Development and exacerbation of asthma have long been associated with exposure and sensitization to allergens. While exposure to respiratory allergens such as German cockroach, mouse, cat, and dust mite is thought to occur largely by inhalation, the best method to measure the exposure remains unclear. Similarly ambiguous are the ideal measurement and allergic or respiratory effects of exposure to fungi. As most people in the industrialized world, especially small children, spend the majority of their time indoors, the home environment is of prime importance. Previous researchers have shown that poor housing quality or maintenance lead to growth of fungi, increased pest populations, and higher concentrations of other allergens and irritants. These elevated levels in turn are associated with greater rates of sensitization and asthma in the occupants. This dissertation focused on exposure measurement, assessment of sensitization to common molds, and estimating the effects of neighborhood-level pest prevalence and housing quality on asthma symptoms. The study subjects and homes were part of either the Puerto Rican Asthma Study (PRAS), a longitudinal cohort study focused on identifying how multiple risk factors affected allergic sensitization and asthma development in U.S.-born Puerto Rican children with atopic mothers, or the Head Start Study (HSS), which examined allergen exposure and sensitization in young children of low-income families. From 199 of these New York City homes, settled dust, high-volume air, and nasal air sample measurements were simultaneously collected and analyzed for cockroach, mouse, dog, cat, rat, and mite allergens. Cockroach and mouse allergens were quantified from all three sample types while the other allergens were measured from air samples only. Ninety-three women from PRAS were tested for sensitization to six species of mold using the halogen immunoassay and four mold species using ImmunoCAP. The results were compared to previously quantified specific-IgE to other inhalant allergens as well as to self-reported allergy and asthma symptoms and demographic characteristics. Finally in a cross-sectional sample of 225 children from both study populations, the impacts of housing conditions and pests on current asthma at both the individual and neighborhood level were examined. Questionnaire data on demographics, housing factors, asthma symptoms, and health behaviors were evaluated with allergic sensitization and environmental sampling results for each child. They were also grouped and contrasted by neighborhood using United States Census neighborhood-level data on reported pest prevalence and housing quality. The effects of individual and neighborhood factors on current asthma symptoms were estimated using a generalized linear model. Allergen concentrations were generally highest in settled dust, followed by high-volumetric air, and then nasal air samples. Mouse allergen was most frequently detected in air samples, followed by dog, cat, and cockroach. No samples contained rat or any of three types of mite allergens above the detection limit. While all three measurements enhanced the exposure picture for mouse allergen, air samples rarely had detectable cockroach allergen despite being detected in settled dust. This led to the conclusion that settled dust sampling is still crucial when assessing a child's exposure to cockroach allergen but may be less important for buoyant mammalian allergens such as mouse. Nearly one-third of the 93 mothers were sensitized to one or more molds as determined by either assay. Being sensitized was positively associated with sensitization to tree, grass, or pigeon allergens but not to other inhalant allergens. Moreover there was no association seen between sensitization to the fungal species and asthma or allergy symptoms. Of note, however, interesting differences between the halogen immunoassay and ImmunoCAP were identified that merit additional investigation. For the 225 children, current asthma symptoms were positively associated with early respiratory infections, presence of environmental tobacco smoke, having higher concentrations of cockroach allergen in bed dust, a higher intensity sensitization level to one or more inhalant allergens, and current asthma in the mothers. After adjusting for individual-level factors (cockroach allergen in bed dust, environmental tobacco smoke, and study population), no effect of neighborhood-level characteristics could be associated with current asthma prevalence. The lack of effect was likely due to a combination of factors including: small sample size, self and other selection biases, and insufficient diversity across the study population and neighborhoods

Background: The physiological and pathophysiological roles of secreted membrane-enclosed vesicles known as extracellular vesicles (EVs) have become increasingly recognized, making the EV field a quickly evolving area of research. EVs and their encapsulated molecular material including microRNAs are key mediators of intercellular communication, making EVs analogous to a message in a bottle. This discovery has fundamentally changed the study of gene regulation, and understanding the central role of EVs and their cargo in health and disease will generate new opportunities for basic biology, diagnostics, and disease treatment. EV release and the packaging of molecular material into EVs can be altered by stressors such as air pollution exposure. Exposure to air pollution is associated with significant morbidity among individuals with asthma, especially children who participate in more frequent outdoor activities and are more susceptible to exposure due to their narrower airways and higher breathing rate. Thus, sensitive biomarkers of air pollution exposure are needed to identify children at risk of worsened symptoms and asthma exacerbations. Given their role in cell-to-cell communication, EVs also represent a plausible molecular mechanism in the etiology of disorders such as aging-related cognitive decline. Individuals with mild cognitive impairment and experiencing increased rates of cognitive decline are more likely to develop Alzheimer’s disease and other dementias, signifying the importance of identifying and treating cognitive impairment early. More precise identification of the neurobiological processes of cognitive decline in aging populations may provide critical insights into the precursors of late-life dementias and identify health interventions that can delay cognitive impairment or therapeutic targets for treatment. This dissertation evaluates the utility of EV-encapsulated microRNAs (EV-miRNAs) as biomarkers of environmental exposure (i.e., air pollution) and assesses their role in disease risk (i.e., cognitive decline) in two separate studies. First, in Chapters 2-3, using a cohort of children with asthma in the greater Boston area, we describe saliva EVs isolated from these children using a high-throughput method and explore the potential of salivary EV-miRNAs as easy-to-measure biomarkers of exposure to fine particulate matter (PM2.5), nitrogen dioxide (NO2), and ozone (O₃). Then, in Chapter 4, we evaluate the association between EV-miRNAs and cognitive function and rates of cognitive decline in a cohort of elderly men and discuss the utility of circulating EV-miRNAs as biomarkers of risk. Furthermore, we discuss the pathways that these EV-miRNAs target if they play a causal role in cognitive decline which could have implications for development of therapeutics. Methods: Drawing from the School Inner-City Asthma Study (SICAS), we isolated salivary EVs and EV-miRNA from children with asthma for analysis in relation to ambient exposure to PM₂.₅, NO₂, and O₃. In accordance with the recommended minimal experimental requirements for the definition of EVs, in Chapter 2 we employ multiple orthogonal methods to describe the EVs that were isolated from cell-free saliva using a high-throughput polymer-based reagent (ExoQuick-TC). In Chapter 3, we utilize EV-miRNA data generated via RNA sequencing and ambient air pollution data estimated using a validated spatiotemporal high-resolution model. We perform differential expression analyses to examine the effect of high exposure to PM₂.₅, NO₂, and O₃ on saliva EV-miRNA abundance. In Chapter 4, we leverage data from the Normative Aging Study, a longitudinal cohort of elderly men, to investigate whether circulating EV-miRNAs are associated with cognitive function and rates of cognitive decline. We used linear models to assess the relationship between plasma EV-miRNAs and cognitive function and linear mixed models to evaluate the relationship between plasma EV-miRNAs and rates of cognitive decline. We performed gene ontology functional enrichment and pathway enrichment analyses to identify the biological pathways that these EV-miRNAs would target if they play causal roles in cognitive decline. Results:In Chapter 2, we demonstrate that EVs can be isolated from human saliva using ExoQuick-TC. The saliva EVs isolated from ExoQuick (N=180) ranged in size but were mostly ~55 nm in diameter and expressed tetraspanins CD9 and CD63, canonical markers for EVs, but did not highly express the tetraspanin CD81. In Chapter 3, in a subset of the SICAS cohort (N=69), we show that relatively high (>19.37 parts per billion) short-term ambient NO2 exposure and relatively high (>38.38 parts per million) prior-day O3 exposure are associated with down-regulation of several saliva EV-miRNAs. We did not detect differential expression of any EV-miRNAs in relation to PM₂.₅ exposure over multiple time windows of exposure. Finally, in Chapter 4, multivariable linear and linear mixed models demonstrated a relationship between several plasma EV-miRNAs and global cognitive function and rates of global cognitive decline, measured by the Mini-Mental State Examination. Functional enrichment and pathway enrichment analyses revealed that the biological pathways targeted by these miRNAs are relevant in neurodegeneration, including pathways regulating synaptic function and plasticity and neuronal death. We found no association between EV-miRNAs and cognitive function or cognitive decline as assessed by cognitive tests measuring specific domains of cognitive function. Conclusion: This work demonstrates the opportunities that EV-miRNAs can create for advancing environmental health research. EV-miRNAs may serve as sensitive biomarkers of environmental exposures as well as biomarkers of risk and may play mechanistic roles in disease. We also make recommendations for integrating EV research into the field of environmental health. Future studies should continue to evaluate the potential of EV-miRNAs and seek to identify EV-miRNAs that can serve as mechanistic biomarkers between exposures and effect across all stages of life to (1) increase our understanding of the consequences of circulating miRNA changes and the contribution of the environment to heterogeneous disorders, (2) advance development of non-invasive diagnostics to allow for longitudinal monitoring, and (3) pave the way for new opportunities for disease prevention and treatment.

Chapter 1. Background: Asthma is the most common chronic childhood disease and is characterized by recurrent airway obstruction, bronchial hyper-responsiveness, and airway inflammation. Asthma is the leading cause of childhood hospitalization and school absenteeism in the United States. The associations between adverse respiratory effects and exposure to indoor nitrogen dioxide (NO2) and other byproducts of combustion such as particulate matter (PM) in particular ultrafine particulates (UFP), Ozone (O3) and Sulfur Dioxide (SO2), have been the focus of many epidemiological studies in recent years. Indoor exposure to NO2 and other pollutants from combustion may increase the risk of acute and chronic respiratory disease, reduce lung function, initiate and exacerbate asthma in children. The levels of exposure to NO2 indoors are of public health concern because children spend nearly 70% of their time indoors at home. According to the 2010 US Census report, approximately 39% of US households use natural gas for cooking, and the primary source of residential NO2 is a gas-fuel cooking appliance. Indoor levels of NO2 where NO2 sources are present can be much higher than outdoors, where the primary source of NO2 is vehicular traffic. Epidemiological studies in developed countries suggest that gas stoves used for cooking and/or heat are associated with an increased risk of asthma and respiratory symptoms in children. While there are numerous, epidemiological studies supporting an association between increased NO2 levels and gas stoves and asthma symptom severity in children, there are other studies that have examined the relationship in homes that did not observe significant associations. A better understanding of how NO2 and other indoor environmental (e.g., environmental tobacco smoke (ETS), allergens) exposures contribute to asthma morbidity in inner city preschool children will allow interventions to more effectively designed and implemented. To date, there are conflicting results on the role of exposure to indoor NO2 and its association with new-onset asthma in young inner-city children. The recent studies assessing the effects of indoor NO2 on asthma morbidity were limited to inner-city children, largely older, who were diagnosed with asthma. A gap in knowledge remains regarding the role indoor NO2 plays on the development of asthma in children not previously diagnosed. The scientific and public health rationale for conducting this dissertation was to describe the association of exposure to indoor NO2 and primary sources with the initiation and exacerbation of asthma symptoms among pre-school children with and without diagnosed asthma. The data analyzed in the current research come from a larger study of Endotoxin, Obesity, and Asthma (EOA) in the New York City Head Start Program, funded in the summer of 2002. The primary research objective of that study was to identify modifiable risk factors associated with asthma and asthma persistence among preschool children from low-income families living in select New York City neighborhoods with high pediatric asthma hospitalization rates. We conducted a cross-sectional analysis of data collected from the study questionnaire and home visit sampling at study enrollment. The analyses were performed in two phases: the first phases used data collected at study enrollment and the second phase used data collected 12-months after study baseline. Henceforth, the dissertation will refer to the first analyses as the baseline study and the second as the follow-up study. The research evaluated the association of NO2 exposure with asthma status among New York City Head Start children with and without asthma at study enrollment and with respiratory symptoms among children with asthma at 12-month follow-up. Chapter 2. Baseline Study: We conducted a cross-sectional analysis of data collected from the study questionnaire and home visit sampling at study enrollment. Specifically, the research sought to evaluate the association of NO2 exposure with asthma status among New York City Head Start children with and without asthma at study enrollment and with respiratory symptoms among children with asthma at enrollment. A total of 503 children were included in the baseline study. A total of 105 children (20.9%) met the criteria for both asthma and allergy, and 67 (13.3%) met the criteria for asthma alone. Girls made up 51.7% and boys, 48.3% of the 503 study participants. Descriptive analyses suggested that asthma/allergy status was associated with: male gender, non-Mexican ethnicity/national origin, presence of a smoker in the child’s home, number of smokers in the child’s home, self-reported parental history of asthma, mother’s education level and sensitization to one or more of the four allergens. Logistic regression models were used to investigate the magnitude and direction (as well as trend) of the association between childhood asthma and indoor NO2 sources in the child’s home. Chapter 3. Follow-up Study: Our follow-up study involved the analysis of the 12-month follow-up data from the study of Endotoxin, Obesity, and Asthma in the New York City Head Start Program funded in the summer of 2002. We focused on assessing the magnitude and direction of the associations of exposure to indoor NO2 levels (based on baseline NO2 measurements) with children’s asthma status and with symptom severity among asthmatics at 1-year follow-up. For the follow-up study, we categorized children by whether their asthma status had changed since baseline. Descriptive analyses were performed looking at key characteristics by “change in asthma status.” Children’s asthma status at baseline and at follow-up, were based on responses to the questionnaire. We analyzed indoor NO2 level measurements at baseline in relation to asthma outcomes on follow-up. We did not have enough data on NO2 levels at follow-up to analyze them in relation to asthma status on follow-up. Unless the family had relocated since baseline and/or reported changes since baseline in the use of gas appliances or the number of smokers in the home, we assumed that baseline NO2 levels in the participating children’s homes were reasonable proxies for current exposures. We looked at the number of children who moved since baseline and whether the move (for example, looking at gas stove status, age of new building) may have impacted indoor NO2 levels. Of the 503 children who were included in the baseline analyses, 47.3% had data on asthma status on follow-up. A total of 238 children (111 male, 127 female) were grouped into the four mutually exclusive outcome categories: 122 (51.3%) did not have asthma at baseline or on follow-up, 34 (14.3%) had asthma on follow-up but not at baseline, 65 (27.3%) had asthma at baseline but not on follow-up, and 17 (7.1%) had asthma at baseline and on follow-up. The mean age at 1-year follow-up was 59.5 months (6.95), and neither age nor gender was associated with asthma. The distribution of ethnicity/national origin among the 238 children remained the same as at baseline; no one ethnicity group experienced disproportionate loss to follow-up, and asthma status remained associated with non-Mexican ethnicity/national origin, although 44.1% with new-onset asthma were of Mexican background. Asthma was also associated with self-reported parental history of asthma and allergy in children, but nearly 80% of children with new-onset asthma had no such parental history of asthma. More parents of children with new-onset (35.3%) or persistent asthma (23.5%) than of other children reported making efforts to reduce risk factors or triggers for asthma exacerbations in the past 12 months. Chapter 4. Dissertation Conclusion : The primary objective of the dissertation research was the examination of the relationship between asthma and asthma severity and exposure to gas cooking and residential NO2. In both our baseline and 12-month follow-up studies, exposure to indoor NO2 was represented by the baseline measurement of NO2 and the NO2 surrogate, gas stove. Asthma status of children was based on parental responses on the questionnaire regarding asthma symptoms and urgent care visits due to respiratory distress over the course of each 12-month period prior to the conducting study questionnaires. For both studies, we did not find an association between exposure to NO2 levels at baseline and asthma status or severity. Our findings contradict the results of most recent studies of both NO2 levels and residential sources of NO2 and their effects on asthma symptoms in very young children. However, it remains difficult to compare our results we those of previous published studies because those studies primarily focused on children who were diagnosed with asthma, whereas our research included preschool aged children with and without asthma. Based on our findings and the fact they conflict with other epidemiological studies, of which there were also conflicting results, we feel that the relationship between asthma symptoms and NO2 exposures remains ambiguous. The lack of consistent results of epidemiological research raises questions that should be the focus of future epidemiological studies. What are the roles of co-pollutants and co-risk factors? Does NO2 work alone or in concert with other indoor pollutants? There exists a real lack of understanding on the possible synergistic effects of exposure to NO2 and other combustion byproducts. Important to furthering our knowledge of the role of exposure to indoor NO2 and asthma is determining whether NO2 acts as a surrogate for co-pollutants that are considered risk factors for asthma and other respiratory conditions. Another focus of future indoor pollution studies should be the development of effective methods and technologies for measuring the constituents of the complex mixture of pollutants in indoor air; these methods and technologies can then be applied in personal monitoring of exposure to indoor pollutants in epidemiological studies that would help to determine with much more accuracy the effects of individual indoor pollutants on asthma and other respiratory symptoms. This knowledge would help in the development of more effective public health and environment policies towards reducing the burden of childhood asthma.

Indoor air quality has become an important health concern due to the number of indoor pollutants and the realization that even minimal exposures to volatile organic compounds may produce direct or indirect adverse health outcomes. Young people are most vulnerable to these poisonous chemicals as they spend much of their times indoors at homes, schools, nurseries and in day care centers. Exposure to volatile organic compounds indoors has been related to asthma and other respiratory symptoms. The adverse effects of air pollution on respiratory health in South Durban have been described in a number of studies. In 2000, a study in the South Durban Basin at Settlers Primary School demonstrated both a high prevalence of respiratory diseases amongst schoolchildren as well as an association between ambient air pollutants and other adverse health outcomes. The South Durban Health Study subsequently undertook a health risk assessment and an epidemiological study investigating this association further on behalf of the eThekwini Municipality. The study highlighted that relatively moderate ambient concentration of N02, NO, PMIO and S02 were strongly and significantly associated with a reduction in lung function among children with persistent asthma. Moreover, attending primary school in South Durban was significantly associated with increased risk from persistent asthma when compared to schools in North Durban. METHODS The descriptive study measured the total volatile organic compound levels within selected homes and schools of asthmatic children in South and North Durban. Recommendations for reducing or mitigating indoor total volatile organic compound exposures were made. The study involved a secondary analysis of data obtained from the South Durban Health Study. The monitoring for total volatile organic compounds within homes and classrooms was undertaken using passive samplers during a 72-hour period and analyzed using a gaschromatography/ mass spectrometry method. Temperature and humidity was assessed using temperature and humidity sensors. Statistical analysis was performed using SPSS version 13. The dataset comprised 140 total volatile organic compound samples from homes and 14 from classrooms. Total volatile organic compounds were measured in microgram per cubic meter (g/m3), temperature in degrees Celsius and relative humidity in percentage of moisture. RESULTS Total volatile organic compounds with levels in households ranging from 17g/m3 to 1440g/m3 and in classrooms ranging from 48g/m3 to 5292g/m3 were measured. The mean levels detected were significantly different in homes and classrooms / Thesis (MMed.)-University of KwaZulu-Natal, Durban, 2008.