Pathogenic differences of cynomolgus macaques after Taï Forest virus infection depend on the viral stock propagation.

Taï Forest virus (TAFV) is a negative-sense RNA virus in the Filoviridae family. TAFV has caused only a single human infection, but several disease outbreaks in chimpanzees have been linked to this virus. Limited research has been done on this human-pathogenic virus. We sought to establish an animal model to assess TAFV disease progression and pathogenicity at our facility. We had access to two different viral stock preparations from different institutions, both originating from the single human case. Type I interferon receptor knockout mice were inoculated with TAFV stock 1 or stock 2 by the intraperitoneal route. Inoculation resulted in 100% survival with no disease regardless of viral stock preparation or infectious dose. Next, cynomolgus macaques were inoculated with TAFV stock 1 or stock 2. Inoculation with TAFV stock 1 resulted in 100% survival and robust TAFV glycoprotein-specific IgG responses including neutralizing antibodies. In contrast, macaques infected with TAFV stock 2 developed disease and were euthanized 8-11 days after infection exhibiting viremia, thrombocytopenia, and increased inflammatory mediators identified by transcriptional analysis. Histopathologic analysis of tissue samples collected at necropsy confirmed classic filovirus disease in numerous organs. Genomic differences in both stock preparations were mapped to several viral genes which may have contributed to disease severity. Taken together, we demonstrate that infection with the two TAFV stocks resulted in no disease in mice and opposing disease phenotypes in cynomolgus macaques, highlighting the impact of viral stock propagation on pathogenicity in animal models.

Monocytes and macrophages in pregnancy: The good, the bad, and the ugly.

A successful human pregnancy requires precisely timed adaptations by the maternal immune system to support fetal growth while simultaneously protecting mother and fetus against microbial challenges. The first trimester of pregnancy is characterized by a robust increase in innate immune activity that promotes successful implantation of the blastocyst and placental development. Moreover, early pregnancy is also a state of increased vulnerability to vertically transmitted pathogens notably, human immunodeficiency virus (HIV), Zika virus (ZIKV), SARS-CoV-2, and Listeria monocytogenes. As gestation progresses, the second trimester is marked by the establishment of an immunosuppressive environment that promotes fetal tolerance and growth while preventing preterm birth, spontaneous abortion, and other gestational complications. Finally, the period leading up to labor and parturition is characterized by the reinstatement of an inflammatory milieu triggering childbirth. These dynamic waves of carefully orchestrated changes have been dubbed the "immune clock of pregnancy." Monocytes in maternal circulation and tissue-resident macrophages at the maternal-fetal interface play a critical role in this delicate balance. This review will summarize the current data describing the longitudinal changes in the phenotype and function of monocyte and macrophage populations in healthy and complicated pregnancies.

Simian Varicella Virus: Molecular Virology and Mechanisms of Pathogenesis.

Simian varicella virus (SVV) was first isolated in 1966 from African green monkeys (Cercopithecus aethiops) imported from Nairobi, Kenya, to the Liverpool School of Tropical Medicine in the United Kingdom (UK) (Clarkson et al., Arch Gesamte Virusforsch 22:219-234, 1967). SVV infection caused severe disease that resulted in a 56% case fatality rate (CFR) in the imported animals within 48 h of the appearance of a varicella-like rash (Clarkson et al., Arch Gesamte Virusforsch 22:219-234, 1967; Hemme et al., Am J Trop Med Hyg 94:1095-1099, 2016). The deceased animals presented with fever, widespread vesicular rash, and multiple hemorrhagic foci throughout the lungs, liver, and spleen (Clarkson et al., Arch Gesamte Virusforsch 22:219-234, 1967). This outbreak was quickly followed by a second outbreak in 47 patas monkeys (Erythrocebus patas) imported from Chad and Nigeria by Glaxo Laboratories (London, England, UK), which quickly spread within the facility (McCarthy et al., Lancet 2:856-857, 1968).

Preexisting Immunity Does Not Prevent Efficacy of Vesicular Stomatitis Virus-Based Filovirus Vaccines in Nonhuman Primates.

Ebola virus (EBOV) and Marburg virus (MARV) made headlines in the past decade, causing outbreaks of human disease in previously nonendemic yet overlapping areas. While EBOV outbreaks can be mitigated with licensed vaccines and treatments, there is not yet a licensed countermeasure for MARV. Here, we used nonhuman primates (NHPs) previously vaccinated with vesicular stomatitis virus (VSV)-MARV and protected against lethal MARV challenge. After a resting period of 9 months, these NHPs were revaccinated with VSV-EBOV and challenged with EBOV, resulting in 75% survival. Surviving NHPs developed EBOV glycoprotein (GP)-specific antibody titers and no viremia or clinical signs of disease. The single vaccinated NHP succumbing to challenge showed the lowest EBOV GP-specific antibody response after challenge, supporting previous findings with VSV-EBOV that antigen-specific antibodies are critical in mediating protection. This study again demonstrates that VSVΔG-based filovirus vaccine can be successfully used in individuals with preexisting VSV vector immunity, highlighting the platform's applicability for consecutive outbreak response.

The architecture of the simian varicella virus transcriptome.

Primary infection with varicella-zoster virus (VZV) causes varicella and the establishment of lifelong latency in sensory ganglion neurons. In one-third of infected individuals VZV reactivates from latency to cause herpes zoster, often complicated by difficult-to-treat chronic pain. Experimental infection of non-human primates with simian varicella virus (SVV) recapitulates most features of human VZV disease, thereby providing the opportunity to study the pathogenesis of varicella and herpes zoster in vivo. However, compared to VZV, the transcriptome and the full coding potential of SVV remains incompletely understood. Here, we performed nanopore direct RNA sequencing to annotate the SVV transcriptome in lytically SVV-infected African green monkey (AGM) and rhesus macaque (RM) kidney epithelial cells. We refined structures of canonical SVV transcripts and uncovered numerous RNA isoforms, splicing events, fusion transcripts and non-coding RNAs, mostly unique to SVV. We verified the expression of canonical and newly identified SVV transcripts in vivo, using lung samples from acutely SVV-infected cynomolgus macaques. Expression of selected transcript isoforms, including those located in the unique left-end of the SVV genome, was confirmed by reverse transcription PCR. Finally, we performed detailed characterization of the SVV homologue of the VZV latency-associated transcript (VLT), located antisense to ORF61. Analogous to VZV VLT, SVV VLT is multiply spliced and numerous isoforms are generated using alternative transcription start sites and extensive splicing. Conversely, low level expression of a single spliced SVV VLT isoform defines in vivo latency. Notably, the genomic location of VLT core exons is highly conserved between SVV and VZV. This work thus highlights the complexity of lytic SVV gene expression and provides new insights into the molecular biology underlying lytic and latent SVV infection. The identification of the SVV VLT homolog further underlines the value of the SVV non-human primate model to develop new strategies for prevention of herpes zoster.

Correlates of SARS-CoV-2 anti-RBD IgG antibody titers among persons experiencing homelessness in Los Angeles.

OBJECTIVES: People experiencing homelessness (PEH) have been especially impacted by the COVID-19 pandemic, likely due to increased vulnerabilities stemming from chronic diseases, substance use, and mental health conditions. DESIGN: A case-control study to assess the presence of antibodies against SARS-CoV-2 among PEH and associations with key variables. SAMPLE: A convenience sample of 97 PEH in Skid Row, Los Angeles. MEASUREMENTS: A structured questionnaire assessing socio-demographic, mental health, drug and alcohol use, health care access, pandemic stress, and other COVID-19-specific questions. RESULTS: We found high anti-receptor binding domain (RBD) IgG titers among five of 15 PEH who reported no prior COVID-19 diagnosis or being vaccinated, suggesting undiagnosed and/or asymptomatic COVID-19. While anti-RBD IgG titers across vaccination categories were not statistically significant (p = .069), participants vaccinated with Janssen had the lowest mean anti-RBD IgG titers. In multivariable analysis, we found negative associations between level of SARS-CoV-2 antibody titers with the Janssen vaccine and depression; thus, a need for integrated care for PEH with depression and COVID-19. CONCLUSIONS: Further research is warranted to confirm the immune response, initial and over time, to SARS-CoV-2 infection and to COVID-19 vaccinations, particularly among PEH whose immune systems may be impacted by multiple health conditions.

Ethanol Consumption Induces Nonspecific Inflammation and Functional Defects in Alveolar Macrophages.

Chronic alcohol drinking is associated with increased susceptibility to viral and bacterial respiratory pathogens. In this study, we use a rhesus macaque model of voluntary ethanol self-administration to study the effects of long-term alcohol drinking on the immunological landscape of the lung. We report a heightened inflammatory state in alveolar macrophages (AMs) obtained from ethanol (EtOH)-drinking animals that is accompanied by increased chromatin accessibility in intergenic regions that regulate inflammatory genes and contain binding motifs for transcription factors AP-1, IRF8, and NFKB p-65. In line with these transcriptional and epigenetic changes at the basal state, AMs from EtOH-drinking animals generate elevated inflammatory mediator responses to lipopolysaccharides and respiratory syncytial virus. However, the transcriptional analysis revealed an inefficient induction of interferon-stimulated genes with EtOH in response to the respiratory syncytial virus, suggesting disruption of antimicrobial defenses. Correspondingly, AMs from EtOH-drinking animals exhibited transcriptional shifts indicative of increased oxidative stress and oxidative phosphorylation, which was coupled with higher cytosolic reactive oxygen species and mitochondrial potential. This heightened oxidative stress state was accompanied by decreased ability to phagocytose bacteria. Bulk RNA and assay for transposase-accessible chromatin sequencing data further revealed reduced expression and chromatin accessibility of loci associated with tissue repair and maintenance with chronic EtOH drinking. Similarly, analysis of single-cell RNA sequencing data revealed shifts in cell states from tissue maintenance to inflammatory responses with EtOH. Collectively, these data provide novel insight into mechanisms by which chronic EtOH drinking increases susceptibility to infection in patients with alcohol use disorders.

Transcriptional Analysis of Lymphoid Tissues from Infected Nonhuman Primates Reveals the Basis for Attenuation and Immunogenicity of an Ebola Virus Encoding a Mutant VP35 Protein.

Infection with Zaire ebolavirus (EBOV), a member of the Filoviridae family, causes a disease characterized by high levels of viremia, aberrant inflammation, coagulopathy, and lymphopenia. EBOV initially replicates in lymphoid tissues and disseminates via dendritic cells (DCs) and monocytes to liver, spleen, adrenal gland, and other secondary organs. EBOV protein VP35 is a critical immune evasion factor that inhibits type I interferon signaling and DC maturation. Nonhuman primates (NHPs) immunized with a high dose (5 × 105 PFU) of recombinant EBOV containing a mutated VP35 (VP35m) are protected from challenge with wild-type EBOV (wtEBOV). This protection is accompanied by a transcriptional response in the peripheral blood reflecting a regulated innate immune response and a robust induction of adaptive immune genes. However, the host transcriptional response to VP35m in lymphoid tissues has not been evaluated. Therefore, we conducted a transcriptional analysis of axillary and inguinal lymph nodes and spleen tissues of NHPs infected with a low dose (2 × 104 PFU) of VP35m and then back-challenged with a lethal dose of wtEBOV. VP35m induced early transcriptional responses in lymphoid tissues that are distinct from those observed in wtEBOV challenge. Specifically, we detected robust antiviral innate and adaptive responses and fewer transcriptional changes in genes with roles in angiogenesis, apoptosis, and inflammation. Two of three macaques survived wtEBOV back-challenge, with only the nonsurvivor displaying a transcriptional response reflecting Ebola virus disease. These data suggest that VP35 is a key modulator of early host responses in lymphoid tissues, thereby regulating disease progression and severity following EBOV challenge.IMPORTANCE Zaire Ebola virus (EBOV) infection causes a severe and often fatal disease characterized by inflammation, coagulation defects, and organ failure driven by a defective host immune response. Lymphoid tissues are key sites of EBOV pathogenesis and the generation of an effective immune response to infection. A recent study demonstrated that infection with an EBOV encoding a mutant VP35, a viral protein that antagonizes host immunity, can protect nonhuman primates (NHPs) against lethal EBOV challenge. However, no studies have examined the response to this mutant EBOV in lymphoid tissues. Here, we characterize gene expression in lymphoid tissues from NHPs challenged with the mutant EBOV and subsequently with wild-type EBOV to identify signatures of a protective host response. Our findings are critical for elucidating viral pathogenesis, mechanisms of host antagonism, and the role of lymphoid organs in protective responses to EBOV to improve the development of antivirals and vaccines against EBOV.

Importance of sex and trauma context on circulating cytokines and amygdalar GABAergic signaling in a comorbid model of posttraumatic stress and alcohol use disorders.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based "2-hit" rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel (NOV) context. Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-vulnerable individuals (≥0.8 g/kg average 2-h ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin, and IL-4 that discriminated between stress context (NOV > FAM > Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment.

Profiling of extracellular vesicle-bound miRNA to identify candidate biomarkers of chronic alcohol drinking in nonhuman primates.

BACKGROUND: Long-term alcohol drinking is associated with numerous health complications including susceptibility to infection, cancer, and organ damage. However, due to the complex nature of human drinking behavior, it has been challenging to identify reliable biomarkers of alcohol drinking behavior prior to signs of overt organ damage. Recently, extracellular vesicle-bound microRNAs (EV-miRNAs) have been found to be consistent biomarkers of conditions that include cancer and liver disease. METHODS: In this study, we profiled the plasma EV-miRNA content by miRNA-Seq from 80 nonhuman primates after 12 months of voluntary alcohol drinking. RESULTS: We identified a list of up- and downregulated EV-miRNA candidate biomarkers of heavy drinking and those positively correlated with ethanol dose. We overexpressed these candidate miRNAs in control primary peripheral immune cells to assess their potential functional mechanisms. We found that overexpression of miR-155, miR-154, miR-34c, miR-450a, and miR-204 led to increased production of the inflammatory cytokines TNFα or IL-6 in peripheral blood mononuclear cells after stimulation. CONCLUSION: This exploratory study identified several EV-miRNAs that could serve as biomarkers of long-term alcohol drinking and provide a mechanism to explain alcohol-induced peripheral inflammation.

The Safety and Immunologic Effectiveness of the Live Varicella-Zoster Vaccine in Patients Receiving Tumor Necrosis Factor Inhibitor Therapy : A Randomized Controlled Trial.

BACKGROUND: The safety and effectiveness of live virus vaccines, such as the varicella-zoster vaccine, are unknown in patients with inflammatory diseases receiving immunomodulatory therapy such as tumor necrosis factor inhibitors (TNFis). OBJECTIVE: To evaluate the safety and immunogenicity of the live attenuated zoster vaccine (ZVL) in patients receiving TNFis. DESIGN: Randomized, blinded, placebo-controlled trial. (ClinicalTrials.gov: NCT02538341). SETTING: Academic and community-based rheumatology, gastroenterology, and dermatology practices. PATIENTS: Adults aged 50 years or older receiving TNFis for any indication. INTERVENTION: Random assignment to ZVL versus placebo. MEASUREMENTS: Glycoprotein enzyme-linked immunosorbent assay (gpELISA) and enzyme-linked immunosorbent spot (ELISpot) from serum and peripheral blood mononuclear cells measured at baseline and 6 weeks after vaccination. Suspected varicella infection or herpes zoster was clinically assessed using digital photographs and polymerase chain reaction on vesicular fluid. RESULTS: Between March 2015 and December 2018, 617 participants were randomly assigned in a 1:1 ratio to receive ZVL (n = 310) or placebo (n = 307) at 33 centers. Mean age was 62.7 years (SD, 7.5); 66.1% of participants were female, 90% were White, 8.2% were Black, and 5.9% were Hispanic. The most common TNFi indications were rheumatoid arthritis (57.6%) and psoriatic arthritis (24.1%); TNFi medications were adalimumab (32.7%), infliximab (31.3%), etanercept (21.2%), golimumab (9.1%), and certolizumab (5.7%). Concomitant therapies included methotrexate (48.0%) and oral glucocorticoids (10.5%). Through week 6, no cases of confirmed varicella infection were found; cumulative incidence of varicella infection or shingles was 0.0% (95% CI, 0.0% to 1.2%). At 6 weeks, compared with baseline, the mean increases in geometric mean fold rise as measured by gpELISA and ELISpot were 1.33 percentage points (CI, 1.17 to 1.51 percentage points) and 1.39 percentage points (CI, 1.07 to 1.82 percentage points), respectively. LIMITATION: Potentially limited generalizability to patients receiving other types of immunomodulators. CONCLUSION: This trial informs safety concerns related to use of live virus vaccines in patients receiving biologics. PRIMARY FUNDING SOURCE: The National Institute of Arthritis and Musculoskeletal and Skin Diseases and the American College of Rheumatology.

Sex and context differences in the effects of trauma on comorbid alcohol use and post-traumatic stress phenotypes in actively drinking rats.

Alcohol use disorder (AUD) and affective disorders are frequently comorbid and share underlying mechanisms that could be targets for comprehensive treatment. Post-traumatic stress disorder (PTSD) has high comorbidity with AUD, but comprehensive models of this overlap are nascent. We recently characterized a model of comorbid AUD and PTSD-like symptoms, wherein stressed rats receive an inhibitory avoidance (IA)-related footshock on two occasions followed by two-bottle choice (2BC) voluntary alcohol drinking. Stressed rats received the second footshock in a familiar (FAM, same IA box as the first footshock) or novel context (NOV, single-chambered apparatus); the FAM paradigm more effectively increased alcohol drinking in males and the NOV paradigm in females. During abstinence, stressed males displayed avoidance-like PTSD symptoms, and females showed hyperarousal-like PTSD symptoms. Rats in the model had altered spontaneous action potential-independent GABAergic transmission in the central amygdala (CeA), a brain region key in alcohol dependence and stress-related signaling. However, PTSD sufferers may have alcohol experience prior to their trauma. Here, we therefore modified our AUD/PTSD comorbidity model to provide 3 weeks of intermittent extended alcohol access before footshock and then studied the effects of NOV and FAM stress on drinking and PTSD phenotypes. NOV stress suppressed the escalation of alcohol intake and preference seen in male controls, but no stress effects were seen on drinking in females. Additionally, NOV males had decreased action potential-independent presynaptic GABA release and delayed postsynaptic GABAA receptor kinetics in the CeA compared to control and FAM males. Despite these changes to alcohol intake and CeA GABA signaling, stressed rats showed broadly similar anxiogenic-like behaviors to our previous comorbid model, suggesting decoupling of the PTSD symptoms from the AUD vulnerability for some of these animals. The collective results show the importance of alcohol history and trauma context in vulnerability to comorbid AUD/PTSD-like symptoms.

Henipavirus W Proteins Interact with 14-3-3 To Modulate Host Gene Expression.

Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus in the Paramyxoviridae family, are recently emerged, highly lethal zoonotic pathogens. The NiV and HeV nonsegmented, negative-sense RNA genomes encode nine proteins, including the W protein. Expressed from the P gene through mRNA editing, W shares a common N-terminus with P and V but has a unique C-terminus. Expressed alone, W modulates innate immune responses by several mechanisms, and elimination of W from NiV alters the course of infection in experimentally infected ferrets. However, the specific host interactions that allow W to modulate innate immunity are incompletely understood. This study demonstrates that the NiV and HeV W proteins interact with all seven isoforms of the 14-3-3 family, regulatory molecules that preferentially bind phosphorylated target proteins to regulate a wide range of cellular functions. The interaction is dependent on the penultimate amino acid residue in the W sequence, a conserved, phosphorylated serine. The cocrystal structure of the W C-terminal binding motif with 14-3-3 provides only the second structure of a complex containing a mode III interactor, which is defined as a 14-3-3 interaction with a phosphoserine/phosphothreonine at the C-termini of the target protein. Transcriptomic analysis of inducible cell lines infected with an RNA virus and expressing either wild-type W or W lacking 14-3-3 binding, identifies new functions for W. These include the regulation of cellular metabolic processes, extracellular matrix organization, and apoptosis.IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus, are recently emerged, highly lethal zoonotic pathogens that cause yearly outbreaks. NiV and HeV each encode a W protein that has roles in regulating host signaling pathways, including antagonism of the innate immune response. However, the mechanisms used by W to regulate these host responses are not clear. Here, characterization of the interaction of NiV and HeV W with 14-3-3 identifies modulation of 14-3-3-regulated host signaling pathways not previously associated with W, suggesting new avenues of research. The cocrystal structure of the NiV W:14-3-3 complex, as only the second structure of a 14-3-3 mode III interactor, provides further insight into this less-well-understood 14-3-3 binding motif.

Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques.

Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013-2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease.IMPORTANCE Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis.

SARS-CoV-2 acquisition and immune pathogenesis among school-aged learners in four diverse schools.

BACKGROUND: Understanding SARS-CoV-2 infection in children is necessary to reopen schools safely. METHODS: We measured SARS-CoV-2 infection in 320 learners [10.5 ± 2.1 (sd); 7-17 y.o.] at four diverse schools with either remote or on-site learning. Schools A and B served low-income Hispanic learners; school C served many special-needs learners, and all provided predominantly remote instruction. School D served middle- and upper-income learners, with predominantly on-site instruction. Testing occurred in the fall (2020), and 6-8 weeks later during the fall-winter surge (notable for a tenfold increase in COVID-19 cases). Immune responses and mitigation fidelity were also measured. RESULTS: We found SARS-CoV-2 infections in 17 learners only during the surge. School A (97% remote learners) had the highest infection (10/70, 14.3%, p < 0.01) and IgG positivity rates (13/66, 19.7%). School D (93% on-site learners) had the lowest infection and IgG positivity rates (1/63, 1.6%). Mitigation compliance [physical distancing (mean 87.4%) and face-covering (91.3%)] was remarkably high at all schools. Documented SARS-CoV-2-infected learners had neutralizing antibodies (94.7%), robust IFN-γ + T cell responses, and reduced monocytes. CONCLUSIONS: Schools can implement successful mitigation strategies across a wide range of student diversity. Despite asymptomatic to mild SARS-CoV-2 infection, children generate robust humoral and cellular immune responses. IMPACT: Successful COVID-19 mitigation was implemented across a diverse range of schools. School-associated SARS-CoV-2 infections reflect regional rates rather than remote or on-site learning. Seropositive school-aged children with asymptomatic to mild SARS-CoV-2 infections generate robust humoral and cellular immunity.

Impaired hypothalamic feedback dysregulates brain glucocorticoid signaling in genetically-selected Marchigian Sardinian alcohol-preferring rats.

Genetically-selected Marchigian Sardinian alcohol-preferring (msP) rats display comorbid symptoms of increased alcohol preference and elevated anxiety-like behavior. Heightened stress sensitivity in msPs is influenced by genetic polymorphisms of the corticotropin-releasing factor receptor in the central nucleus of the amygdala (CeA), as well as reduced influence of anti-stress mechanisms that normally constrain the stress response. Given this propensity for stress dysregulation, in this study, we expand on the possibility that msPs may display differences in neuroendocrine processes that normally terminate the stress response. We utilized behavioral, biochemical, and molecular assays to compare basal and restraint stress-induced changes in the hypothalamic-pituitary-adrenal (HPA) axis of male and female msPs relative to their nonselected Wistar counterparts. The results showed that msPs display deficits in marble-burying behavior influenced by environmental factors and procedures that modulate arousal states in a sex-dependent manner. Whereas male msPs display evidence of dysregulated neuroendocrine function (higher adrenocorticotropic hormone levels and subthreshold reductions in corticosterone), females display restraint-induced elevations in corticosterone levels that were persistently higher in msPs. A dexamethasone challenge reduced the circulation of these stress hormones, although the reduction in corticosterone was generally attenuated in msP versus Wistar rats. Finally, we found evidence of diminished stress-induced glucocorticoid receptor (GR) phosphorylation in the hypothalamic paraventricular nucleus of msPs, as well as innate increases in phosphorylated GR levels in the CeA of male msPs. Collectively, these findings suggest that negative feedback processes regulating HPA responsiveness are diminished in msP rats, possibly underlying differences in the expression of anxiety-like behaviors.

Chronic ethanol consumption alters lamina propria leukocyte response to stimulation in a region-dependent manner.

Chronic heavy alcohol consumption, also referred to as chronic heavy drinking (CHD), results in intestinal injury characterized by increased permeability, dysbiosis, nutrient malabsorption, potentially higher susceptibility to infection, and increased risk of colorectal cancer. However, our understanding of the mechanisms by which CHD results in intestinal damage remains incomplete. Here, we investigated the impact of chronic drinking on transcriptional and functional responses of lamina propria leukocytes (LPLs) isolated from the 4 major gut sections. Although no significant differences were detected between LPLs isolated from the ethanol and control groups at resting state within each major gut section, our analysis uncovered key regional differences in composition and function of LPLs independent of alcohol consumption. However, in response to phorbol myristate acetate and ionomycin, duodenal LPLs from ethanol-drinking animals generated a dampened response, whereas jejunal and ileal LPLs from ethanol-drinking animals produced a heightened response. Transcriptional responses following stimulation were pronounced in ileal and duodenal LPLs from the ethanol-drinking group but less evident in jejunal and colonic LPLs compared with controls, suggesting a more significant impact of alcohol on these gut regions. The altered intestinal LPL function detected in our study reveals remarkable region specificity and novel insight into potential mechanisms of intestinal injury associated with CHD.-Barr, T., Lewis, S. A., Sureshchandra, S., Doratt, B., Grant, K. A., Messaoudi, I. Chronic ethanol consumption alters lamina propria leukocyte response to stimulation in a region-dependent manner.

Maternal Pregravid Obesity Remodels the DNA Methylation Landscape of Cord Blood Monocytes Disrupting Their Inflammatory Program.

Prepregnancy maternal obesity is associated with adverse outcomes for the offspring, including increased incidence of neonatal bacterial sepsis and necrotizing enterocolitis. We recently reported that umbilical cord blood (UCB) monocytes from babies born to obese mothers generate a reduced IL-6/TNF-α response to TLR 1/2 and 4 ligands compared to those collected from lean mothers. These observations suggest altered development of the offspring's immune system, which in turn results in dysregulated function. We therefore investigated transcriptional and epigenetic differences within UCB monocytes stratified by prepregnancy maternal body mass index. We show that UCB monocytes from babies born to obese mothers generate a dampened response to LPS stimulation compared with those born to lean mothers, at the level of secreted immune mediators and transcription. Because gene expression profiles of resting UCB monocytes from both groups were comparable, we next investigated the role of epigenetic differences. Indeed, we detected stark differences in methylation levels within promoters and regulatory regions of genes involved in TLR signaling in resting UCB monocytes. Interestingly, the DNA methylation status of resting cells was highly predictive of transcriptional changes post-LPS stimulation, suggesting that cytosine methylation is one of the dominant mechanisms driving functional inadequacy in UCB monocytes obtained from babies born to obese mothers. These data highlight a potentially critical role of maternal pregravid obesity-associated epigenetic changes in influencing the function of an offspring's monocytes at birth. These findings further our understanding of mechanisms that explain the increased risk of infection in neonates born to mothers with high prepregnancy body mass index.

Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering Gene Expression in Circulating Leukocytes.

Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections, whereas moderate alcohol consumption decreases the incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations > 80 mg/dl) suppressed, whereas moderate alcohol consumption (blood ethanol concentrations < 50 mg/dl) enhanced, T and B cell responses to modified vaccinia Ankara vaccination in a nonhuman primate model of voluntary ethanol consumption. To uncover the molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post-modified vaccinia Ankara vaccination, the earliest time point at which we detected differences in T cell and Ab responses. Overall, chronic heavy alcohol consumption reduced the expression of immune genes involved in response to infection and wound healing and increased the expression of genes associated with the development of lung inflammatory disease and cancer. In contrast, chronic moderate alcohol consumption upregulated the expression of genes involved in immune response and reduced the expression of genes involved in cancer. To uncover mechanisms underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate the expression of mRNAs differentially expressed in our data set.

Long-lasting effect of obesity on skeletal muscle transcriptome.

BACKGROUND: Reduced physical activity and increased intake of calorically-dense diets are the main risk factors for obesity, glucose intolerance, and type 2 diabetes. Chronic overnutrition and hyperglycemia can alter gene expression, contributing to long-term obesity complications. While caloric restriction can reduce obesity and glucose intolerance, it is currently unknown whether it can effectively reprogram transcriptome to a pre-obesity level. The present study addressed this question by the preliminary examination of the transcriptional dynamics in skeletal muscle after exposure to overnutrition and following caloric restriction. RESULTS: Six male rhesus macaques of 12-13 years of age consumed a high-fat western-style diet for 6 months and then were calorically restricted for 4 months without exercise. Skeletal muscle biopsies were subjected to longitudinal gene expression analysis using next-generation whole-genome RNA sequencing. In spite of significant weight loss and normalized insulin sensitivity, the majority of WSD-induced (n = 457) and WSD-suppressed (n = 47) genes remained significantly dysregulated after caloric restriction (FDR ≤0.05). The MetacoreTM pathway analysis reveals that western-style diet induced the sustained activation of the transforming growth factor-ß gene network, associated with extracellular matrix remodeling, and the downregulation of genes involved in muscle structure development and nutritional processes. CONCLUSIONS: Western-style diet, in the absence of exercise, induced skeletal muscle transcriptional programing, which persisted even after insulin resistance and glucose intolerance were completely reversed with caloric restriction.