The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection

Infection of human B cells with wild-type Epstein-Barr virus (EBV) in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). Latent Membrane Protein 2 (LMP2) is expressed early after infection and previous research has suggested a possible role in this process. Therefore, we generated recombinant EBV with knockouts of either or both protein isoforms, LMP2A and LMP2B (Δ2A, Δ2B, Δ2A/Δ2B) to study the effect of LMP2 in early B cell infection. Infection of B cells with Δ2A and Δ2A/Δ2B viruses led to a marked decrease in activation and proliferation relative to wild-type (wt) viruses, and resulted in higher percentages of apoptotic B cells. Δ2B virus infection showed activation levels comparable to wt, but fewer numbers of proliferating B cells. Early B cell infection with wt, Δ2A and Δ2B viruses did not result in changes in latent gene expression, with the exception of elevated LMP2B transcript in Δ2A virus infection. Infection with Δ2A and Δ2B viruses did not affect viral latency, determined by changes in LMP1/Zebra expression following BCR stimulation. However, BCR stimulation of Δ2A/Δ2B cells resulted in decreased LMP1 expression, which suggests loss of stability in viral latency. Long-term outgrowth assays revealed that LMP2A, but not LMP2B, is critical for efficient long-term growth of B cells in vitro. The lowest levels of activation, proliferation, and LCL formation were observed when both isoforms were deleted. These results suggest that LMP2A appears to be critical for efficient activation, proliferation and survival of EBV-infected B cells at early times after infection, which impacts the efficient long-term growth of B cells in culture. In contrast, LMP2B did not appear to play a significant role in these processes, and long-term growth of infected B cells was not affected by the absence of this protein. © 2013 Wasil et al

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI &lt;18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For school&#x2;aged children and adolescents, we report thinness (BMI &lt;2 SD below the median of the WHO growth reference) and obesity (BMI &gt;2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit

Proliferation of B cells infected with wt and LMP2 KO viruses.

<p>5×10⁴ B cells infected with recombinant wt and LMP2 KO EBV at MOI 1. EGFP expression and proliferation was examined at 4, 7 and 14 days post-infection using inverted fluorescent microscope.</p

Efficient Proliferation and Survival of EBV-infected B cells require LMP2.

<p>1×10⁶ purified B cells were labeled with Violet Tracer (Invitrogen) and infected with recombinant wt and LMP2 KO EBV at MOI 1. B cells were harvested at 4, 7 and 14 days post-infection and stained for Annexin V. During fixation step, 10 μl counting beads (CountBright Absolute Counting Beads, Invitrogen) were added to each sample. During acquisition, the event gate was set to 5000 beads, which normalized the acquisition volume between samples and allowed for accurate, absolute counts of proliferating B cells. (A) Gating strategy for proliferation and apoptosis analysis. Doublets and dead cells were excluded. Next, lymphocytes and EGFP+ cells were selected for proliferation and apoptosis analysis. (B) Representative donor for proliferation and apoptosis data. (C) Proliferation and (D) Apoptosis data points are an average of 3 independent experiments ± SEM. Statistical significance was determined using a Two-Way ANOVA test and Bonferroni Post-test (each data point for LMP2 KO viruses was compared to wild-type infection to determine statistical significance). P-value (*) <0.05, p-value (**) <0.01 and p-value (****) <0.0001.</p

Primers and Probes for Real Time PCR analysis of EBV gene expression.

*<p>All probes were conjugated with 5′ FAM and a 3′ TAMRA quencher.</p

EBV Gene Expression in early EBV infection with LMP2 KO viruses.

<p>1×10⁶ purified B cells were infected with recombinant wt and LMP2 KO EBV at MOI 1. (A) RNA was harvested for analysis at 12, 24, 48, 72, 96, 120 and 168 hours post-infection and analyzed by Real Time PCR to determine the effect of LMP2 on latent gene expression. Specifically, we examined the latent genes LMP2A, LMP2B (B), EBNA1 (C), EBNA2 (D), LMP1 (E) for changes in early gene expression. (F) In addition to latent genes, the lytic gene BZLF1 or Zebra, an immediate early lytic transactivation gene, was examined. β₂Microglobulin mRNA was amplified for EBV gene normalization. Each data point is an average of three independent experiments ± SEM. Statistical significance determined using Two-way ANOVA and Bonferroni Post-test (each data point for LMP2 knockout viruses was compared to wild-type infection to determine statistical significance). P-value (****) <0.0001.</p

Schematic representation of role for LMP2 in early EBV infection leading to B cell proliferation.

<p>Schematic representation of role for LMP2 in early EBV infection leading to B cell proliferation.</p

LMP2A is critical for sufficient activation of B cells.

<p>1×10⁶ purified B cells infected with recombinant wt and LMP2 KO EBV at MOI 1. B cells harvested at 4, 8 and 16 days post-infection for analysis of activation marker expression. (A) Expression of CD23 (marker of B cell activation) and CD71 (marker of lymphocyte activation and proliferation) assessed by flow cytometry and analyzed using FlowJo 7.6.1 software. (B) Expression of CD23 and (C) CD71 quantified and expressed as percent positive for surface markers. Each data point is an average of 2 independent experiments ± SEM. Statistical significance was determined using Two-way ANOVA and Bonferroni Post-test (each data point for LMP2 KO viruses was compared to wild-type infection to determine statistical significance). P-value (**) <0.01 for Δ2A CD23 expression at 4 days post-infection. P-value (***) <0.001 for Δ2A/Δ2B CD23 expression at 4 days post-infection and Δ2A CD71 expression at 4 days post-infection. P-value (****) <0.0001 for Δ2A CD71 expression at 4 days post-infection, and Δ2A and Δ2A/Δ2B viruses for both CD23 and CD71 expression at 8 and 16 days post-infection.</p

Recombinant BACs have deleted LMP2 regions and retain latent gene expression.

<p>(A) LMP2 knockout EBV BACs were created by deletion of LMP2A and LMP2B transcription initiating exons (Exon 1 and Exon 1′). LoxP sites were inserted, flanking each initial exon, and recombined via introduction of Cre Recombinase expression plasmid (pKD46) into DH10B bacteria containing floxed BACs. (B) Recombinant wt and Δ2B BACs contain LMP2A Exon 1, which is demonstrated by presence of 522 bp PCR band. Δ2A and Δ2A/Δ2B BACs have deleted Exon 1, which is demonstrated by absence of PCR band due to removal of the forward primer-binding site. (C) Recombinant wt and Δ2A BACs contain Exon 1′, which is demonstrated by 374 bp PCR band. Δ2B and Δ2A/Δ2B BACs have deleted Exon 1′, evidenced by the presence of 167 bp PCR deletion band. (D) Recombinant wt and LMP2 knockout EBV BACs transfected into HEK 293 cells. After establishment of BAC-containing cell lines, each cell line was assessed for latent gene expression using Real Time PCR. Δ2B cell lines were negative for LMP2B transcript, Δ2A cell lines were negative for LMP2A transcript, and Δ2A/Δ2B cell lines were negative for both transcripts. Expression of latent genes was similar for wt and LMP2 knockout BACs.</p