Latent Toxoplasma gondii infections are associated with elevated biomarkers of inflammation and vascular injury.

BACKGROUND: Toxoplasma gondii is a protozoan parasite that infects cats as definitive hosts and other warm-blooded animals including humans as intermediate hosts. It forms infectious cysts in the brain, muscle and other tissues establishing life-long latent infection. Approximately 10% of the US population is infected. While latent infections are largely asymptomatic, they are associated with neurological deficits and elevated risks of neuropsychiatric diseases. METHODS: This cross-sectional epidemiological study investigated associations of T. gondii infections with biomarkers of inflammation and vascular injury: soluble intercellular adhesion molecule 1 (ICAM-1), soluble vascular cell adhesion molecule 1 (VCAM-1), C-reactive protein (CRP), and serum amyloid A (SAA). Serum samples from 694 adults in the Raleigh-Durham-Chapel Hill, North Carolina metropolitan area were tested for IgG antibody response to T. gondii, and for the above biomarkers using commercially available assays. RESULTS: T. gondii seroprevalence rate in this sample was 9.7%. Seropositivity was significantly associated with 11% (95% confidence limits 4, 20%) greater median levels of VCAM-1 (p = 0.003), and marginally significantly with 9% (1, 17%), and 36% (1, 83%) greater median levels of ICAM-1, and CRP, respectively (p = 0.04 for each) after adjusting for sociodemographic and behavioral covariates, while the 23% (- 7, 64%) adjusted effect on SAA was not statistically significant (p = 0.15). CONCLUSIONS: Latent infections with T. gondii are associated with elevated biomarkers of chronic inflammation and vascular injury that are also known to be affected by ambient air pollution.

Greater tree cover near residence is associated with reduced allostatic load in residents of central North Carolina.

BACKGROUND: Among urban residents, increased contacts with nature are associated with reduced morbidity and mortality. The concept of allostatic load, a biomarker-based composite measure of physiological dysregulation, can be applied to study subclinical benefits of exposure, and to elucidate pathways leading to improved health. OBJECTIVE: This research explored associations between residential vegetated land cover and an allostatic load index calculated using the statistical distance measure known as Mahalanobis distance. METHODS: This cross-sectional population-based study involved 186 adult residents of the Durham-Chapel Hill, North Carolina metropolitan area. Measures of tree and grass cover within 500 m of residence were derived from the U.S. Environmental Protection Agency's EnviroAtlas land cover database. Fifteen biomarkers of immune, neuroendocrine, and metabolic functions were analyzed in serum samples. Regression analysis was conducted using generalized additive models with thin-plate spline functions of geographic coordinates, adjusting for modelled traffic air pollution from local sources and sociodemographic covariates. RESULTS: The second and third tertiles of distance-weighted tree cover were associated with 14% (95% Confidence Limits 20%; 8%) and 15% (21%; 8%) reduction in adjusted median allostatic load, respectively, compared to the first tertile. The same tertiles of tree cover were also associated with 0.16 (0.03; 0.76) and 0.04 (0.01; 0.35) adjusted odds ratios of having allostatic load index above the 90th percentile of the sample distribution. Grass cover was inversely correlated with tree cover and was not associated with reduced allostatic load. CONCLUSIONS: Subclinical beneficial health effects of green spaces demonstrated in this study are consistent with reduced susceptibility to acute environmental and social stressors, and reduced risks of morbidity and mortality.

Environmental risk factors for Toxoplasma gondii infections and the impact of latent infections on allostatic load in residents of Central North Carolina.

BACKGROUND: Toxoplasma gondii infection can be acquired through ingestion of infectious tissue cysts in undercooked meat or environmental oocysts excreted by cats. This cross-sectional study assessed environmental risk factors for T. gondii infections and an association between latent infections and a measure of physiologic dysregulation known as allostatic load. METHODS: Serum samples from 206 adults in the Durham-Chapel Hill, North Carolina area were tested for immunoglobulin (IgG) responses to T. gondii using commercial ELISA kits. Allostatic load was estimated as a sum of 15 serum biomarkers of metabolic, neuroendocrine and immune functions dichotomized at distribution-based cutoffs. Vegetated land cover within 500 m of residences was estimated using 1 m resolution data from US EPA's EnviroAtlas. RESULTS: Handling soil with bare hands at least weekly and currently owning a cat were associated with 5.3 (95% confidence limits 1.4; 20.7) and 10.0 (2.0; 50.6) adjusted odds ratios (aOR) of T. gondii seropositivity, respectively. There was also a significant positive interaction effect of handling soil and owning cats on seropositivity. An interquartile range increase in weighted mean vegetated land cover within 500 m of residence was associated with 3.7 (1.5; 9.1) aOR of T. gondii seropositivity. Greater age and consumption of undercooked pork were other significant predictors of seropositivity. In turn, T. gondii seropositivity was associated with 61% (13%; 130%) greater adjusted mean allostatic load compared to seronegative individuals. In contrast, greater vegetated land cover around residence was associated with significantly reduced allostatic load in both seronegative (p < 0.0001) and seropositive (p = 0.004) individuals. CONCLUSIONS: Residents of greener areas may be at a higher risk of acquiring T. gondii infections through inadvertent ingestion of soil contaminated with cat feces. T. gondii infections may partially offset health benefits of exposure to the natural living environment.

Signal transduction by different forms of the γδ T cell-specific pattern recognition receptor WC1.

WC1 coreceptors are scavenger receptor cysteine-rich (SRCR) family members, related to T19 in sheep, SCART in mice, and CD163c-α in humans, and form a 13-member subfamily in cattle exclusively expressed on γδ T cells. Subpopulations of γδ T cells are defined by anti-WC1 mAbs and respond to different pathogen species accordingly. In this study, variegated WC1 gene expression within subpopulations and differences in signaling and cell activation due to endodomain sequences are described. The endodomains designated types I to III differ by a 15- or 18-aa insert in type II and an additional 80 aa containing an additional eight tyrosines for type III. Anti-WC1 mAbs enhanced cell proliferation of γδ T cells when cross-linked with the TCR regardless of the endodomain sequences. Chimeric molecules of human CD4 ectodomain with WC1 endodomains transfected into Jurkat cells showed that the tyrosine phosphorylation of the type II was the same as that of the previously reported archetypal sequence (type I) with only Y24EEL phosphorylated, whereas for type III only Y199DDV and Y56TGD were phosphorylated despite conservation of the Y24EEL/Y24QEI and Y199DDV/I tyrosine motifs among the three types. Time to maximal phosphorylation was more rapid with type III endodomains and sustained longer. Differences in tyrosine phosphorylation were associated with differences in function in that cross-linking of type III chimeras with TCR resulted in significantly greater IL-2 production. Identification of differences in the signal transduction through the endodomains of WC1 contributes to understanding the functional role of the WC1 coreceptors in the γδ T cell responses.

Biological and behavioral factors modify urinary arsenic metabolic profiles in a U.S. population.

BACKGROUND: Because some adverse health effects associated with chronic arsenic exposure may be mediated by methylated arsenicals, interindividual variation in capacity to convert inorganic arsenic into mono- and di-methylated metabolites may be an important determinant of risk associated with exposure to this metalloid. Hence, identifying biological and behavioral factors that modify an individual's capacity to methylate inorganic arsenic could provide insights into critical dose-response relations underlying adverse health effects. METHODS: A total of 904 older adults (≥45 years old) in Churchill County, Nevada, who chronically used home tap water supplies containing up to 1850 µg of arsenic per liter provided urine and toenail samples for determination of total and speciated arsenic levels. Effects of biological factors (gender, age, body mass index) and behavioral factors (smoking, recent fish or shellfish consumption) on patterns of arsenicals in urine were evaluated with bivariate analyses and multivariate regression models. RESULTS: Relative contributions of inorganic, mono-, and di-methylated arsenic to total speciated arsenic in urine were unchanged over the range of concentrations of arsenic in home tap water supplies used by study participants. Gender predicted both absolute and relative amounts of arsenicals in urine. Age predicted levels of inorganic arsenic in urine and body mass index predicted relative levels of mono- and di-methylated arsenic in urine. Smoking predicted both absolute and relative levels of arsenicals in urine. Multivariate regression models were developed for both absolute and relative levels of arsenicals in urine. Concentration of arsenic in home tap water and estimated water consumption were strongly predictive of levels of arsenicals in urine as were smoking, body mass index, and gender. Relative contributions of arsenicals to urinary arsenic were not consistently predicted by concentrations of arsenic in drinking water supplies but were more consistently predicted by gender, body mass index, age, and smoking. CONCLUSIONS: These findings suggest that analyses of dose-response relations in arsenic-exposed populations should account for biological and behavioral factors that modify levels of inorganic and methylated arsenicals in urine. Evidence of significant effects of these factors on arsenic metabolism may also support mode of action studies in appropriate experimental models.

Factors associated with self-reported health: implications for screening level community-based health and environmental studies.

BACKGROUND: Advocates for environmental justice, local, state, and national public health officials, exposure scientists, need broad-based health indices to identify vulnerable communities. Longitudinal studies show that perception of current health status predicts subsequent mortality, suggesting that self-reported health (SRH) may be useful in screening-level community assessments. This paper evaluates whether SRH is an appropriate surrogate indicator of health status by evaluating relationships between SRH and sociodemographic, lifestyle, and health care factors as well as serological indicators of nutrition, health risk, and environmental exposures. METHODS: Data were combined from the 2003-2006 National Health and Nutrition Examination Surveys for 1372 nonsmoking 20-50 year olds. Ordinal and binary logistic regression was used to estimate odds ratios and 95 % confidence intervals of reporting poorer health based on measures of nutrition, health condition, environmental contaminants, and sociodemographic, health care, and lifestyle factors. RESULTS: Poorer SRH was associated with several serological measures of nutrition, health condition, and biomarkers of toluene, cadmium, lead, and mercury exposure. Race/ethnicity, income, education, access to health care, food security, exercise, poor mental and physical health, prescription drug use, and multiple health outcome measures (e.g., diabetes, thyroid problems, asthma) were also associated with poorer SRH. CONCLUSION: Based on the many significant associations between SRH and serological assays of health risk, sociodemographic measures, health care access and utilization, and lifestyle factors, SRH appears to be a useful health indicator with potential relevance for screening level community-based health and environmental studies.

Time outdoors and residential greenness are associated with reduced systemic inflammation and allostatic load.

Contacts with nature are linked with reduced morbidity and mortality. Hypothesized pathways include relaxation, physical activity, and improved immune function. This cross-sectional study of 320 adults in central North Carolina assessed health benefits of residential greenness using allostatic load (AL) and systemic inflammation (INFL) indices, composite biomarker-based measures of physiological dysregulation and inflammation, respectively. Distance-to-residence weighted tree cover and vegetated land cover measures were estimated within 500 m of each residence; 37 biomarkers of immune, neuroendocrine, cardiovascular, and metabolic functions were dichotomized at distribution or health-based cut-offs. AL was calculated as a sum of potentially unhealthy values of all biomarkers; INFL was based on a subset of 18 immune biomarkers. Regression analysis used generalized additive models for Poisson-distributed outcome. An interquartile range (IQR) increase in tree cover was associated with 0.89 (95 % Confidence Limits 0.82; 0.97) and 0.90 (0.79; 1.03)-fold change in AL and INFL, respectively. Greater daily outdoor time was associated with reduced AL and INFL, while leisure screen time, problems with sleeping, and common chronic infections were linked with increased AL and INFL. Among 138 individuals spending more than 1 h outdoors daily, an IQR increase in tree cover was associated with 0.76 (0.67; 0.86) and 0.81 (0.65; 1.02)-fold changes in AL and INFL, respectively. Among individuals with residential tree cover above the 50th percentile, spending more than 3 h outdoors daily was associated with 0.54 (0.37; 0.78) and 0.28 (0.15; 0.54)-fold changes in AL and INFL, respectively, compared to spending less than 30 min outdoors; there were no significant effects in the low tree cover stratum. Consistent but weaker effects were observed for vegetated land cover. Interaction effects of tree and vegetative cover and time spent outdoors on AL and INFL were statistically significant. This biomarker-based approach can help to assess public health benefits of green spaces.

Biological and behavioral factors modify biomarkers of arsenic exposure in a U.S. population.

Although consumption of drinking water contaminated with inorganic arsenic is usually considered the primary exposure route, aggregate exposure to arsenic depends on direct consumption of water, use of water in food preparation, and the presence in arsenicals in foods. To gain insight into the effects of biological and behavioral factors on arsenic exposure, we determined arsenic concentrations in urine and toenails in a U.S. population that uses public or private water supplies containing inorganic arsenic. Study participants were 904 adult residents of Churchill County, Nevada, whose home tap water supplies contained <3 to about 1200 µg of arsenic per liter. Biomarkers of exposure for this study were summed urinary concentrations of inorganic arsenic and its methylated metabolites (speciated arsenical), of all urinary arsenicals (total arsenical), and of all toenail arsenicals (total arsenical). Increased tap water arsenic concentration and consumption were associated with significant upward trends for urinary speciated and total and toenail total arsenical concentrations. Significant gender differences in concentrations of speciated and total arsenicals in urine and toenails reflected male-female difference in water intake. Both recent and higher habitual seafood consumption significantly increased urinary total but not speciated arsenical concentration. In a stepwise general linear model, seafood consumption significantly predicted urinary total arsenical but not urinary speciated or toenail total arsenical concentrations. Smoking behavior significantly predicted urinary speciated or total arsenical concentration. Gender, tap water arsenic concentration, and primary drinking water source significantly predicted urinary speciated and total concentrations and toenail total arsenical concentrations. These findings confirm the primacy of home tap water as a determinant of arsenic concentration in urine and toenails. However, biological and behavioral factors can modify exposure-response relations for these biomarkers. Refining estimates of the influence of these factors will permit better models of dose-response relations for this important environmental contaminant.

Greener residential environment is associated with increased bacterial diversity in outdoor ambient air.

In urban areas, exposure to greenspace has been found to be beneficial to human health. The biodiversity hypothesis proposed that exposure to diverse ambient microbes in greener areas may be one pathway leading to health benefits such as improved immune system functioning, reduced systemic inflammation, and ultimately reduced morbidity and mortality. Previous studies observed differences in ambient outdoor bacterial diversity between areas of high and low vegetated land cover but didn't focus on residential environments which are important to human health. This research examined the relationship between vegetated land and tree cover near residence and outdoor ambient air bacterial diversity and composition. We used a filter and pump system to collect ambient bacteria samples outside residences in the Raleigh-Durham-Chapel Hill metropolitan area and identified bacteria by 16S rRNA amplicon sequencing. Geospatial quantification of total vegetated land or tree cover was conducted within 500 m of each residence. Shannon's diversity index and weighted UniFrac distances were calculated to measure α (within-sample) and ß (between-sample) diversity, respectively. Linear regression for α-diversity and permutational analysis of variance (PERMANOVA) for ß-diversity were used to model relationships between vegetated land and tree cover and bacterial diversity. Data analysis included 73 ambient air samples collected near 69 residences. Analysis of ß-diversity demonstrated differences in ambient air microbiome composition between areas of high and low vegetated land (p = 0.03) and tree cover (p = 0.07). These relationships remained consistent among quintiles of vegetated land (p = 0.03) and tree cover (p = 0.008) and continuous measures of vegetated land (p = 0.03) and tree cover (p = 0.03). Increased vegetated land and tree cover were also associated with increased ambient microbiome α-diversity (p = 0.06 and p = 0.03, respectively). To our knowledge, this is the first study to demonstrate associations between vegetated land and tree cover and the ambient air microbiome's diversity and composition in the residential ecosystem.

Dramatic improvements in beach water quality following gull removal.

Gulls are often cited as important contributors of fecal contamination to surface waters, and some recreational beaches have used gull control measures to improve microbial water quality. In this study, gulls were chased from a Lake Michigan beach using specially trained dogs, and water quality improvements were quantified. Fecal indicator bacteria and potentially pathogenic bacteria were measured before and during gull control using culture methods and quantitative polymerase chain reaction (qPCR). Harassment by dogs was an effective method of gull control: average daily gull populations fell from 665 before to 17 during intervention; and a significant reduction in the density of a gull-associated marker was observed (p < 0.001). Enterococcus spp. and Escherichia coli densities were also significantly reduced during gull control (p < 0.001 and p = 0.012, respectively for culture methods; p = 0.012 and p = 0.034, respectively for qPCR). Linear regression results indicate that a 50% reduction in gulls was associated with a 38% and 29% decrease in Enterococcus spp. and E. coli densities, respectively. Potentially human pathogenic bacteria were detected on 64% of days prior to gull control and absent during gull intervention, a significant reduction (p = 0.005). This study demonstrates that gull removal can be a highly successful beach remedial action to improve microbial water quality.

Allele-Specific Knockdown of Mutant Huntingtin Protein via Editing at Coding Region Single Nucleotide Polymorphism Heterozygosities.

Huntington's disease (HD) is a devastating, autosomal dominant neurodegenerative disease caused by a trinucleotide repeat expansion in the huntingtin (HTT) gene. Inactivation of the mutant allele by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 based gene editing offers a possible therapeutic approach for this disease, but permanent disruption of normal HTT function might compromise adult neuronal function. Here, we use a novel HD mouse model to examine allele-specific editing of mutant HTT (mHTT), with a BAC97 transgene expressing mHTT and a YAC18 transgene expressing normal HTT. We achieve allele-specific inactivation of HTT by targeting a protein coding sequence containing a common, heterozygous single nucleotide polymorphism (SNP). The outcome is a marked and allele-selective reduction of mHTT protein in a mouse model of HD. Expression of a single CRISPR-Cas9 nuclease in neurons generated a high frequency of mutations in the targeted HD allele that included both small insertion/deletion (InDel) mutations and viral vector insertions. Thus, allele-specific targeting of InDel and insertion mutations to heterozygous coding region SNPs provides a feasible approach to inactivate autosomal dominant mutations that cause genetic disease.

Evaluation of genetic susceptibility to childhood allergy and asthma in an African American urban population.

BACKGROUND: Asthma and allergy represent complex phenotypes, which disproportionately burden ethnic minorities in the United States. Strong evidence for genomic factors predisposing subjects to asthma/allergy is available. However, methods to utilize this information to identify high risk groups are variable and replication of genetic associations in African Americans is warranted. METHODS: We evaluated 41 single nucleotide polymorphisms (SNP) and a deletion corresponding to 11 genes demonstrating association with asthma in the literature, for association with asthma, atopy, testing positive for food allergens, eosinophilia, and total serum IgE among 141 African American children living in Detroit, Michigan. Independent SNP and haplotype associations were investigated for association with each trait, and subsequently assessed in concert using a genetic risk score (GRS). RESULTS: Statistically significant associations with asthma were observed for SNPs in GSTM1, MS4A2, and GSTP1 genes, after correction for multiple testing. Chromosome 11 haplotype CTACGAGGCC (corresponding to MS4A2 rs574700, rs1441586, rs556917, rs502581, rs502419 and GSTP1 rs6591256, rs17593068, rs1695, rs1871042, rs947895) was associated with a nearly five-fold increase in the odds of asthma (Odds Ratio (OR) = 4.8, p = 0.007). The GRS was significantly associated with a higher odds of asthma (OR = 1.61, 95% Confidence Interval = 1.21, 2.13; p = 0.001). CONCLUSIONS: Variation in genes associated with asthma in predominantly non-African ethnic groups contributed to increased odds of asthma in this African American study population. Evaluating all significant variants in concert helped to identify the highest risk subset of this group.

Allergens in household dust and serological indicators of atopy and sensitization in Detroit children with history-based evidence of asthma.

BACKGROUND: Home exposure to allergens is an important factor in the development of sensitization and subsequent exacerbations of allergic asthma. We investigated linkages among allergen exposure, immunological measurements, and asthma by examining (1) reservoir dust allergen levels in homes, (2) associations between presence of allergens in homes and sensitization status of resident children, and (3) associations between asthma status and total IgE, atopy (by Phadiatop), and positive allergen-specific tests. METHODS: The study protocol was approved by Institutional Review Boards (IRBs) of the University of North Carolina Chapel Hill; Westat, Inc.; and the US Environmental Protection Agency Human Research Protocol Office. Data were collected from questionnaires, serum analyses, and household vacuum dust. Children (n = 205) were predominately African American (AA) (85.4%) and 51.6% were asthmatic. Sera from 185 children and home dust samples (n = 141) were analyzed for total and specific IgE antibodies to allergens from cat and dog dander, cockroach, dust mites, mice, rats, and molds. RESULTS: Sixty percent of the homes had detectable levels of three or more dust allergens. The proportions of children with positive allergen-specific IgE tests were dust mite (32%), dog (28%), cat (23%), cockroach (18%), mouse (5%), rat (4%), and molds (24-36%). Children testing positive to a single allergen also had positive responses to other allergens. Those children with positive serum tests for cat, dog, and dust mite lived in homes with detectable levels of cat (51%), dog (90%), and dust mite (Der f 1) (92%) allergens. Correlations between children's specific IgE levels and dust levels were linearly related for dog (p < .04), but not for cat (p = .12) or dust mite (Der f 1) (p = .21). Odds ratios (95% CI) for the associations between asthma and serum-specific IgE were over 1.0 for cat, dog, dust mite (Der f 1), cockroach, and four types of molds. House dust allergen exposure levels, however, exhibited no differences between asthmatic and non-asthmatic homes. CONCLUSIONS: Both the co-occurrence of multiple allergens in dust and the high frequency of multiple allergen sensitizations indicate that a broad-based intervention aimed at reducing multiple allergens (pets, pests, and molds) would be more successful than any approach that aimed at reducing one type of allergen.

Mechanistic indicators of childhood asthma (MICA) study: piloting an integrative design for evaluating environmental health.

BACKGROUND: Asthma is a common complex disease responsible for considerable morbidity and mortality, particularly in urban minority populations. The Mechanistic Indicators of Childhood Asthma study was designed to pilot an integrative approach in children's health research. The study incorporates exposure metrics, internal dose measures, and clinical indicators to decipher the biological complexity inherent in diseases such as asthma and cardiovascular disease with etiology related to gene-environment interactions. METHODS/DESIGN: 205 non-asthmatic and asthmatic children, (9-12 years of age) from Detroit, Michigan were recruited. The study includes environmental measures (indoor and outdoor air, vacuum dust), biomarkers of exposure (cotinine, metals, total and allergen specific Immunoglobulin E, polycyclic aromatic hydrocarbons, volatile organic carbon metabolites) and clinical indicators of health outcome (immunological, cardiovascular and respiratory). In addition, blood gene expression and candidate SNP analyses were conducted. DISCUSSION: Based on an integrative design, the MICA study provides an opportunity to evaluate complex relationships between environmental factors, physiological biomarkers, genetic susceptibility and health outcomes. PROJECT APPROVAL: IRB Number 05-EPA-2637: The human subjects' research protocol was reviewed by the Institutional Review Board (IRB) of the University of North Carolina; the IRB of Westat, Inc., the IRB of the Henry Ford Health System; and EPA's Human Subjects' Research Review Official.

Environmental risk factors associated with pulmonary isolation of nontuberculous mycobacteria, a population-based study in the southeastern United States.

The prevalence of pulmonary nontuberculous mycobacteria (NTM) disease is increasing in the United States. Associations were evaluated among residents of central North Carolina between pulmonary isolation of NTM and environmental risk factors including: surface water, drinking water source, urbanicity, and exposures to soils favorable to NTM growth. Reports of pulmonary NTM isolation from patients residing in three counties in central North Carolina during 2006-2010 were collected from clinical laboratories and from the State Laboratory of Public Health. This analysis was restricted to patients residing in single family homes with a valid residential street address and conducted at the census block level (n = 13,495 blocks). Negative binomial regression models with thin-plate spline smoothing function of geographic coordinates were applied to assess effects of census block-level environmental characteristics on pulmonary NTM isolation count. Patients (n = 507) resided in 473 (3.4%) blocks within the study area. Blocks with >20% hydric soils had 26.8% (95% confidence interval (CI): 1.8%, 58.0%), p = 0.03, higher adjusted mean patient counts compared to blocks with ≤20% hydric soil, while blocks with >50% acidic soil had 24.8% (-2.4%, 59.6%), p = 0.08 greater mean patient count compared to blocks with ≤50% acidic soil. Isolation rates varied by county after adjusting for covariates. The effects of using disinfected public water supplies vs. private wells, and of various measures of urbanicity were not significantly associated with NTM. Our results suggest that proximity to certain soil types (hydric and acidic) could be a risk factor for pulmonary NTM isolation in central North Carolina.

A Multiplex Noninvasive Salivary Antibody Assay for SARS-CoV-2 Infection and Its Application in a Population-Based Survey by Mail.

Noninvasive salivary antibody immunoassays can enable low-cost epidemiological surveillance of infections. This study involved developing and validating a multiplex suspension immunoassay on the Luminex platform to measure immunoglobulin G (IgG) responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid and spike (S) proteins, and the spike protein's S1 and S2 subunits and receptor binding domain. Multiple versions of these recombinant proteins acquired from commercial and noncommercial sources were evaluated. Assay development and validation utilized saliva and serum samples from coronavirus disease 2019 (COVID-19) cases procured from commercial sources and negative controls from a prepandemic survey. Saliva was also collected in a demonstration survey by mail involving adult individuals in the United States who were diagnosed with SARS-CoV-2 infection 15 to 80 days prior to sample collection. The survey had an 83% valid sample return rate (192 samples from 38 states). Most COVID-19 cases (93%) reported mildly symptomatic or asymptomatic infections. The final salivary assay based on the best-performing spike and nucleocapsid proteins had a sensitivity of 87.1% (95% bootstrap confidence interval, 82.1 to 91.7%) and specificity of 98.5% (95.0 to 100%) using 227 and 285 saliva samples, respectively. The same assay had 95.9% (92.8 to 98.9%) sensitivity and 100% (98.4 to 100%) specificity in serum (174 and 285 serum samples, respectively). Salivary and serum antibody responses to spike and nucleocapsid proteins were strongly correlated in 22 paired samples (r = 0.88 and r = 0.80, respectively). Antibody responses peaked at approximately 50 days postonset; greater illness severity was associated with stronger responses. This study demonstrated that a salivary antibody assay can be used in large-scale population surveys by mail to better characterize public health impacts of COVID-19. IMPORTANCE Given the enormous impacts of the COVID-19 pandemic, developing tools for population surveillance of infection is of paramount importance. This article describes the development of a multiplex immunoassay on a Luminex platform to measure salivary immunoglobulin G responses to the spike protein, its two subunits and receptor binding domain, and the nucleocapsid protein of SARS-CoV-2. The assay validation utilized serum and saliva samples from prepandemic controls and recent COVID-19 cases. A survey by mail targeting recent COVID-19 cases across the United States also demonstrated the utility of safe, at-home self-collection of saliva. By incorporating multiple SARS-CoV-2 proteins, this assay may differentiate responses to natural SARS-CoV-2 infections from responses to most vaccines. Application of this noninvasive immunoassay in COVID-19 surveillance can help provide estimates of cumulative incidence rates of symptomatic and asymptomatic infections in various communities and subpopulations, temporal patterns of antibody responses, and risk factors for infection.

Recreational water exposure and waterborne infections in a prospective salivary antibody study at a Lake Michigan beach.

In a prospective observational study, seroconversion to a specific pathogen can serve as a marker of an incident infection, whether or not that infection is symptomatic or clinically diagnosed. While self-reported symptoms can be affected by reporting bias, seroconversion is likely to be free of this bias as it is based on objective measurements of antibody response. Non-invasive salivary antibody tests can be used instead of serum tests to detect seroconversions in prospective studies. In the present study, individuals and families were recruited at a Lake Michigan beach in Wisconsin in August 2011. Data on recreational water exposure and baseline saliva samples (S1) were collected at recruitment. Follow-up data on gastrointestinal symptoms were collected via a telephone interview approximately 10 days post-recruitment. Follow-up saliva samples were self-collected approximately 2 weeks (S2) and 30-40 days post-recruitment (S3) and mailed to the study laboratory. Samples were analyzed for immunoglobulin (Ig) G responses to recombinant antigens of three noroviruses and Cryptosporidium, as well as protein purification tags as internal controls, using an in-house multiplex suspension immunoassay on the Luminex platform. Responses were defined as ratios of antibody reactivities with a target protein and its purification tag. Seroconversions were defined as at least four-fold and three-fold increases in responses in S2 and S3 samples compared to S1, respectively. In addition, an S2 response had to be above the upper 90% one-sided prediction limit of a corresponding spline function of age. Among 872 study participants, there were seven (0.8%) individuals with seroconversions, including six individuals with seroconversions to noroviruses and two to Cryptosporidium (one individual seroconverted to both pathogens). Among 176 (20%) individuals who reported swallowing lake water, there were six (3.4%) seroconversions compared to one (0.14%) seroconversion among the remaining 696 individuals: the crude and age-standardized risk differences per 1000 beachgoers were 32.7 (95% confidence limits 5.7; 59.6) and 94.8 (4.6; 276), respectively. The age-adjusted odds ratio of seroconversion in those who swallowed water vs. all others was 49.5 (4.5; 549), p = 0.001. Individuals with a norovirus seroconversion were more likely to experience vomiting symptoms within 4 days of the index beach visit than non-converters with an odds ratio of 34 (3.4, 350), p = 0.003. This study contributed further evidence that recreational water exposure is associated with symptomatic and asymptomatic waterborne infections, and that salivary antibody assays can be used in epidemiological surveys of norovirus and Cryptosporidium infections.

The gad2 promoter is a transcriptional target of estrogen receptor (ER)alpha and ER beta: a unifying hypothesis to explain diverse effects of estradiol.

Estradiol (E(2)) regulates a wide range of neural functions, many of which require activation of estrogen receptor alpha (ERalpha) and/or ERbeta, ligand-gated transcriptional regulators. Surprisingly, very few neural gene targets of ERs have been identified, and these cannot easily explain the myriad effects of E(2). GABA regulates most of the same neural functions as E(2), and GABAergic neurons throughout the brain contain ER. Therefore, we examined whether E(2) directly regulates expression of glutamic acid decarboxylase 2 (gad2), the enzyme primarily responsible for GABA synthesis for synaptic release. Using dual luciferase assays, we found that E(2), but not other gonadal steroids, stimulated the activity of a 2691 bp rat gad2 promoter reporter construct. Activation required either ERalpha or ERbeta, and ERbeta did not repress ERalpha-mediated transactivation. Site-directed mutagenesis studies identified three estrogen response elements (EREs) with cell-specific functions. An ERE at -711 upstream of the gad2 translational start site was essential for transactivation in both MCF-7 breast cancer cells and SN56.B5.G4 neural cells, but an ERE at -546 enhanced transcription only in neural cells. A third ERE at -1958 was inactive in neural cells but exerted potent transcriptional repression in E(2)-treated MCF-7 cells. Chromatin immunoprecipitation assays in mouse GABAergic N42 cells confirmed that E(2) induced ERalpha binding to a DNA fragment containing sequences corresponding to the -546 and -711 EREs of the rat promoter. Based on these data, we propose that direct transcriptional regulation of gad2 may explain, at least in part, the ability of E(2) to impact such a diverse array of neural functions.

Increased plasma reactive oxidant levels and their relationship to blood cells, total IgE, and allergen-specific IgE levels in asthmatic children.

Asthma is a disorder characterized by inflammation of the airways. Oxidative stress may play a role in the pathophysiology of several diseases including asthma. Characterizing biomarkers of oxidative stress in the context of other systemic measures of immune function or inflammation could provide insight regarding underlying mechanisms inducing asthma. We evaluated whether oxidative stress in the form of plasma reactive oxidants differs between asthmatic and non-asthmatic children and elucidate relationships between plasma reactive oxidants and other asthma-related immunological markers. Plasma reactive oxidants, white blood cell counts, total serum immunoglobulin E(IgE), and a multi-allergen-specific IgE screen were measured in 74 asthmatic and 74 non-asthmatic children(9 to 13 years of age) from the Detroit, Michigan area. Plasma reactive oxidants were measured using a lucigenin-based chemiluminescence assay. Plasma reactive oxidants, eosinophils, and neutrophils(absolute counts and percent of total white blood cell counts), total IgE, and allergen-specific IgE levels were elevated in asthmatics after adjusting for age, gender, and ethnicity. IgE(total or allergen-specific), eosinophils and neutrophils were not significantly associated with plasma reactive oxidant levels. The association between plasma reactive oxidants and asthma status was similar when eosinophils, neutrophils, total IgE, or allergen-specific IgE were included as possible confounders in multivariate logistic regression models. In conclusion, plasma reactive oxidants are elevated in asthmatics and appear to be an independent predictor of asthma status. Measurement of plasma reactive oxidants may be a useful adjunct diagnostic tool and potential mechanistic indicator relevant to the study of asthma and asthma exacerbation.

Human Cytomegalovirus Infections Are Associated With Elevated Biomarkers of Vascular Injury.

Background: Human cytomegalovirus (HCMV) infects ~50% of adults in the United States. HCMV infections may cause vascular inflammation leading to cardiovascular disease, but the existing evidence is inconsistent. Objective: We investigated demographic predictors of HCMV infection and explored associations between HCMV infection status, the intensity of anti-HCMV Immunoglobulin G (IgG) antibody response, and biomarkers of inflammation and endothelial function which are known predictors of cardiovascular disease. Methods: We conducted a cross-sectional study of 694 adults residing in the Raleigh-Durham-Chapel Hill, NC metropolitan area. Serum samples were tested for IgG antibody response to HCMV, and for biomarkers of vascular injury including soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), C-reactive protein (CRP), and serum amyloid A (SAA). Associations between HCMV and biomarker levels were analyzed using two approaches with HCMV serostatus modeled as a binary variable and as an ordinal variable with five categories comprised of seronegative individuals and quartiles of anti-HCMV antibody responses in seropositive individuals. Results: HCMV seroprevalence in the study population was 56%. Increased body mass index, increased age, female gender, racial/ethnic minority status, and current smoking were significantly associated with HCMV seropositivity in a multivariate regression analysis. HCMV seropositivity was also associated with 9% (95% confidence interval 4-15%) and 20% (0.3-44%) increases in median levels of sICAM-1 and CRP, respectively, after adjusting for covariates. The association between HCMV seropositivity and median levels of sVCAM-1 and SAA were positive but not statistically significant. Significant positive associations were observed between the intensity of anti-HCMV IgG responses and levels of sICAM-1 and sVCAM-1 (p-values 0.0008 and 0.04 for linear trend, respectively). To our knowledge, this is the first epidemiological study to show a relationship between anti-HCMV IgG responses and vascular injury biomarkers sICAM-1 and sVCAM-1 in the general population. Conclusion: HCMV infections are associated with vascular injury and inflammation biomarkers in adult residents of North Carolina.