Recommendations for accelerating open preprint peer review to improve the culture of science.

Peer review is an important part of the scientific process, but traditional peer review at journals is coming under increased scrutiny for its inefficiency and lack of transparency. As preprints become more widely used and accepted, they raise the possibility of rethinking the peer-review process. Preprints are enabling new forms of peer review that have the potential to be more thorough, inclusive, and collegial than traditional journal peer review, and to thus fundamentally shift the culture of peer review toward constructive collaboration. In this Consensus View, we make a call to action to stakeholders in the community to accelerate the growing momentum of preprint sharing and provide recommendations to empower researchers to provide open and constructive peer review for preprints.

T Cells in Atherosclerosis: Key Players in the Pathogenesis of Vascular Disease.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-rich plaques within arterial walls. T cells play a pivotal role in the pathogenesis of atherosclerosis in which they help orchestrate immune responses and contribute to plaque development and instability. Here, we discuss the recognition of atherosclerosis-related antigens that may trigger T cell activation together with additional signaling from co-stimulatory molecules and lesional cytokines. Although few studies have indicated candidates for the antigen specificity of T cells in atherosclerosis, further research is needed. Furthermore, we describe the pro-atherogenic and atheroprotective roles of diverse subsets of T cells such as CD4+ helper, CD8+ cytotoxic, invariant natural killer, and γδ T cells. To classify and quantify T cell subsets in atherosclerosis, we summarize current methods to analyze cellular heterogeneity including single cell RNA sequencing and T cell receptor (TCR) sequencing. Further insights into T cell biology will help shed light on the immunopathology of atherosclerosis, inform potential therapeutic interventions, and pave the way for precision medicine approaches in combating cardiovascular disease.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Social connection and gene regulation during the COVID-19 pandemic: Divergent patterns for online and in-person interaction.

BACKGROUND: Social connection has been linked to reduced disease risk and enhanced antiviral immunity, but it is unclear whether online social connections have similar effects to those previously documented for in-person/offline social relationships, or whether online connections can substitute for in-person social relations when the latter are restricted. We examined this question in the context of the COVID-19 pandemic, focusing specifically on an immune system gene regulation profile known as the conserved transcriptional response to adversity (CTRA), which is characterized by up-regulation of proinflammatory genes and down-regulation of genes linked to innate antiviral responses and antibody production. METHODS: We analyzed CTRA RNA profiles in blood samples from 142 healthy young adults (69% female, 87% white) during the "social distancing" period of the COVID-19 pandemic. Mixed effect linear models quantified the relation of CTRA gene expression to measures of in-person social connection (number of friends, social eudaimonia, loneliness) and online psychosocial connection (online loneliness, perceived social value in online leisure and educational contexts, and internet use) while controlling for demographic and health factors. RESULTS: Multiple indicators of in-person and generalized social connection were associated with lower CTRA gene expression, whereas no measure of online social connection showed any significant association with CTRA gene expression. CONCLUSION: Experiences of in-person social connection are associated with reduced CTRA gene expression during a period of restricted social interaction. In contrast, online social relationships show no such association. Digitally mediated social relations do not appear to substantially offset the absence of in-person/offline social connection in the context of immune cell gene regulation.

Use of protective antigen of Bacillus anthracis as a model recombinant antigen to evaluate toll-like receptors 2, 3, 4, 7 and 9 agonists in mice using established functional antibody assays, antigen-specific antibody assays and cellular assays.

Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax™), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M-052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.

Internet gaming, embodied distress, and psychosocial well-being: A syndemic-syndaimonic continuum.

We examine internet gaming-related suffering as a novel syndemic most prevalent among contemporary emerging adults. Synthetic analysis of our prior research on internet gaming and health affirms how social factors and mental and physical wellness mutually condition each other in this online play context. Employing biocultural anthropological mixed methods, we focus on statistical interactions between intensive gaming and social well-being in relation to genomic markers of immune function. We show that among gamers with low social well-being, intensive game play is associated with compromised immunity markers, but among those with robust social connection, that same play correlates with decreased activation of stress-related immunity activation. The apparently beneficial interaction of higher social well-being and intensive game play resonates with an emerging body of research showing how positive practices-in this case, engaged and pleasurable videogame play-can increase resilience to the negative linked psychological and genomic responses to precarity. Based on these findings, we argue, in relation to gaming behaviors, a syndemics analysis could usefully be expanded by attending to both sides of the synergistic interaction between two social conditions: not just exacerbation of dysfunction in relation to their combined effect, but also non-additive enhancement of health that may stem from such combinations. We draw on literature emphasizing the relevance to health of "eudaimonic" well-being-psychosocial processes that transcend immediate self-gratification and involve the pursuit of meaningful and pro-social goals. On that basis, we propose the term "syndaimonics" to capture synergies between social context and mental flourishing, which, in this context and presumably others, can illuminate sources of health resilience and overall improved psychosocial wellbeing.

Influenza Antigens NP and M2 Confer Cross Protection to BALB/c Mice against Lethal Challenge with H1N1, Pandemic H1N1 or H5N1 Influenza A Viruses.

Influenza hemagglutinin (HA) is considered a major protective antigen of seasonal influenza vaccine but antigenic drift of HA necessitates annual immunizations using new circulating HA versions. Low variation found within conserved non-HA influenza virus (INFV) antigens may maintain protection with less frequent immunizations. Conserved antigens of influenza A virus (INFV A) that can generate cross protection against multiple INFV strains were evaluated in BALB/c mice using modified Vaccinia virus Ankara (MVA)-vectored vaccines that expressed INFV A antigens hemagglutinin (HA), matrix protein 1 (M1), nucleoprotein (NP), matrix protein 2 (M2), repeats of the external portion of M2 (M2e) or as tandem repeats (METR), and M2e with transmembrane region and cytoplasmic loop (M2eTML). Protection by combinations of non-HA antigens was equivalent to that of subtype-matched HA. Combinations of NP and forms of M2e generated serum antibody responses and protected mice against lethal INFV A challenge using PR8, pandemic H1N1 A/Mexico/4108/2009 (pH1N1) or H5N1 A/Vietnam/1203/2004 (H5N1) viruses, as demonstrated by reduced lung viral burden and protection against weight loss. The highest levels of protection were obtained with NP and M2e antigens delivered as MVA inserts, resulting in broadly protective immunity in mice and enhancement of previous natural immunity to INFV A.

Evaluation of the AV7909 Anthrax Vaccine Toxicity in Sprague Dawley Rats Following Three Intramuscular Administrations.

AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

Cell-specific and divergent roles of the CD40L-CD40 axis in atherosclerotic vascular disease.

Atherosclerosis is a major underlying cause of cardiovascular disease. Previous studies showed that inhibition of the co-stimulatory CD40 ligand (CD40L)-CD40 signaling axis profoundly attenuates atherosclerosis. As CD40L exerts multiple functions depending on the cell-cell interactions involved, we sought to investigate the function of the most relevant CD40L-expressing cell types in atherosclerosis: T cells and platelets. Atherosclerosis-prone mice with a CD40L-deficiency in CD4+ T cells display impaired Th1 polarization, as reflected by reduced interferon-γ production, and smaller atherosclerotic plaques containing fewer T-cells, smaller necrotic cores, an increased number of smooth muscle cells and thicker fibrous caps. Mice with a corresponding CD40-deficiency in CD11c+ dendritic cells phenocopy these findings, suggesting that the T cell-dendritic cell CD40L-CD40 axis is crucial in atherogenesis. Accordingly, sCD40L/sCD40 and interferon-γ concentrations in carotid plaques and plasma are positively correlated in patients with cerebrovascular disease. Platelet-specific deficiency of CD40L does not affect atherogenesis but ameliorates atherothrombosis. Our results establish divergent and cell-specific roles of CD40L-CD40 in atherosclerosis, which has implications for therapeutic strategies targeting this pathway.

Immunoinflammatory, Thrombohaemostatic, and Cardiovascular Mechanisms in COVID-19.

The global coronavirus disease 2019 (COVID-19) pandemic has deranged the recent history of humankind, afflicting more than 27 million individuals to date. While the majority of COVID-19 patients recuperate, a considerable number of patients develop severe complications. Bilateral pneumonia constitutes the hallmark of severe COVID-19 disease but an involvement of other organ systems, namely the cardiovascular system, kidneys, liver, and central nervous system, occurs in at least half of the fatal COVID-19 cases. Besides respiratory failure requiring ventilation, patients with severe COVID-19 often display manifestations of systemic inflammation and thrombosis as well as diffuse microvascular injury observed postmortem. In this review, we survey the mechanisms that may explain how viral entry and activation of endothelial cells by severe acute respiratory syndrome coronavirus 2 can give rise to a series of events including systemic inflammation, thrombosis, and microvascular dysfunction. This pathophysiological scenario may be particularly harmful in patients with overt cardiovascular disease and may drive the fatal aspects of COVID-19. We further shed light on the role of the renin-angiotensin aldosterone system and its inhibitors in the context of COVID-19 and discuss the potential impact of antiviral and anti-inflammatory treatment options. Acknowledging the comorbidities and potential organ injuries throughout the course of severe COVID-19 is crucial in the clinical management of patients affecting treatment approaches and recovery rate.

Glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) drives atherosclerosis in mice and is associated with an unstable plaque phenotype and cerebrovascular events in humans.

AIMS: GITR-a co-stimulatory immune checkpoint protein-is known for both its activating and regulating effects on T-cells. As atherosclerosis bears features of chronic inflammation and autoimmunity, we investigated the relevance of GITR in cardiovascular disease (CVD). METHODS AND RESULTS: GITR expression was elevated in carotid endarterectomy specimens obtained from patients with cerebrovascular events (n = 100) compared to asymptomatic patients (n = 93) and correlated with parameters of plaque vulnerability, including plaque macrophage, lipid and glycophorin A content, and levels of interleukin (IL)-6, IL-12, and C-C-chemokine ligand 2. Soluble GITR levels were elevated in plasma from subjects with CVD compared to healthy controls. Plaque area in 28-week-old Gitr-/-Apoe-/- mice was reduced, and plaques had a favourable phenotype with less macrophages, a smaller necrotic core and a thicker fibrous cap. GITR deficiency did not affect the lymphoid population. RNA sequencing of Gitr-/-Apoe-/- and Apoe-/- monocytes and macrophages revealed altered pathways of cell migration, activation, and mitochondrial function. Indeed, Gitr-/-Apoe-/- monocytes displayed decreased integrin levels, reduced recruitment to endothelium, and produced less reactive oxygen species. Likewise, GITR-deficient macrophages produced less cytokines and had a reduced migratory capacity. CONCLUSION: Our data reveal a novel role for the immune checkpoint GITR in driving myeloid cell recruitment and activation in atherosclerosis, thereby inducing plaque growth and vulnerability. In humans, elevated GITR expression in carotid plaques is associated with a vulnerable plaque phenotype and adverse cerebrovascular events. GITR has the potential to become a novel therapeutic target in atherosclerosis as it reduces myeloid cell recruitment to the arterial wall and impedes atherosclerosis progression.

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis.

Actin dynamics generate forces to deform the membrane and overcome the cell's high turgor pressure during clathrin-mediated endocytosis (CME) in yeast, but precise molecular details are still unresolved. Our previous models predicted that actin filaments of the endocytic meshwork continually polymerize and disassemble, turning over multiple times during an endocytic event, similar to other actin systems. We applied single-molecule speckle tracking in live fission yeast to directly measure molecular turnover within CME sites for the first time. In contrast with the overall ~20 s lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 s, and similarly high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins' motions. These results indicate that endocytic proteins turn over up to five times during the formation of an endocytic vesicle, and suggest revising quantitative models of force production.

Positive mental well-being and immune transcriptional profiles in highly involved videogame players.

Previous research has identified a link between experiencing life as meaningful and purposeful-what is referred to as "eudaimonia"-and reduced expression of a stress-induced gene profile known as the "conserved transcriptional response to adversity" (CTRA). In the current study, we examine whether similar links between eudaimonic well-being and CTRA reduction occur in a sample of 56 individuals with a particularly strong engagement with virtual worlds: avid online videogame players. Results consistently linked higher eudaimonic well-being, and more specifically the social well-being subdomain of eudaimonia, to lower levels of CTRA gene expression. That favorable psychobiological relationship between eudaimonia and CTRA appeared most strongly among individuals reporting high levels of positive psychosocial involvement with gaming. Findings are consistent with the hypothesis that committed social/recreational activity may help damp CTRA expression especially among persons who are already experiencing some kind of threshold of positive eudaimonic experience.

Repeat-Dose Toxicity Study of a Lyophilized Recombinant Protective Antigen-Based Anthrax Vaccine Adjuvanted With CpG 7909.

A recombinant protective antigen (rPA) anthrax vaccine candidate (rPA7909) was developed as a next-generation vaccine indicated for postexposure prophylaxis of disease resulting from suspected or confirmed Bacillus anthracis exposure. The lyophilized form of rPA7909-vaccinated candidate contains 75 µg purified rPA, 750 µg aluminum (as Alhydrogel adjuvant), and 250 µg of an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide CpG 7909 in a 0.5 mL phosphate-buffered suspension. General toxicity and local reactogenicity were evaluated in Sprague Dawley rats vaccinated with the full human dose of rPA7909 by intramuscular injection. Animals were immunized on study days 1, 15, and 29. Control groups were administered diluent only or adjuvant control (excipients, CpG 7909, and Alhydrogel adjuvant in diluent) intramuscularly at the same dose volume and according to the same schedule used for rPA7909. Toxicity was assessed based on the results of clinical observations, physical examinations, body weights, injection site reactogenicity, ophthalmology, clinical pathology (hematology, coagulation, and serum chemistry), organ weights, and macroscopic and microscopic pathology evaluation. The immune response to rPA7909 vaccination was confirmed by measuring serum anti-PA immunoglobulin G levels. The rPA7909 vaccine produced no apparent systemic toxicity and only transient reactogenicity at the injection site. The injection site reaction from animals receiving the adjuvant control was very similar to those receiving rPA7909 with respect to the inflammation. The inflammatory response observed in the injection site and the draining lymph nodes was consistent with expected immune stimulation. The overall results indicated a favorable safety profile for rPA7909.

The cross-cultural expression of internet gaming distress in North America, Europe, and China.

We compare the forms online gaming-related distress takes cross-culturally, and examine how much such distress resembles the World Health Organization's (WHO) "Gaming disorder," understood to be an "addiction." Our preliminary exploratory factor analysis (EFA) in North America (n = 2025), Europe (n = 1198), and China (n = 841) revealed a constant four-factor structure across the three regions, with classic "addiction" symptoms always clustering together on the first and most important factor, though with some variability in regional factors' exact item composition. In the present study, we use second-order confirmatory factor analysis (CFA) to further examine this factor structure and the cultural similarities and differences. Specifically, we focus on confirming the regional structure and composition of an ethnographically developed 21-item gaming distress scale, which contains a wider symptoms pool than typical gaming disorder scales, and thus allows us to better separate generalized gaming distress's "addictive" from other culturally-influenced "problem" experiences and behaviors in each regional case. We use propensity score matching to separate the impact on gaming-related distress of regional culture from demographic variables (North America/Europe: n = 1043 pairs; North America/China: n = 535 pairs). Although our results support current WHO formulations of gaming-related distress as an addictive disorder, we show how cultural forces can shape how "addictive" and "problem" gaming are experienced and thus psychiatrically presented in different parts of the world. In particular, generalized gaming distress's addictive and problematic dimensions seem to be shaped by culture-specific expressions of achievement motivations, social connection and disconnection, and unique psychosomatic experiences.

PD-L1 expression on nonclassical monocytes reveals their origin and immunoregulatory function.

The role of nonclassical monocytes (NCMs) in health and disease is emerging, but their location and function within tissues remain poorly explored. Imaging of NCMs has been limited by the lack of an established single NCM marker. Here, we characterize the immune checkpoint molecule PD-L1 (CD274) as an unequivocal marker for tracking NCMs in circulation and pinpoint their compartmentalized distribution in tissues by two-photon microscopy. Visualization of PD-L1+ NCMs in relation to bone marrow vasculature reveals that conversion of classical monocytes into NCMs requires contact with endosteal vessels. Furthermore, PD-L1+ NCMs are present in tertiary lymphoid organs (TLOs) under inflammatory conditions in both mice and humans, and NCMs exhibit a PD-L1-dependent immunomodulatory function that promotes T cell apoptosis within TLOs. Our findings establish an unambiguous tool for the investigation of NCMs and shed light on their origin and function.

Amino Acids and Their Metabolism in Atherosclerosis.

As a leading cause of death worldwide, cardiovascular disease is a global health concern. The development and progression of atherosclerosis, which ultimately gives rise to cardiovascular disease, has been causally linked to hypercholesterolemia. Mechanistically, the interplay between lipids and the immune system during plaque progression significantly contributes to the chronic inflammation seen in the arterial wall during atherosclerosis. Localized inflammation and increased cell-to-cell interactions may influence polarization and proliferation of immune cells via changes in amino acid metabolism. Specifically, the amino acids l-arginine (Arg), l-homoarginine (hArg) and l-tryptophan (Trp) have been widely studied in the context of cardiovascular disease, and their metabolism has been established as key regulators of vascular homeostasis, as well as immune cell function. Cyclic effects between endothelial cells, innate, and adaptive immune cells exist during Arg and hArg, as well as Trp metabolism, that may have distinct effects on the development of atherosclerosis. In this review, we describe the current knowledge surrounding the metabolism, biological function, and clinical perspective of Arg, hArg, and Trp in the context of atherosclerosis.