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Despite the availability of effective vaccines against COVID-19, booster vaccinations are needed to maintain vaccine-induced protection against variant strains and breakthrough infections. This study aimed to investigate the efficacy, safety, and immunogenicity of the Ad26.COV2.S vaccine (Janssen) as primary vaccination plus a booster dose.ENSEMBLE2 is a randomised, double-blind, placebo-controlled, phase 3 trial including crossover vaccination after emergency authorisation of COVID-19 vaccines. Adults aged at least 18 years without previous COVID-19 vaccination at public and private medical practices and hospitals in Belgium, Brazil, Colombia, France, Germany, the Philippines, South Africa, Spain, the UK, and the USA were randomly assigned 1:1 via a computer algorithm to receive intramuscularly administered Ad26.COV2.S as a primary dose plus a booster dose at 2 months or two placebo injections 2 months apart. The primary endpoint was vaccine efficacy against the first occurrence of molecularly confirmed moderate to severe-critical COVID-19 with onset at least 14 days after booster vaccination, which was assessed in participants who received two doses of vaccine or placebo, were negative for SARS-CoV-2 by PCR at baseline and on serology at baseline and day 71, had no major protocol deviations, and were at risk of COVID-19 (ie, had no PCR-positive result or discontinued the study before day 71). Safety was assessed in all participants; reactogenicity, in terms of solicited local and systemic adverse events, was assessed as a secondary endpoint in a safety subset (approximately 6000 randomly selected participants). The trial is registered with ClinicalTrials.gov, NCT04614948, and is ongoing.Enrolment began on Nov 16, 2020, and the primary analysis data cutoff was June 25, 2021. From 34 571 participants screened, the double-blind phase enrolled 31 300 participants, 14 492 of whom received two doses (7484 in the Ad26.COV2.S group and 7008 in the placebo group) and 11 639 of whom were eligible for inclusion in the assessment of the primary endpoint (6024 in the Ad26.COV2.S group and 5615 in the placebo group). The median (IQR) follow-up post-booster vaccination was 36·0 (15·0-62·0) days. Vaccine efficacy was 75·2% (adjusted 95% CI 54·6-87·3) against moderate to severe-critical COVID-19 (14 cases in the Ad26.COV2.S group and 52 cases in the placebo group). Most cases were due to the variants alpha (B.1.1.7) and mu (B.1.621); endpoints for the primary analysis accrued from Nov 16, 2020, to June 25, 2021, before the global dominance of delta (B.1.617.2) or omicron (B.1.1.529). The booster vaccine exhibited an acceptable safety profile. The overall frequencies of solicited local and systemic adverse events (evaluated in the safety subset, n=6067) were higher among vaccine recipients than placebo recipients after the primary and booster doses. The frequency of solicited adverse events in the Ad26.COV2.S group were similar following the primary and booster vaccinations (local adverse events, 1676 [55·6%] of 3015 vs 896 [57·5%] of 1559, respectively; systemic adverse events, 1764 [58·5%] of 3015 vs 821 [52·7%] of 1559, respectively). Solicited adverse events were transient and mostly grade 1-2 in severity.A homologous Ad26.COV2.S booster administered 2 months after primary single-dose vaccination in adults had an acceptable safety profile and was efficacious against moderate to severe-critical COVID-19. Studies assessing efficacy against newer variants and with longer follow-up are needed.Janssen ResearchDevelopment.

While hypertension is classed as a long-term condition—implying that it is incurable—it can be prevented, managed and even reversed through lifestyle changes, argues Brian Webster.

Nursing vacancies are high across the UK, with some nurses considering leaving the profession. Evidence suggests that employers, including the NHS, need to be more flexible about working times to support employees' work-life balance and job satisfaction. Self-rostering is one approach that has the potential to enhance nurses' work-life balance and job satisfaction, enabling scope for greater autonomy. This could in turn lead to fewer nurses leaving the profession and contribute to making nursing more attractive as a career. This literature review focused on nurses in the NHS and found that self-rostering had a positive effect on their work-life balance and job satisfaction. However, a move to self-rostering can pose challenges and it should be assessed for suitability before implementation. Given the nursing vacancy crisis in the UK and many nurses' intentions to leave the profession, the potential benefits of self-rostering for nurses cannot be overlooked.

BackgroundAdult immunization rates are belowHealthy People 2020targets. Our objective was to evaluate the effectiveness of a multicomponent intervention to improve adult immunization rates.MethodsThis prospective interventional before-and-after non-randomized study was conducted through the American Academy of Family Physicians National Research Network with 43 primary care physicians from a large multi-specialty healthcare organization (multicomponent intervention groupn = 23; comparator groupn = 20) in the United States. The multicomponent intervention included provider reminders, quarterly provider-level performance reports, provider education, patient visual aid materials, and standing orders on adult pneumococcal, influenza, and zoster immunizations. We assessed individual and comparative provider-level vaccination rates and missed opportunities detailing concordance with targets established by Healthy People 2020 for pneumococcal, influenza, and zoster immunizations.ResultsVaccination rates increased after 12 months in intervention and comparator groups respectively for: a). influenza from 44.4 ± 16.7 to 51.3% ± 12.9% (by 6.9 percentage points,p = 0.001) and from 35.1 ± 19.1 to 41.3% ± 14.2%, (by 6.2 percentage points,p = 0.01); b). pneumococcal vaccinations in older adults from 62.8 ± 17.6 to 81.4% ± 16.6% (by 18.6 percentage points, forp p p p = 0.001). Pneumococcal vaccinations in adults at risk did not change from baseline in intervention group (35.7 ± 19.6 to 34.5% ± 19.0%,p = 0.3) and improved slightly in comparator group (24.3 ± 20.1 to 28.2% ± 20.0%,p = 0.003). Missed opportunities reduced after 12 months, most noticeably, for: a). for influenza from 57.7 to 48.6% (by 9.1 percentage points,p p p p ConclusionsMulticomponent interventions show promise in improving vaccination rates and reducing missed opportunities in older adults for pneumococcal and zoster vaccines and vaccination against influenza. Provider reminders remain the most effective strategy when delivered either as a component of these interventions or alone.

Simple Summary Chimeric antigen receptor (CAR) T cell therapy can be associated with substantial side effects primarily due to intense immune activation following treatment, or target antigen recognition on off-tumor tissue. Consequently, temporal and tunable control of CAR T cell activity is of major importance for the clinical translation of innovative CAR designs. This work demonstrates the transcriptional regulation of an anti-CD20 CAR in primary T cells using a drug inducible zinc finger-based transcription factor. The switch system enables titratable induction of CAR expression and CAR T cell effector function with the clinically relevant inducer drug tamoxifen and its metabolites both in vitro and in vivo, whereby CAR activity is strictly dependent on the presence of the inducer drug. The results obtained can readily be transferred to other CARs for which an improved control of expression is required. Abstract Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor β receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-β on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.

espanolEn este trabajo se desarrollo un protocolo de censo a gran escala para obtener datos actuales de las aves nocturnas y que sirva de base para futuros trabajos. El area de estudio, Euskadi, se dividio en cuadriculas UTM de 5x5 km de las que se escogieron 65 que representaban todos los tipos de vegetacion. Dentro de cada cuadricula se seleccionaron 8 Unidades de Censo (UCs), separadas entre si por una distancia minima de 1 km. En cada UC se llevo a cabo un protocolo de censo dividido en tres fases: 5 min de escucha de voces espontaneas, 5 min de reclamo de una sola especie y 5 min de escuchas en silencio, que se repitio mensualmente desde enero hasta julio de 2018. Solo se utilizo el reclamo de una especie por censo, de acuerdo con los picos anuales especificos de respuesta al reclamo, y unicamente para las estrigiformes. El carabo comun resulto ser la especie mas abundante y ampliamente distribuida de Euskadi, siguiendole bastante lejos la lechuza comun, el autillo europeo y el mochuelo europeo; mientras que el buho chico y el buho real fueron escasos y con una distribucion localizada, y el buho campestre y el mochuelo boreal resultaron ser especies raras. El chotacabras europeo aparecio en el 31,1% de las UCs, mientras que no se detectaron chotacabras cuellirrojos, ni alcaravanes comunes, y tan solo tres machos de chocha perdiz en el periodo reproductor. Finalmente, las codornices comunes fueron detectadas en el 3,8% de las UCs y solo en el censo de julio EnglishWe developed a large-scale survey protocol to secure current data on nocturnal birds as well as to obtain a reliable method for future surveys. We surveyed nocturnal birds in 65 5x5 km UTM squares that represented all the vegetation types of the Basque Country. We selected eight Survey Points (SPs) in each square, considering a minimum distance of 1 km between SPs. In each SP we developed a three-phase survey protocol for censusing nocturnal species: a 5-min wait for spontaneous voices, a 5-min broadcast of recorded vocalisations of only one species, and a 5-min wait in silence, which was repeated on a monthly basis from January to July 2018. According to the annual maximum peak of response to playback broadcasts in our study area, we broadcasted recorded vocalisations of only one owl species per survey period. The tawny owl was the most abundant and widely distributed owl species in the study area, followed a long way behind by the barn owl, Eurasian scops owl, and little owl, whereas the long-eared owl and eagle owl resulted scarce and patchily distributed, and the short-eared owl and boreal owl were rare species. European nightjars appeared in 31.1% of the SPs, whereas no records of red-necked nightjars and stone-curlews were obtained and only three woodcocks were detected during the breeding season. Common quails were detected in 3.8% of the SPs and only in July euskaraLan honetan azalera handiko guneetan burutzen diren zenbaketen protokolo bat garatu da, gaueko hegaztien datu berriak lortzeko eta etorkizuneko lanen oinarri izateko. Ikerketa area, Euskadi, 5 x 5 km-ko UTM lauki sareetan banatu zen, landaredi mota guztiak ordezkatzen zituzten 65 lauki sare aukeratu zirelarik. Lauki sare bakoitzean zortzi Zenbaketa Unitate (ZU) hautatu ziren, bata bestetik kilometro bateko tartearekin. ZU bakoitzean hiru fasetan banatutako zenbaketa protokoloa gauzatu zen: 5 min hegaztien kantuak entzunez, 5 min espezie apeua erabiliz eta 5 min isilean entzunez, protokoloa hilean behin gauzatu zen 2018ko urtarriletik uztailera bitarte. Zenbaketa bakoitzean apeua espezie bakar batentzat erabili zen, betiere apeuaren erantzun espezifikorako urteko momentu egokiena baliatuz, eta soilik estrigiformeentzat. Urubi arrunta izan da Euskadi mailan ugariena, bai kopuruan nahiz banaketa arean, alde handiz hurrenak hontz zuria, apo-hontz europarra eta mozolo europarra; aldiz, hontz ertaina eta hontz handia nahiko eskasak, eta banaketa area nahiko mugatua erakutsi dute, eta espezie urrienak landako hontza eta elurretako mozoloa. Zata europarra ZUetako % 31,5ean azaldu zen; aldiz, ez dira topatu zata lepogorria, ez eta atalar arrunta, eta ugaltze garaia izanda soilik hiru oilagor ar. Amaitzeko, galeper arrunta soilik ZUetatik %3,8an antzeman zen eta uztaileko zenbaketan baino ez.

Seeking to improve student enrolment, engagement, and retention, Kingston University began a pre-arrival shared reading scheme in 2014–2015, sending a free book to every student about to start at the university and making copies available to staff in all roles and departments across the institution. A number of associated events were organized and outcomes monitored through a variety of project-specific and institutional metrics. Continuing with the scheme in 2015–2016, Kingston University and Edinburgh Napier University joined together as research partners. Edinburgh Napier, having participated in the process of choosing a book for all to read, made the same single title available to their students and staff. In this paper the processes and outcomes of the collaboration are reported, including the differences in project implementation in the two institutions and what they learned from each other. Recommendations are made for how universities can work together on projects of mutual desirability, pointing out particular associated sensitivities, in this case when managing a long-distance collaboration, and what can be learned for the future.

Type I interferon (IFN-I) is critical for antiviral defense, and plasmacytoid dendritic cells (pDCs) are a predominant source of IFN-I during virus infection. pDC-mediated antiviral responses are stimulated upon physical contact with infected cells, during which immunostimulatory viral RNA is transferred to pDCs, leading to IFN production via the nucleic acid sensor TLR7. Using dengue, hepatitis C, and Zika viruses, we demonstrate that the contact site of pDCs with infected cells is a specialized platform we term the interferogenic synapse, which enables viral RNA transfer and antiviral responses. This synapse is formed via αLβ2 integrin-ICAM-1 adhesion complexes and the recruitment of the actin network and endocytic machinery. TLR7 signaling in pDCs promotes interferogenic synapse establishment and provides feed-forward regulation, sustaining pDC contacts with infected cells. This interferogenic synapse may allow pDCs to scan infected cells and locally secrete IFN-I, thereby confining a potentially deleterious response.

SummaryType I interferon (IFN-I) is critical for protection against viral infections. Plasmacytoid dendritic cells (pDCs) massively produce IFN-I against viruses. Physical contacts are required for pDC-mediated sensing of cells infected by genetically distant viruses. How and why these contacts are established remains enigmatic. Using dengue, hepatitis C, zika viruses, we demonstrate that the pDC/infected cell interface is a specialized platform for viral immunostimulatory-RNA transfer, which we named interferogenic synapse and required for pDC-mediated antiviral response. This synapse is an exquisitely differentiated territory with polarized adhesion complexes and regulators of actin network and endocytosis. Toll-like receptor 7-induced signaling in pDCs promotes the interferogenic synapse establishment, thus providing a feed-forward regulation that sustains contacts with infected cells. We propose that the interferogenic synapse is crucial to pDC function as it allows scanning of infected cells to locally secrete IFN-I at the infection site, thereby confining a response potentially deleterious to the host.HighlightspDCs adhere to infected cells via αLβ2 integrin/ICAM-1Regulators of actin network and endocytosis polarize at contactTLR7-induced signaling potentiates pDC polarityInfected cells activate pDCs by interferogenic synapse

Background We sought to characterize how the term “missed opportunities” is reported in the literature in the context of immunization rates and to assess how missed opportunities can be operationalized. Methods Peer-reviewed literature searches were conducted in April – May, 2015, to answer: “What methods research studies used to operationalize missed opportunities to vaccinate?” A meta-narrative review methodology was used. Results Seven studies met inclusion criteria. The methodologies for quantifying missed opportunities fell into two general categories based on: 1. the number of healthcare encounters per patient without appropriate vaccination services, defined as a number of visits per patient with no vaccination related services (Missed opportunities per patient); 2. vaccination status as “non-vaccinated” among a group of patients who had a healthcare encounter where the vaccination should/could have had happened (Missed opportunities per population). Conclusions Our study provided an initial overview of the methods reported in the literature, and concluded that the quantifiable missed opportunity holds promise as a measurable outcome (variable) for research and quality improvement projects aimed to increase adult immunization recommendation and uptake in primary care. Electronic supplementary material The online version of this article (10.1186/s12875-017-0694-1) contains supplementary material, which is available to authorized users.

Cells infected with hepatitis C virus (HCV) become refractory to further infection by HCV (T. Schaller et al., J. Virol. 81:4591–4603, 2007; D. M. Tscherne et al., J. Virol. 81:3693–3703, 2007). This process, termed superinfection exclusion, does not involve downregulation of surface viral receptors but instead occurs inside the cell at the level of RNA replication. The originally infecting virus may occupy replication niches or sequester host factors necessary for viral growth, preventing effective growth of viruses that enter the cell later. However, there appears to be an additional level of intracellular competition between viral genomes that occurs at or shortly following mitosis. In the setting of cellular division, when two viral replicons of equivalent fitness are present within a cell, each has an equal opportunity to exclude the other. In a population of dividing cells, the competition between viral genomes proceeds apace, randomly clearing one or the other genome from cells in the span of 9 to 12 days. These findings demonstrate a new mechanism of intracellular competition between HCV strains, which may act to further limit HCV's genetic diversity and ability to recombine in vivo .

The term ‘Simulated Patient’ (SP) commonly refers to people trained to portray the role of a patient, relative, carer or co-worker in healthcare education. 1 This project aimed to develop, pilot and evaluate a bespoke, evidence-based training programme for Trainers and SPs in the North West of England. The regional SP Programme incorporates the ‘SP Train-The-Trainer’ (SP3T) and ‘Train-The-SP’ (2TSP) e-learning and workshops. A pragmatic, mixed-methods design facilitated a comprehensive exploration of the SP Programme. University ethical approval was obtained. Data collection methods included: A regional survey (n = 89 from 24 different organisations) Electronic evaluation questionnaire featuring open and closed questions; SP3T (n = 18) and 2TSP (n = 34) SP3T participant focus groups (n = 18) to explore course delivery methods and resources Usability testing of the SP Database and SP Passport in healthcare organisations (n = 5). The survey identified variances in SP terminology, recruitment, payment, contracts, risk assessment, training and quality assurance procedures. Survey findings were used to develop the SP Common Framework and SP3T package. 2 The SP3T package 2 was positively reviewed by all pilot participants (SP trainers). The depth of information and content was considered appropriate for novice and experienced simulation facilitators. The 2TSP package 3 was found to be stimulating, useful, flexible, and provided a good level of activity. SP Database testing confirmed usability in organisations with diverse IT infrastructures. The SP Passport was valued to track, record and monitor SP training and involvement. The SP Programme is now implemented throughout the region. The SP Common Framework and SP Programme are designed to empower simulation trainers to effectively work with SPs in education and training, thus promoting educational excellence. It is envisaged that engagement with well-trained SPs will improve education for the health and social care workforce and ultimately lead to improvements in patient safety and public engagement. References Nestel D, Bearman M. Chapter 1: Introduction to simulated patient methodology. In: Nestel D, Bearman M, eds. Simulated patient methodology: theory, evidence and practice. West Sussex: Wiley Blackwell, 2015: 1 –4. Gough S, Greene L, Nestel D, Hellaby M, MacKinnon R, Natali A, Roberts S, Tuttle N, Webster B. Simulated patients: a standardised, quality assured approach to training and implementation. Final Project Report. Manchester: Health Education North West, 2015. Greene L, Gough S. Simulated Patients: blending performing arts pedagogy and healthcare education. Final Project Report. Manchester: Health Education North West, 2015.

All cells possess signaling pathways designed to trigger antiviral responses, notably characterized by type I interferon (IFN) production, upon recognition of invading viruses. Especially, host sensors recognize viral nucleic acids. Nonetheless, virtually all viruses have evolved potent strategies that preclude host responses within the infected cells. The plasmacytoid dendritic cell (pDC) is an immune cell type known as a robust type I IFN producer in response to viral infection. Evidence suggests that such functionality of the pDCs participates in viral clearance. Nonetheless, their contribution, which is likely complex and varies depending on the pathogen, is still enigmatic for many viruses. pDCs are not permissive to most viral infections, and consistently, recent examples suggest that pDCs respond to immunostimulatory viral RNA transferred via noninfectious and/or noncanonical viral/cellular carriers. Therefore, the pDC response likely bypasses innate signaling blockages induced by virus within infected cells. Importantly, the requirement for cell-cell contact is increasingly recognized as a hallmark of the pDC-mediated antiviral state, triggered by evolutionarily divergent RNA viruses.

Background: Purulent skin and soft tissue infections (SSTIs) requiring medical attention are often managed in primary care. The prevalence of SSTIs caused by community-acquired Methicillin-resistant Staphylococcus aureus (CA-MRSA) has been increasing rapidly, including in otherwise healthy individuals. The Centers for Disease Control and Prevention (CDC) issued guidelines to improve the management of SSTIs in primary care. Purpose: In primary care settings, to assess the prevalence of CA-MRSA using an electronic chart audit and then evaluate SSTI management strategies consistent with CDC guidelines. Methods: A practical intervention that compared a historical cohort to an intervention cohort of patients seen for SSTI in 16 primary care practices in two health care systems. The intervention included a ready-made kit for I & D procedures, MRSA information for clinicians, a patient information handout, provider education, and patient follow-up. Results: A total of 3112 SSTI cases (cellulitis or purulent) were observed during the preintervention period and 1406 cases during the intervention. For purulent infections in the intervention period (n = 148), univariate and multivariate analyses showed no significant improvement in the rate of I & D procedures or cultures obtained but showed increased use of antibiotics overall and agents that typically cover MRSA strains (OR, 2.183; 95% CI, 1.443 to 3.303 and 2.624; 95% CI, 1.500 to 4.604, respectively). For infections that were cellulitis with or without purulence (n = 1258), overall rates in the use of antibiotics and those that cover MRSA increased significantly, but secular trends could not be ruled out as an explanation for this increase. Conclusion: In SSTIs, this intervention resulted in increased use of antibiotics, including antibiotics that typically cover MRSA strains, but did not demonstrate increased rates of recommended drainage procedures. It is replicable and portable, and may improve antibiotic selection in other settings.

The 3' maturation of chloroplast pre-mRNAs in Chlamydomonas proceeds via endonucleolytic cleavage, exonucleolytic trimming of the upstream cleavage product, and rapid degradation of the downstream moiety. However, the cis elements and trans factors remain to be characterized in detail. In the case of atpB, a 300 nucleotide processing determinant (PD), consisting of an inverted repeat (IR) and endonuclease cleavage site (ECS), directs 3' maturation. To further characterize the PD, 15 variants were examined in vivo in ectopic contexts. This revealed that the IR, and nucleotides 15-37 downstream of the ECS stimulate processing. A candidate trans factor for 3' maturation was subsequently functionally analyzed. This factor is encoded by the nuclear locus MCD4, and the mcd4 mutant was known to accumulate abnormally 3'-processed chloroplast mRNAs. When the mcd4 mutation was crossed into strains containing reporter genes with insertions of several PD versions, processing was reduced in some cases. This caused accumulation of RNA sequences downstream of the PD, which are normally degraded. From these data, it can be suggested that MCD4 facilitates the endonucleolytic cleavage step in 3' end maturation of atpB and perhaps other mRNAs, by interacting with the IR, RNA downstream of the IR, or with proteins bound there.

Lymph nodes grow rapidly and robustly at the initiation of an immune response, and this growth is accompanied by growth of the blood vessels. Although the vessels are critical for supplying nutrients and for controlling cell trafficking, the regulation of lymph node vascular growth is not well understood. We show that lymph node endothelial cells begin to proliferate within 2 d of immunization and undergo a corresponding expansion in cell numbers. Endothelial cell proliferation is dependent on CD11c+ dendritic cells (DCs), and the subcutaneous injection of DCs is sufficient to trigger endothelial cell proliferation and growth. Lymph node endothelial cell proliferation is dependent on vascular endothelial growth factor (VEGF), and DCs are associated with increased lymph node VEGF levels. DC-induced endothelial cell proliferation and increased VEGF levels are mediated by DC-induced recruitment of blood-borne cells. Vascular growth in the draining lymph node includes the growth of high endothelial venule endothelial cells and is functionally associated with increased cell entry into the lymph node. Collectively, our results suggest a scenario whereby endothelial cell expansion in the draining lymph node is induced by DCs as part of a program that optimizes the microenvironment for the ensuing immune response.

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3′ untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo .

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web., Author Summary Hepatitis C Virus (HCV) belongs to the Flaviviridae family, and is an enveloped, positive, single-stranded RNA virus containing a 9.6 kb genome. Plus-strand RNA viruses induce a highly regulated process of membrane rearrangements and novel vesicle formation in infected cells (the “membranous web” for HCV) to create a suitable environment for RNA replication as well as for the assembly and release of new virions. HCV assembly occurs close to lipid droplets (LDs), cell organelles involved in fat storage and utilization. The viral non-structural protein NS5A plays a critical role in both viral RNA replication that occurs within the membranous web and viral assembly at LDs. We identified the host protein TIP47 as a novel host interaction partner for NS5A. TIP47 is a LD-binding protein also known to function as cargo in late-endosome-to-Golgi vesicular transport. Our data support a model where the recruitment of TIP47 by NS5A is required for viral RNA replication, indicating that LDs play a previously unrecognized role not only in viral assembly but also in RNA replication.

Lymph node expansion during immune responses is accompanied by rapid vascular expansion. The re-establishment of quiescence and stabilization of the newly expanded vasculature and the regulatory mechanisms involved have not been well studied. We show that although initiation of vascular expansion in immune-stimulated nodes is associated with upregulated endothelial cell proliferation, increased high endothelial venule trafficking efficiency and VCAM-1 expression, and disrupted perivascular fibroblastic reticular cell organization, the re-establishment of vascular quiescence and stabilization postexpansion is characterized by reversal of these phenomena. Although CD11cmed cells are associated with the initiation of vascular expansion, CD11chiMHC class II (MHC II)med dendritic cells (DCs) accumulate later, and their short-term depletion in mice abrogates the re-establishment of vascular quiescence and stabilization. CD11chiMHC IImed cells promote endothelial cell quiescence in vitro and, in vivo, mediate quiescence at least in part by mediating reduced lymph node vascular endothelial growth factor. Disrupted vascular quiescence and stabilization in expanded nodes is associated with attenuated T cell-dependent B cell responses. These results describe a novel mechanism whereby CD11chiMHC IImed DCs regulate the re-establishment of vascular quiescence and stabilization after lymph node vascular expansion and suggest that these DCs function in part to orchestrate the microenvironmental alterations required for successful immunity.

Characterization of the earliest neuropathologic features of Alzheimer disease (AD) indicates that synaptic degeneration accompanied by tau hyperphosphorylation and amyloid deposition might be an important feature. The mechanisms involved are unclear; however, dysregulation of signaling cascades such as the extracellular signal-regulated kinase (ERK) pathway might play a role. In this context, the main objective of this study was to determine whether ERK hyperactivation occurs in early stages of AD. We compared the patterns of total and phosphorylated ERK (pERK) expression in the midfrontal cortex of patients clinically and neuropathologically characterized with early, intermediate, or advanced AD. Immunocytochemical and Western blot analysis showed that in early AD, there was extensive activation of ERK in astroglial cells in the white matter accompanied by intense astrogliosis. In contrast, in patients with more advanced AD, pERK immunoreactivity was associated with neuronal cell bodies and dystrophic neurites around plaques. Levels of astroglial pERK immunoreactivity in the white matter were strongly correlated with scores of cognitive performance (Blessed, Mini-Mental Status Examination, and Clinical Dementia Rating) and with the severity of AD neuropathology (Braak stage). These findings suggest that astroglial ERK activation may be an important early response to the onset of AD pathology. Identification of cell signaling events unique to early AD may provide therapeutic targets for the prevention or delay of dementia.

Antibiotic overuse in the primary care setting is common. Our objective was to evaluate the effect of a clinical pathway-based intervention on antibiotic use.Eight primary care clinics were randomized to receive clinical pathways for upper respiratory infection, acute bronchitis, acute rhinosinusitis, pharyngitis, acute otitis media, urinary tract infection, skin infections, and pneumonia and patient education materials (study group) versus no intervention (control group). Generalized linear mixed effects models were used to assess trends in antibiotic prescriptions for non-pneumonia acute respiratory infections and broad-spectrum antibiotic use for all 8 conditions during a 2-year baseline and 1-year intervention period.In the study group, antibiotic prescriptions for non-pneumonia acute respiratory infections decreased from 42.7% of cases at baseline to 37.9% during the intervention period (11.2% relative reduction) (P.0001) and from 39.8% to 38.7%, respectively, in the control group (2.8% relative reduction) (P=.25). Overall use of broad-spectrum antibiotics in the study group decreased from 26.4% to 22.6% of cases, respectively (14.4% relative reduction) (P.0001) and from 20.0% to 19.4%, respectively, in the control group (3.0% relative reduction) (P=.35). There were significant differences in the trends of prescriptions for acute respiratory infections (P.0001) and broad-spectrum antibiotic use (P=.001) between the study and control groups during the intervention period, with greater declines in the study group.This intervention was associated with declining antibiotic prescriptions for non-pneumonia acute respiratory infections and use of broad-spectrum antibiotics over the first year. Evaluation of the impact over a longer study period is warranted.

Lymph node expansion during immune responses is accompanied by rapid vascular expansion. The maturation of the newly expanded vasculature and the regulatory mechanisms involved have not been well studied. We show that while initiation of vascular expansion in immune-stimulated nodes is associated with upregulated endothelial cell proliferation, increased high endothelial venule trafficking efficiency and VCAM-1 expression, and disrupted perivascular fibroblastic reticular cell organization, the reestablishment of vascular quiescence and stabilization after expansion is characterized by reversal of these phenomena. While CD11c medium cells are associated with the initiation of vascular expansion, CD11c hi MHCII med dendritic cells accumulate later and their short-term depletion in mice abrogates the reestablishment of vascular quiescence and stabilization. CD11c hi MHCII med cells promote endothelial cell quiescence in vitro and, in vivo, mediate quiescence at least in part by mediating reduced lymph node VEGF. Disrupted vascular quiescence and stabilization in expanded nodes is associated with attenuated T cell-dependent B cell responses. These results describe a novel mechanism whereby CD11c hi MHCII med dendritic cells regulate the reestablishment of vascular quiescence and stabilization after lymph node vascular expansion and suggest that these dendritic cells function in part to orchestrate the microenvironmental alterations required for successful immunity.

Events occurring between the time of thermalization of electrons generated in radiolyzed water and the appearance of the fully developed absorption spectrum of e-aq were studied. First, the significance of including diffuse orbitals in ab-initio calculations on solvated electrons is illustrated by reference to H- and the structures of the oxonium family H3O+, H3O, H3O-. Five mechanisms proposed for the formation of e-sub aq/ were then examined: (a) formation via the radical intermediate H3O; (b) direct electron capture by a water molecule; (c) selective electron attachment, and attachment through a favorable vibrational mode of an n-mer; (d) attachment via the excited state H2O( 3B1); (e) attachment arising from medium interactions. Finally attention is directed to the structure of the hydrated electron.