Dynamic activity in cis-regulatory elements of leukocytes identifies transcription factor activation and stratifies COVID-19 severity in ICU patients.

Transcription factor programs mediating the immune response to coronavirus disease 2019 (COVID-19) are not fully understood. Capturing active transcription initiation from cis-regulatory elements such as enhancers and promoters by capped small RNA sequencing (csRNA-seq), in contrast to capturing steady-state transcripts by conventional RNA-seq, allows unbiased identification of the underlying transcription factor activity and regulatory pathways. Here, we profile transcription initiation in critically ill COVID-19 patients, identifying transcription factor motifs that correlate with clinical lung injury and disease severity. Unbiased clustering reveals distinct subsets of cis-regulatory elements that delineate the cell type, pathway-specific, and combinatorial transcription factor activity. We find evidence of critical roles of regulatory networks, showing that STAT/BCL6 and E2F/MYB regulatory programs from myeloid cell populations are activated in patients with poor disease outcomes and associated with COVID-19 susceptibility genetic variants. More broadly, we demonstrate how capturing acute, disease-mediated changes in transcription initiation can provide insight into the underlying molecular mechanisms and stratify patient disease severity.

Highly mutated antibodies capable of neutralizing N276 glycan-deficient HIV after a single immunization with an Env trimer.

Elicitation of HIV broadly neutralizing antibodies (bnAbs) is challenging because unmutated bnAb precursors are rare and seldom bind HIV envelope glycoprotein (Env) trimers. One strategy to initiate bnAb responses is to use germline-targeting (GT) immunogens with high affinity to bnAb-class precursor B cells and then shepherd affinity maturation with booster immunogens that successively look more like native Env. In a mouse model where the frequency of VRC01-precursor (VRC01gHL) B cells mimics that of humans, we show that following a GT HIV Env trimer protein prime, VRC01-class B cells in the germinal center (GC) acquire high-affinity VRC01-class B cell somatic hypermutations (SHMs). Many GC-derived VRC01gHL antibodies robustly bind N276 glycan-deficient Env trimers and neutralize several N276 glycan-deficient tier 2 HIV strains. These results are encouraging for GT Env trimer vaccine designs and demonstrate accumulation of substantial SHMs, including deletions, uncommon point mutations, and functional bnAb features, after a single immunization.

Severity of Daytime Sleepiness and Parkinsonian-Like Symptoms in Korean Adults Aged 50-64 Years.

BACKGROUND AND PURPOSE: While excessive daytime sleepiness can predate Parkinson's disease in late-life, its association with parkinsonian-like (P-L) symptoms in middle age are unknown. Since neurodegeneration can appear decades before a diagnosis of Parkinson's disease, identifying clinical features associated with this early progression is important. The purpose of this study was to determine the association of daytime sleepiness with P-L symptoms in a population-based sample of middle-aged Korean adults. METHODS: During 2013 and 2014, daytime sleepiness and P-L symptoms were assessed in 2,063 males and females aged 50-64 years who were participating in the Korean Genome and Epidemiology Study. The severity of daytime sleepiness was quantified by the score on the Epworth Sleepiness Scale (ESS). Self-reported P-L symptoms included nine motor disorders commonly associated with Parkinson's disease. Participants with parkinsonism and related conditions are excluded. RESULTS: The prevalence of excessive daytime sleepiness (ESS score >10) was 7.0%. The frequencies of P-L symptoms ranged from 0.5% (for "trouble buttoning buttons") to 18.4% (for "handwriting smaller than it once was"). After adjustment for covariates and multiple testing, the relative odds of P-L symptoms comparing the 80th and 20th percentiles of ESS scores was 1.6 (p=0.001) for "voice is softer than it once was," 2.1 (p<0.001) for "balance when walking is poor," and 1.5 (p=0.002) for "loss of facial expression." The prevalence of excessive daytime sleepiness increased from 6.3% to 19.8% when the number of symptoms increased from zero to three (p=0.004). CONCLUSIONS: In Korean adults aged 50-64 years, daytime sleepiness is significantly associated with P-L symptoms. Whether coexisting daytime sleepiness and P-L symptoms predate extrapyramidal and other impairments in later life warrants further investigation.

Sleep Duration, Sleep Apnea, and Gray Matter Volume.

This study aimed to evaluate the effect of sleep duration on brain structures in the presence versus absence of sleep apnea in middle-aged and older individuals. The study investigated a population-based sample of 2,560 individuals, aged 49-80 years. The presence of sleep apnea and self-reported sleep duration were examined in relation to gray matter volume (GMV) in total and lobar brain regions. We identified ranges of sleep duration associated with maximal GMV using quadratic regression and bootstrap sampling. A significant quadratic association between sleep duration and GMV was observed in total and lobar brain regions of men with sleep apnea. In the fully adjusted model, optimal sleep durations associated with peak GMV between brain regions ranged from 6.7 to 7.0 hours. Shorter and longer sleep durations were associated with lower GMV in total and 4 sub-regions of the brain in men with sleep apnea.

Increased Peripheral Blood Neutrophil Activation Phenotypes and Neutrophil Extracellular Trap Formation in Critically Ill Coronavirus Disease 2019 (COVID-19) Patients: A Case Series and Review of the Literature.

BACKGROUND: Increased inflammation has been well defined in coronavirus disease 2019 (COVID-19), while definitive pathways driving severe forms of this disease remain uncertain. Neutrophils are known to contribute to immunopathology in infections, inflammatory diseases, and acute respiratory distress syndrome, a primary cause of morbidity and mortality in COVID-19. Changes in neutrophil function in COVID-19 may give insight into disease pathogenesis and identify therapeutic targets. METHODS: Blood was obtained serially from critically ill COVID-19 patients for 11 days. Neutrophil extracellular trap formation (NETosis), oxidative burst, phagocytosis, and cytokine levels were assessed. Lung tissue was obtained immediately postmortem for immunostaining. PubMed searches for neutrophils, lung, and COVID-19 yielded 10 peer-reviewed research articles in English. RESULTS: Elevations in neutrophil-associated cytokines interleukin 8 (IL-8) and interleukin 6, and general inflammatory cytokines IFN-inducible protien-19, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin 1ß, interleukin 10, and tumor necrosis factor, were identified both at first measurement and across hospitalization (P < .0001). COVID-19 neutrophils had exaggerated oxidative burst (P < .0001), NETosis (P < .0001), and phagocytosis (P < .0001) relative to controls. Increased NETosis correlated with leukocytosis and neutrophilia, and neutrophils and NETs were identified within airways and alveoli in lung parenchyma of 40% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected lungs available for examination (2 of 5). While elevations in IL-8 and absolute neutrophil count correlated with disease severity, plasma IL-8 levels alone correlated with death. CONCLUSIONS: Literature to date demonstrates compelling evidence of increased neutrophils in the circulation and lungs of COVID-19 patients. Importantly, neutrophil quantity and activation correlates with severity of disease. Similarly, our data show that circulating neutrophils in COVID-19 exhibit an activated phenotype with enhanced NETosis and oxidative burst.

Profiling Transcription Initiation in Peripheral Leukocytes Reveals Severity-Associated Cis-Regulatory Elements in Critical COVID-19.

The contribution of transcription factors (TFs) and gene regulatory programs in the immune response to COVID-19 and their relationship to disease outcome is not fully understood. Analysis of genome-wide changes in transcription at both promoter-proximal and distal cis-regulatory DNA elements, collectively termed the 'active cistrome,' offers an unbiased assessment of TF activity identifying key pathways regulated in homeostasis or disease. Here, we profiled the active cistrome from peripheral leukocytes of critically ill COVID-19 patients to identify major regulatory programs and their dynamics during SARS-CoV-2 associated acute respiratory distress syndrome (ARDS). We identified TF motifs that track the severity of COVID- 19 lung injury, disease resolution, and outcome. We used unbiased clustering to reveal distinct cistrome subsets delineating the regulation of pathways, cell types, and the combinatorial activity of TFs. We found critical roles for regulatory networks driven by stimulus and lineage determining TFs, showing that STAT and E2F/MYB regulatory programs targeting myeloid cells are activated in patients with poor disease outcomes and associated with single nucleotide genetic variants implicated in COVID-19 susceptibility. Integration with single-cell RNA-seq found that STAT and E2F/MYB activation converged in specific neutrophils subset found in patients with severe disease. Collectively we demonstrate that cistrome analysis facilitates insight into disease mechanisms and provides an unbiased approach to evaluate global changes in transcription factor activity and stratify patient disease severity.

Association of pericardial adipose tissue with left ventricular structure and function: a region-specific effect?

BACKGROUND: The independent role of pericardial adipose tissue (PAT) as an ectopic fat associated with cardiovascular disease (CVD) remains controversial. This study aimed to determine whether PAT is associated with left ventricular (LV) structure and function independent of other markers of general obesity. METHODS: We studied 2471 participants (50.9 % women) without known CVD from the Korean Genome Epidemiology Study, who underwent 2D-echocardiography with tissue Doppler imaging (TDI) and computed tomography measurement for PAT. RESULTS: Study participants with more PAT were more likely to be men and had higher cardiometabolic indices, including blood pressure, glucose, and cholesterol levels (all P < 0.001). Greater pericardial fat levels across quartiles of PAT were associated with increased LV mass index and left atrial volume index (all P < 0.001) and decreased systolic (P = 0.015) and early diastolic (P < 0.001) TDI velocities, except for LV ejection fraction. These associations remained after a multivariable-adjusted model for traditional CV risk factors and persisted even after additional adjustment for general adiposity measures, such as waist circumference and body mass index. PAT was also the only obesity index independently associated with systolic TDI velocity (P < 0.001). CONCLUSIONS: PAT was associated with subclinical LV structural and functional deterioration, and these associations were independent of and stronger than with general and abdominal obesity measures.

A longitudinal observational population-based study of brain volume associated with changes in sleep timing from middle to late-life.

STUDY OBJECTIVES: Sleep behaviors are related to brain structure and function, but the impact of long-term changes in sleep timing on brain health has not been clearly addressed. The purpose of this study was to examine the association of longitudinal changes in sleep timing from middle to late-life with gray matter volume (GMV), an important marker of brain aging. METHODS: We enrolled 1798 adults (aged 49-82 years, men 54.6%) who underwent magnetic resonance imaging (MRI) between 2011 and 2014. Midsleep time (MST) on free days corrected for sleep debt on workdays was adopted as a marker of sleep timing. Data on MST were available at the time of MRI assessment and at examinations that were given 9 years earlier (2003-2004). Longitudinal changes in MST over the 9-year period were derived and categorized into quartiles. Subjects in quartile 1 were defined as "advancers" (MST advanced ≥ 1 h) while those in quartile 4 were defined as "delayers" (MST delayed ≥ 0.2 h). Quartiles 2-3 defined a reference group (MST change was considered modest). The relationship of GMV with MST changes over 9 years was investigated. RESULTS: Nine-year change in MST were significantly associated with GMV. Compared to the reference group, advancers had smaller GMVs in the frontal and temporal regions. A delay in MST was also associated with smaller cerebellar GMV. CONCLUSIONS: In middle-to-late adulthood, the direction of change in MST is associated with GMV. While advancers and delayers in MST tend to present lower GMV, associations appear to differ across brain regions.

Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity.

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4+ and CD8+ T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4+ and CD8+ T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4+ and CD8+ T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4+ T cell, CD8+ T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.

B cells expressing authentic naive human VRC01-class BCRs can be recruited to germinal centers and affinity mature in multiple independent mouse models.

Animal models of human antigen-specific B cell receptors (BCRs) generally depend on "inferred germline" sequences, and thus their relationship to authentic naive human B cell BCR sequences and affinities is unclear. Here, BCR sequences from authentic naive human VRC01-class B cells from healthy human donors were selected for the generation of three BCR knockin mice. The BCRs span the physiological range of affinities found in humans, and use three different light chains (VK3-20, VK1-5, and VK1-33) found among subclasses of naive human VRC01-class B cells and HIV broadly neutralizing antibodies (bnAbs). The germline-targeting HIV immunogen eOD-GT8 60mer is currently in clinical trial as a candidate bnAb vaccine priming immunogen. To attempt to model human immune responses to the eOD-GT8 60mer, we tested each authentic naive human VRC01-class BCR mouse model under rare human physiological B cell precursor frequency conditions. B cells with high (HuGL18HL) or medium (HuGL17HL) affinity BCRs were primed, recruited to germinal centers, and they affinity matured, and formed memory B cells. Precursor frequency and affinity interdependently influenced responses. Taken together, these experiments utilizing authentic naive human VRC01-class BCRs validate a central tenet of germline-targeting vaccine design and extend the overall concept of the reverse vaccinology approach to vaccine development.

Multifaceted Effects of Antigen Valency on B Cell Response Composition and Differentiation In Vivo.

How antigen valency affects B cells in vivo during immune responses is not well understood. Here, using HIV immunogens with defined valencies ranging from 1 to 60, we investigated the role of antigen valency during different phases of B cell responses in vivo. Highly multimerized immunogens preferentially rapidly activated cognate B cells, with little affinity discrimination. This led to strong early induction of the transcription factors IRF4 (interferon regulatory factor 4) and Bcl6, driving both early extrafollicular plasma cell and germinal center responses, in a CD4+ T-cell-dependent manner, involving B cells with a broad range of affinities. Low-valency antigens induced smaller effector B cell responses, with preferential recruitment of high-affinity B cells. Thus, antigen valency has multifaceted effects on B cell responses and can dictate affinity thresholds and competitive landscapes for B cells in vivo, with implications for vaccine design.

Efficacy of a Multimodal Online Lifestyle Intervention for Depressive Symptoms and Quality of Life in Individuals With a History of Major Depressive Disorder.

Background Major depressive disorder (MDD) is a complex bio-psycho-social syndrome that affects millions of individuals and is one of the leading causes of impaired quality of life (QOL). In addition to the symptoms of depression and low mood, many individuals with MDD also suffer from isolation without the sense of a supportive, surrounding community. Given the challenges of treating individuals with MDD, social isolation and a lack of communal connection, this randomized controlled trial was designed to determine the efficacy of a multimodal, online and community-based lifestyle intervention for improving depressive symptoms and QOL in individuals with a history of MDD. Materials and methods The study enrolled 71 female or male participants between the ages of 20 and 64 with a self-reported BMI between 18.4 and 34.9 kg/m2 and a history of MDD. Individuals were randomized to either participate in a 44-day multimodal, online, community-based lifestyle intervention or placed on a wait list where they would complete the intervention at a later date. The multimodal intervention involved a self-directed learning program where individuals were guided to make lifestyle changes including adopting a whole-foods diet, increasing movement, and adopting stress management and mindfulness practices. All participants completed the 36-Item Short Form Health Survey (SF-36), the Cleveland Clinic Center for Functional Medicine's Medical Symptoms Questionnaire (MSQ), and the Patient Health Questionnaire-9 (PHQ-9) before and after the online program to assess health-related QOL, overall symptom burden, and depressive symptom burden, respectively.  Results A total of 37 participants were randomized to participate in the multimodal intervention with 26 completing all three study questionnaires at both study time points; 34 participants were randomized to the wait list control group with 27 completing all three study questionnaires at both study time points. There were no clinically or statistically significant differences between the control group or the intervention group at baseline. The control group showed no clinically nor statistically significant changes in the MSQ, PHQ-9 or any of the eight subdomains of the SF-36 from the beginning to the end of the 10-week study period. When compared to the control group, the intervention group showed statistically and clinically significant improvements in median (M) scores of the SF-36 subdomains of vitality and mental health, and clinically but not statistically significant improvements in the subdomain of emotional role functioning. There were additional statistically and clinically significant improvements in the mean score of the MSQ and M scores of the PHQ-9 (treatment pre-intervention M = 10.5, inter-quartile range [IQR] = 14, to treatment post-intervention M = 5, IQR = 8.25; control pre-intervention M = 15, IQR = 8, to control post-intervention M = 13.5, IQR = 12.5). Conclusions  Our randomized controlled study provides evidence for the role of a multimodal, online and community-based lifestyle intervention to improve depressive symptoms, QOL, and total symptom burden in individuals with a history of MDD. Given the growing challenges of effectively supporting individuals suffering with MDD, it appears critical to further explore the utilization of novel, multimodal and self-directed lifestyle interventions.

Factors in B cell competition and immunodominance.

The majority of all vaccines work by inducing protective antibody responses. The mechanisms by which the B cells responsible for producing protective antibodies are elicited to respond are not well understood. Interclonal B cell competition to complex antigens, particularly in germinal centers, has emerged as an important hurdle in designing effective vaccines. This review will focus on recent advances in understanding the roles of B cell precursor frequency, B cell receptor affinity for antigen, antigen avidity, and other factors that can substantially alter the outcomes of B cell responses to complex antigens. Understanding the interdependence of these fundamental factors that affect B cell responses can inform current vaccine design efforts for pathogens with complex proteins as candidate immunogens such as HIV, influenza, and coronaviruses.

Rapid isolation of potent SARS-CoV-2 neutralizing antibodies and protection in a small animal model.

The development of countermeasures to prevent and treat COVID-19 is a global health priority. In under 7 weeks, we enrolled a cohort of SARS-CoV-2-recovered participants, developed neutralization assays to interrogate serum and monoclonal antibody responses, adapted our high throughput antibody isolation, production and characterization pipeline to rapidly screen over 1000 antigen-specific antibodies, and established an animal model to test protection. We report multiple highly potent neutralizing antibodies (nAbs) and show that passive transfer of a nAb provides protection against high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs define protective epitopes to guide vaccine design.

Outcomes of Childhood Preventive Intervention Across 2 Generations: A Nonrandomized Controlled Trial.

Importance: Trials of preventive interventions for children that were implemented in the 1980s have reported sustained positive outcomes on behavioral and health outcomes into adulthood, years after the end of the intervention. This present study examines whether intervention in childhood may show sustained benefits across generations. Objective: To examine possible intervention outcomes on the offspring of individuals (now parents) who participated in the Raising Healthy Children preventive intervention as children in the elementary grades. Design, Setting, and Participants: This nonrandomized controlled trial was conducted in public elementary schools serving high-crime areas in Seattle, Washington. The panel originated in Seattle but was followed up locally and in out-of-state locations over time. Data analyzed in this study were collected from September 1980 to June 2011, with follow-up of the firstborn offspring (aged 1 through 22 years) of 182 parents who had been in the full intervention vs control conditions in childhood. Their children were assessed across 7 waves in 2 blocks (2002-2006 and 2009-2011). Data were analyzed for this article from September 2018 through January 2019. Interventions: In grades 1 through 6, the Raising Healthy Children intervention provided elementary school teachers with methods of classroom management and instruction, first-generation (G1) parents with skills to promote opportunities for children's active involvement in the classroom and family, and second-generation (G2) child with social and emotional skills training. Main Outcomes and Measures: Outcomes examined in the third-generation (G3) offspring were self-regulation (emotion, attention, and behavioral regulation), cognitive capabilities, and social capabilities. Risk behaviors, including substance use and delinquency, were examined from age 6 years to study completion. Early onset of sexual activity was examined from age 13 years to study completion. Intent-to-treat analyses controlled for potential confounding factors. Results: A total of 182 G3 children were included in this analysis (72 in the full intervention and 110 in the control condition; mean age at first wave of data collection, 7 [range, 1-13] years). Significant differences in the offspring of intervention parents were observed across 4 domains: improved early child developmental functioning (ages 1-5 years; significant standardized ß range, 0.45-0.56), lower teacher-rated behavioral problems (ages 6-18 years; significant standardized ß range, -0.39 to -0.46), higher teacher-rated academic skills and performance (ages 6-18 years; significant standardized ß range, 0.34-0.49), and lower child-reported risk behavior (ages 6-18 years; odds ratio for any drug use [alcohol, cigarettes, or marijuana], 0.27 [95% CI, 0.10-0.73]). Conclusions and Relevance: To our knowledge, this is the first study to report significant intervention differences in the offspring of participants in a universal childhood preventive intervention. Cost-benefit analyses have examined the benefits of childhood intervention in the target generation. The present study suggests that additional benefits can be realized in the next generation as well. Trial Registration: ClinicalTrials.gov Identifier: NCT04075019.

Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model.

Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained weight and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.

Neck Circumference and Cerebral Gray Matter Volume.

BACKGROUND: While obesity is linked with brain atrophy and dementia incidence, associations with regional adiposity are uncertain. Our goal was to determine whether cerebral gray matter volume is related to neck circumference (NC), a measure of regional adiposity having unique relationships with metabolic disorders and cardiovascular disease. METHODS: Magnetic resonance imaging and NC were cross-sectionally assessed from 2011 to 2014 in a population-based sample of 2916 men and women in the Korean Genome and Epidemiology Study. RESULTS: For men, NC was inversely associated with total and regional gray matter in the frontal, temporal, and occipital lobes after adjusting for age and intracranial brain volume. Associations were especially strong in the presence of diabetes. With further adjustment for indices of body composition and other characteristics, total and frontal gray matter in diabetic men were lowered by 6.1 mL (95% confidence interval: 2.5-9.7, P=0.004) and 2.9 mL (95% confidence interval: 1.0-4.9, P=0.017), respectively, per SD increase in NC (2.3 cm). For men without diabetes, and in all women, associations were less apparent. CONCLUSIONS: In men with diabetes, NC was inversely associated with cerebral gray matter volume. The link between neck anthropometry and brain aging in diabetic men warrants further consideration.

Author Correction: A Engineered immunogen binding to alum adjuvant enhances humoral immunity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.