EBNA2-EBF1 complexes promote MYC expression and metabolic processes driving S-phase progression of Epstein-Barr virus-infected B cells.

Epstein-Barr virus (EBV) is a human tumor virus which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen 2 (EBNA2) is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA-binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor 1 (EBF1), a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. The α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F, as well as multiple metabolic processes linked to cell cycle progression are impaired in EBVΔα1-infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV-infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification.

PLK1-dependent phosphorylation restrains EBNA2 activity and lymphomagenesis in EBV-infected mice.

While Epstein-Barr virus (EBV) establishes a life-long latent infection in apparently healthy human immunocompetent hosts, immunodeficient individuals are at particular risk to develop lymphoproliferative B-cell malignancies caused by EBV. A key EBV protein is the transcription factor EBV nuclear antigen 2 (EBNA2), which initiates B-cell proliferation. Here, we combine biochemical, cellular, and in vivo experiments demonstrating that the mitotic polo-like kinase 1 (PLK1) binds to EBNA2, phosphorylates its transactivation domain, and thereby inhibits its biological activity. EBNA2 mutants that impair PLK1 binding or prevent EBNA2 phosphorylation are gain-of-function mutants. They exhibit enhanced transactivation capacities, accelerate the proliferation of infected B cells, and promote the development of monoclonal B-cell lymphomas in infected mice. Thus, PLK1 coordinates the activity of EBNA2 to attenuate the risk of tumor incidences in favor of the establishment of latency in the infected but healthy host.

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells.

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2.

Replacing the Clinical Institute Withdrawal Assessment-Alcohol revised with the modified Richmond Agitation and Sedation Scale for alcohol withdrawal to support management of alcohol withdrawal symptoms: potential impact on length of stay and complications.

PURPOSE: We evaluated impact on length of stay and possible complications of replacing the Clinical Institute Withdrawal Assessment-Alcohol Revised (CIWA-Ar) scale with a slightly modified Richmond Agitation and Sedation Scale (mRASS-AW) to support managing patients admitted with alcohol withdrawal symptoms in a community hospital. Since mRASS-AW is viewed as easier and quicker to use than CIWA-Ar, provided use of mRASS-AW does not worsen outcomes, it could be a safe alternative in a busy ED environment and offer an opportunity to release nursing time to care. METHODS: Retrospective time-series analysis of mean quarterly length of stay. All analyses exclusively used our hospital's administrative discharge diagnoses database. During April 1st 2012 to December 14th 2014, the CIWA-Ar was used in the ED and in-patient units to guide benzodiazepine dosing decisions for alcohol withdrawal symptoms. After this point, CIWA-Ar was replaced with mRASS-AW. Data was evaluated until December 31st 2020. PRIMARY OUTCOME: mean quarterly length of stay. SECONDARY OUTCOMES: delirium, intensive care unit (ICU) admission, other post-admission complications, mortality. RESULTS: N = 1073 patients. No association between length of stay and scale switch (slope change 0.3 (95% CI - 0.03 to 0.6), intercept change, 0.06 (- 0.03 to 0.2). CIWA-Ar (n = 317) mean quarterly length of stay, 5.7 days (95% 4.2-7.1), mRASS-AW (n = 756) 5.0 days (95% CI 4.3-5.6). Incidence of delirium, ICU admission or mortality was not different. However, incidence of other post-admission complications was higher with CIWA-Ar (6.6%) than mRASS-AW (3.4%) (p = 0.020). CONCLUSIONS: This was the first study to compare patient outcomes associated with using mRASS-AW for alcohol withdrawal symptoms outside the ICU. Replacing CIWA-Ar with mRASS-AW did not worsen length of stay or complications. These findings provide some evidence that mRASS-AW could be considered an alternative to CIWA-Ar and potentially may provide an opportunity to release nursing time to care.

ABSTRAIT: BUT: Nous avons évalué l'impact sur la durée du séjour et les complications possibles du remplacement de l'échelle Clinical Institute Withdrawal Assessment- Alcohol Revised (CIWA-Ar) par une échelle d'agitation et de sédation de Richmond légèrement modifiée (mRASS-AW) soutenir la prise en charge des patients admis avec des symptômes de sevrage d'alcool dans un hôpital communautaire. Étant donné que le mRASS-AW est considéré comme plus facile et plus rapide à utiliser que le CIWA-Ar, à condition que l'utilisation du mRASS-AW n'aggrave pas les résultats, il pourrait s'agir d'une solution de rechange sécuritaire dans un environnement de SU occupé et offrir une occasion de libérer du temps pour les soins infirmiers. MéTHODES: Analyse rétrospective de séries chronologiques de la durée moyenne trimestrielle du séjour. Toutes les analyses utilisaient exclusivement la base de données des diagnostics de sortie administrative de notre hôpital. Entre le 1er avril 2012 et le 14 décembre 2014, le CIWA-Ar a été utilisé dans les unités de soins intensifs et de soins aux patients hospitalisés pour guider les décisions de dosage des benzodiazépines pour les symptômes de sevrage de l'alcool. Après ce point, CIWA-Ar a été remplacé par mRASS-AW. Les données ont été évaluées jusqu'au 31 décembre 2020. Résultat principal : durée moyenne trimestrielle du séjour. Résultats secondaires : délire, admission en unité de soins intensifs (USI), autres complications post-admission, mortalité. RéSULTATS: N = 1073 patients. Aucune association entre la durée de séjour et le changement d'échelle (changement de pente 0,3 (IC à 95 % -0,03 à 0,6), changement d'interception, 0,06 (-0,03 à 0,2). CIWA-Ar (n = 317) durée moyenne trimestrielle du séjour, 5,7 jours (95 % 4,2 à 7,1), mRASS-AW (n = 756) 5,0 jours (95 % IC 4,3 à 5,6). L'incidence du délire, de l'admission aux soins intensifs ou de la mortalité n'était pas différente. Cependant, l'incidence d'autres complications post-admission était plus élevée avec CIWA-Ar (6,6%) que mRASS-AW (3,4%) (p = 0,020). CONCLUSIONS: Il s'agissait de la première étude à comparer les résultats des patients associés à l'utilisation du mRASS-AW pour les symptômes de sevrage alcoolique en dehors des soins intensifs. Le remplacement de CIWA-Ar par mRASS-AW n'a pas aggravé la durée du séjour ou les complications. Ces résultats fournissent certaines preuves que le mRASS-AW pourrait être considéré comme une alternative au CIWA-Ar et pourrait potentiellement fournir une occasion de libérer du temps de soins infirmiers.

Bacille Calmette-Guérin Vaccine Strain Modulates the Ontogeny of Both Mycobacterial-Specific and Heterologous T Cell Immunity to Vaccination in Infants.

Differences in Bacille Calmette-Guérin (BCG) immunogenicity and efficacy have been reported, but various strains of BCG are administered worldwide. Since BCG immunization may also provide protection against off-target antigens, we sought to identify the impact of different BCG strains on the ontogeny of vaccine-specific and heterologous vaccine immunogenicity in the first 9 months of life, utilizing two African birth cohorts. A total of 270 infants were studied: 84 from Jos, Nigeria (vaccinated with BCG-Bulgaria) and 187 from Cape Town, South Africa (154 vaccinated with BCG-Denmark and 33 with BCG-Russia). Infant whole blood was taken at birth, 7, 15, and 36 weeks and short-term stimulated (12 h) in vitro with BCG, Tetanus and Pertussis antigens. Using multiparameter flow cytometry, CD4+ T cell memory subset polyfunctionality was measured by analyzing permutations of TNF-α, IL-2, and IFN-γ expression at each time point. Data was analyzed using FlowJo, SPICE, R, and COMPASS. We found that infants vaccinated with BCG-Denmark mounted significantly higher frequencies of BCG-stimulated CD4+ T cell responses, peaking at week 7 after immunization, and possessed durable polyfunctional CD4+ T cells that were in a more early differentiated memory stage when compared with either BCG-Bulgaria and BCG-Russia strains. The latter responses had lower polyfunctional scores and tended to accumulate in a CD4+ T cell naïve-like state (CD45RA+CD27+). Notably, BCG-Denmark immunization resulted in higher magnitudes and polyfunctional cytokine responses to heterologous vaccine antigens (Tetanus and Pertussis). Collectively, our data show that BCG strain was the strongest determinant of both BCG-stimulated and heterologous vaccine stimulated T cell magnitude and polyfunctionality. These findings have implications for vaccine policy makers, manufacturers and programs worldwide and also suggest that BCG-Denmark, the first vaccine received in many African infants, has both specific and off-target effects in the first few months of life, which may provide an immune priming benefit to other EPI vaccines.