE allele carrier status., Conclusions: The APOE epsilon 2 allele is associated with lower serum cholesterol concentration. However, the APOE polymorphism does not seem to influence the cholesterol-raising effect of coffee.

Some methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms are associated with hyperhomocysteinemia. Trials have shown a plasma homocysteine raising effect of coffee. We determined the effect of a daily intake of 600 ml coffee and a supplementation of 200 microg folic acid or placebo on plasma homocysteine (tHcy) with respect to the MTHFR C677T and A1298C polymorphisms. One hundred and twenty healthy, non-smoking men (22%) and women (78%) aged 29-65 years, took part in a controlled, randomized, blinded study with two intervention periods: i) a coffee-free period of three weeks, ii) 600 ml coffee/day and a supplement of 200 microg folic acid/d or placebo for four weeks. The results showed that tHcy at baseline was significantly higher for the 677TT genotype group compared to the 677CC genotype group (p=0.0045) and that this group responded with significantly larger increase in tHcy upon coffee exposure than the 677CC and 677CT genotype groups (p=0.0045 and p=0.0041, respectively). Supplementation with 200 microg folic acid compared to placebo reduced the tHcy increasing effect of coffee in the 677TT genotype group. The A1298C polymorphism did not affect tHcy concentration significantly at any stage in the study. In conclusion, the homocysteine increasing effect of coffee is particularly seen in individuals with the homozygous 677TT genotype. Supplementation with 200 microg folic acid/d decreases this tHcy increment.

Background: Genetic polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) and transcobalamin (TC) genes influence homocysteine metabolism which in turn may influence the risk of spontaneous abortion. It was hypothesized that there may be a significant interaction between MTHFR and TC genotypes which affects the pathogenesis of spontaneous abortion., Methods and Results: A total of 76 fetal tissue samples from spontaneous abortions between weeks 6 and 20 of pregnancy, and 114 control samples from healthy blood donors were genotyped for the MTHFR 677C>T and 776C>G polymorphisms. Subjects with combined MTHFR 677TT/TC 776GG and combined MTHFR 677TT/TC 776CG genotypes gave an odds ratio for spontaneous abortion of 3.8 (95% confidence interval 1.4-9.9, P = 0.005)., Conclusions: Embryos that have combined MTHFR 677TT and TC 776CG or 776GG genotypes; genotypes that individually are associated with impaired homocysteine metabolism in adults, are at increased risk for spontaneous abortion compared with embryos that have only one of these genotypes.