Susan L. Ewart - US grants

Dr. Ewart proposes to investigate the mechanism(s) underlying airway hyperresponsiveness (AHR). This response is well recognized as an essential feature of asthma and other obstructive airway disorders. A genetic predisposition to nonspecific AHR can be demonstrated in humans and in many animal models. While multiple mechanisms may play a role in AHR, building evidence suggests T lymphocyte mediated bronchial inflammation is likely to be a common factor in the development of this response. The general hypothesis governing this proposal is that AHR is regulated by a gene associated with the inflammatory response. The goal is to map the gene that regulates AHR and to begin to elucidate the molecular mechanisms by which alleles at this locus determine AHR in vivo. A natural animal model in which a single autosomal recessive gene (Ach locus) directs acetylcholine mediated AHR in inbred strains of mice is proposed. The first objective will be to evaluate potential mechanisms by which T Iymphocytes mediate AHR. The qualitative and functional relationships between lymphokine production and AHR will, be characterized using reverse transcriptase polymersse chain reaction and transgenic methodologies. The second objective is to describe the detailed map location for the major gene (Ach locus) regulating airway responsiveness in this modeL Random polymorphic DNA markers and potential candidate genes will be evaluated for linkage with the AHR phenotype. These studies can be used to elucidate primary mechanisms establishing the phenotype of AHR, rule out disease mechanisms and suggest hypotheses for localizing human genes controlling AHR.

Dr. Ewart proposes to investigate the mechanism(s) underlying airway hyperresponsiveness (AHR). This response is well recognized as an essential feature of asthma and other obstructive airway disorders. A genetic predisposition to nonspecific AHR can be demonstrated in humans and in many animal models. While multiple mechanisms may play a role in AHR, building evidence suggests T lymphocyte mediated bronchial inflammation is likely to be a common factor in the development of this response. The general hypothesis governing this proposal is that AHR is regulated by a gene associated with the inflammatory response. The goal is to map the gene that regulates AHR and to begin to elucidate the molecular mechanisms by which alleles at this locus determine AHR in vivo. A natural animal model in which a single autosomal recessive gene (Ach locus) directs acetylcholine mediated AHR in inbred strains of mice is proposed. The first objective will be to evaluate potential mechanisms by which T Iymphocytes mediate AHR. The qualitative and functional relationships between lymphokine production and AHR will, be characterized using reverse transcriptase polymersse chain reaction and transgenic methodologies. The second objective is to describe the detailed map location for the major gene (Ach locus) regulating airway responsiveness in this modeL Random polymorphic DNA markers and potential candidate genes will be evaluated for linkage with the AHR phenotype. These studies can be used to elucidate primary mechanisms establishing the phenotype of AHR, rule out disease mechanisms and suggest hypotheses for localizing human genes controlling AHR.

DESCRIPTION (Adapted from the applicant's abstract): A genetic predisposition to the development of asthma along with exposure to environmental factors such as allergens are required for the development of clinical symptoms. Airway hyperresponsiveness, airway inflammation, and elevated serum IgE are integral components of the asthma phenotype. Abhr1(lod=4.2) and Abhr2(lod=3.7) are quantitative genes that control susceptibility to airway hyperresponsiveness in the progeny of inbred mouse strains(A/J and C3H/HeJ) with significantly different susceptibilities to allergen-induced airway hyperresponsiveness. These genes are located on murine chromosome 2 near the genes for GATA-3 and interleukin-1 receptor antagonist. GATA-3 is essential for Th2-driven inflammation and has been shown to be increased in the airways of asthmatics. Interleukin (IL-1) is a potent proinflammatory cytokine and has been implicated in chronic diseases, including asthma. Thus, based on compelling evidence for linkage and relevant mechanisms of action, we hypothesize that genes encoding GATA-3 and/or interleukin-1 receptor antagonist contribute to allergen-induced airway hyperresponsiveness in our murine model. The overall objective of the current study is to understand the molecular mechanisms that cause airway hyperresponsiveness. The investigators will fine map the location of the gene(s) causing allergen-induced airway hyperresponsiveness in our mouse model by refining quantitative associations between allergen-induced airway responsiveness and DNA marker genotypes using crosses between A/J and C3H/HeJ mice. They will investigate the role of positional candidate genes for antigen-induced airway hyperresponsiveness by 1) determining polymorphisms in genes encoding GATA-3 and interleukin-1 receptor antagonist; 2) determining whether these polymorphisms result in altered message or protein levels, and lastly determining whether polymorphisms are associated with allergen-induced airway hyperresponsiveness via consegregation and functional studies. The results of these studies may lead to better prevention and treatment of asthma.

DESCRIPTION (provided by applicant): Dramatic increases in the incidence and severity of allergic asthma, eczema, hay fever and food allergy have been seen recently, particularly in children;every fourth child in the U.S. is affected. Manifestations of these allergic diseases are varied and progress over time. Given this chronological variation, the assessment of children at a single time point may not yield an accurate picture of allergic disease. We have a unique cohort of over 1,000 children characterized for asthma and allergy at birth (in 1989-1990) and ages 1, 2, 4, and 10 years. Using longitudinal data from this cohort we propose to identify time-dependent patterns of allergic disorders, which we term allergic trajectories. Genetic predisposition is a key factor underlying asthma and allergy, yet our knowledge of susceptibility genes for these inflammatory disorders is limited. To gain greater understanding of the mechanisms of allergic asthma and other allergies, we will investigate candidate genes thought to mediate allergic inflammation, IL4, IL4R, IL13, IL1 and IL1RN, in DNA samples collected from our cohort of children. We will use a novel approach to perform genetic association studies by using allergic trajectories as phenotypes instead of relying on parameters assessed at a single time point. In addition to genetic susceptibility, environmental exposures are thought to influence both the presence and severity of allergic diseases. However, the nature of the interaction between genes and the environment remains unclear. Thus, we will assess gene x environment interactions between selected candidate genes and common environmental exposures in cohort children. We have assembled a highly experienced multidisciplinary research team with expertise in genetics, epidemiology, clinical medicine (allergy and immunology) and biostatistics. With this team we will be able to build on a strong foundation of a thoroughly characterized childhood cohort study population by expanding the epidemiologic analyses of asthma and allergy, and beginning genetic association analyses of candidate genes. By determining genetic and environmental risk factors in this cohort, we will contribute information valuable to both health care professionals and parents regarding management practices to minimize risks to susceptible children.

DESCRIPTION (provided by applicant): Health disparities continue to exist in our society. While the problem is multi-faceted and multi-factorial, lack of representation of the underserved groups in the health force is a critical deficiency, and one that can be addressed by research education programs. Experiential learning by active engagement in research is the best way to nurture the next generation of scientists and health care professionals. Therefore, this proposal focuses on recruitment and comprehensive mentored research education training of individuals from disadvantaged and underrepresented groups in the framework of a summer research program on the campus of Michigan State University. Six undergraduate and two graduate-professional students will be recruited to participate in the program. The students will receive close mentoring by faculty and participate in research focused on mechanisms of hypertension or airway biology and diseases. These research areas are prime examples where health disparities exist across various groups underrepresented in the biomedical enterprise. In addition, these diseases are a major concern both locally in Michigan and across the nation. Training faculty have been carefully selected and have been chosen on their mentoring record and ability to serve as excellent role models for the students. A comprehensive research education program has been designed to include seminars on specific research topics, as well as on health disparities in general. Workshops have been included to assist with the preparation of the students for pursuing further graduate and medical education. The students will have multiple opportunities to present their work and increase their communication skills. A supportive and intensive training environment will provide the students an excellent foundation to build on as they consider their career choices. The students will be tracked, and the program will be rigorously evaluated and modified as necessary to improve outcome. The overall goal of the program is to increase the number of qualified individuals from disadvantaged and underrepresented groups entering research careers in areas relevant to cardiovascular and pulmonary disorders. PUBLIC HEALTH RELEVANCE: Health disparities are population-specific differences in the presence of disease, health outcomes, or access to health and are a significant concern in our society. Lack of representation of underserved groups in the health force has been identified as a critical gap by both government and health professionals as inclusive research teams have broader perspectives in setting research agendas, recruiting subjects into clinical research studies, and working more effectively to deliver health care and reduce health disparities. This proposal seeks to address this gap by recruiting, training, and actively engaging underrepresented groups in experiential learning in fields of significant public health concern: hypertension and lung disease. (End of Abstract)

DESCRIPTION (provided by applicant): The need to establish a highly-qualified and diverse research work force in the biomedical and clinical sciences is well-recognized nationally. In doing so we must enlist the most talented individuals in the research and health care enterprise. The objective of the proposed training program is to contribute to the research workforce by providing research training to graduate veterinarians. The rationale for targeting veterinarians for research training is that they possess a uniquely-informed perspective from which to advance the various fields of biomedical research. This stems from the comparative approach that is used in veterinary medical education, as well as in the practice of veterinary medicine. Furthermore, the educational depth and diversity acquired through the veterinary education that our trainees will have upon entering our research training program provides a strong science and medical knowledge base upon which to build. The proposed research training program will be at the postdoctoral level, as trainees will hold a doctor of veterinary medicine (DVM or equivalent) degree. Funds are requested for 3 trainees and trainees are expected to be in the program for 3 years. The training will take place in the form of PhD level graduate education in the Comparative Medicine and Integrative Biology graduate program, in which trainees will conduct original research that will form the basis of a PhD dissertation. Traditional graduate education will be augmented with specific activities that will bolster trainees' readiness for research careers. Trainees will have the option of selecting as mentors (major advisors) a wide array of faculty trainers with nationally recognized expertise in respiratory diseases, cancer, neuroscience, inflammation, genetics, and enteric and infectious diseases.

DESCRIPTION (provided by applicant): Recognizing the need for the diverse skills needed in our biomedical enterprise, the College of Veterinary Medicine at Michigan State University, MSU-CVM, has invested in the training of the next generation of veterinarians in research experiences that will make them valuable contributors to basic, clinical and translational research. The yearlong predoctoral research program has been one of these successful efforts. The goal of the current training grant application is to continue to capture the enthusiasm and imagination of students in the middle of their DVM training and prime their appreciation and capacity for basic research before they recommence their clinical training. Allowing them to gain appreciation of basic research will increase their capacity for posing and answering critical questions to improve individual and public health, and increase their ability to pursue careers in research. We are seeking continued support for this program, now in its eight year of funding. Previous trainees have participated in combined DVM-MS or DVM-PhD programs and/or continued onto further clinical training. They have presented original research data in meetings and peer-reviewed publications. We propose to continue this program for highly qualified veterinary scholars and provide them special research training in the context of the Comparative Medicine and Integrative Biology MS Program, which will provide a nurturing and supportive environment. Our active engagement in summer research programs, both in our college and nationally, will enable us to continue to recruit outstanding students into this program. Special efforts will be made to recruit and nurture an inclusive pool of young scientist. The trainees will receive mentoring from faculty who are outstanding scientist dedicated to training the next generation of scientists. Training in the responsible conduct of research and building project management skills will be additional important components of the program. The training program will enhance our ability to meet the needs for biomedical researchers and improve human health.

Project Summary: Goal: Our goal is to determine the role of the host microbiome in providing resistance or enhancing susceptibility to allergic diseases. Our hypothesis is that alterations in the gut microbiome caused during early education of the immune system can affect the risk of allergic sensitization due either to shifts in microbiome composition after antibiotic exposure or to exposure to specific enteric pathogens. We also expect that certain commensal microbes/microbiomes are protective and may provide an evidence-based intervention strategy. Preliminary data: We have collected fecal microbiota samples from children in an ongoing prospective longitudinal study of offspring of individuals in the Isle of Wight (IOW) 1989 birth cohort, 30?40% of whom have allergy. We have exciting data in a small sample of infants ? 1 year old, where gut Enterobacteriaceae, primarily Escherichia/Shigella and Klebsiella, were robustly shown to be the predominant bacterial group associated with eczema; eczemic children often later develop asthma. Also, Bacteroidaceae predominance was associated with protection against eczema. In other work, we have shown that a human gut microbiota delivered as a fecal transplant can shift the immune response of C57BL/6 mice to an enteric pathogen from a Th1/17 bias to an autoimmune Th2 bias. Proposed work: We propose to create murine models of microbiome-related allergic sensitization by colonizing C57BL/6 mice with gut microbiota from IOW infants of known allergic status and testing development of bronchial hyper-responsiveness of the mice after challenge with a known human allergen. The results can then be compared to observed clinical outcomes in the IOW cohort infants. Once developed, these murine models can be used in future studies to determine gut microbiome-mediated mechanisms underlying risk of and protection from allergic sensitization, for example, by using data from IOW microbiome samples to identify particular bacteria associated with allergy, to isolate them and verify their identities using standard microbiological methods, and to study mechanisms leading to their effects on allergic sensitization in mice. Expected results: We expect to determine the ability of the gut microbiome to mediate allergic responses or protection in the Hu-microbiota mouse models and as a ?proof-of-concept? for use of the models to study this process in humans. An additional strength of these studies is that the clinical relevance of findings in the mice can be assessed in light of epidemiologic and microbiome data obtained in the IOW study. Impact and significance: We expect to provide new animal models for investigation of allergic diseases and protection from allergy as well as to identify potential new strategies to prevent initiation of allergies. Because both pathogenic and protective responses by microbiome members could be explored in these models, the results will pave the way for determining which members of the gut microbiome are major influences on the development of allergic disease and thus are targets that can be manipulated to prevent allergy.

Project Summary/Abstract Population-specific differences in the presence of disease, health outcomes, or access to healthcare are termed ?health disparities? and they are a significant concern in our society. Lack of representation of the underserved groups?which includes minorities, financially disadvantaged, and disabled individuals?in the biomedical research and healthcare workforce has been identified as a critical factor underlying health disparities. Indeed, disparities in the demographics of individuals entering careers in biomedical research are well documented. Biomedical knowledge and healthcare will benefit from broader inclusion of underrepresented individuals engaged in biomedical research as inclusive research teams have broader perspectives in setting research agendas, can more effectively recruit diverse subjects into clinical research studies, and work more effectively to deliver healthcare and reduce health disparities. Experiential learning by active engagement in research is an effective way to nurture the next generation of scientists and health care researchers. Veterinarians conduct research important to human health through comparative studies and animal models; unfortunately the racial and ethnic diversity in the veterinary profession is no better than that of the biomedical research workforce in general. Thus, this proposal is designed to recruit and comprehensively immerse both undergraduate and veterinary medical students from underrepresented groups in summer research experiences on the campus of Michigan State University. These research experiences will be focused on three major areas: hypertension, airway disease and blood cells and components in tissue pathology. The objective of our education research program is to increase the number of students from underrepresented groups who pursue biomedical research-related educational goals and career paths relevant to the mission areas of the NHLBI. The strategy to meet this objective involves 1) providing student participants an immersive research experience that exposes them to the various aspects of a research career, 2) cultivating participants' science identity by facilitating the development of mentor and peer networks, and creating a community that supports the participants' research-related interests, and 3) fostering the participants' successes so as to give them the confidence needed to pursue a research-related career. This strategy will be implemented through the following specific aims: (1) engage undergraduate and veterinary medical students in high-quality mentored research in the areas of heart, lung, and blood diseases; (2) provide comprehensive exposure to the various aspects of biomedical research through an organized program of activities; (3) bolster the trainees' understanding of the critical concepts of responsible conduct of research; (4) recruit trainees from populations underrepresented in the biomedical sciences; and (5) conduct comprehensive evaluations to determine the effectiveness of our programming in enhancing diversity in health-related biomedical research.

ABSTRACT The objective of this professional student short-term research training grant is to provide predoctoral training opportunities to 14 veterinary students annually who have completed their first or second year with immersive experiential learning by carrying out mentored biomedical research, enriched by seminars and events that are designed to inspire them for careers combining their medical training and research. The goal of the program is to capture the enthusiasm and imaginations of DVM students early in their training to prime their appreciation and capacity for basic and translational research. Students will be engaged in hypothesis- driven discovery and problem-solving research in a high-quality laboratory setting for 12 weeks during the summer. Only the most outstanding researchers/mentors from have been included as trainees in the proposed research training program. Participating faculty have been grouped into the following areas of research excellence: 1) Antimicrobial Resistance and Microbial Pathogenesis, 2) Vector Borne Diseases and Disease Ecology, 3) Immunity and Immune Modulation 4) Airway Biology and Disese 5) Comparative Toxicology and 5) Animal Models, Sponaneous Diseases in Animals and Translational Studies, serving as convergent foci. A week long orientation program, and special events have been designed to guide the students for success in this experiential learning opportunity. Training in responsible conduct of research and data reproducibility and reliability are integral parts of the program with special sessions and seminars reinforcing these principles. Recruitment efforts planned are directed not only to DVM students in our college, but to veterinary students from other U.S. colleges of veterinary medicine and are designed to ensure that students underrepresented in the biomedical sciences have access to this research experience. All students participating in the program are required to present their research results at the annual College of Veterinary Medicine Phi Zeta Research Day, and encouraged to present at the National Veterinary Scholars Symposium and other national professional meetings, and to publish their work with their mentors, as appropriate. Increasing our human capital in veterinary scientists is critical to the protection of public health and advancement of science that benefits animals and humans, both as individuals and as populations. Summer research experiences provide a critical step into the research enterprise to the veterinary students early in their training and foster interest and develop aptitude for further training in the biomedical sciences, serving as a steping stone for research intensive careers as biomedical scientists.

PROJECT SUMMARY Pregnancy is an important transition period that parous women (those who have given birth) undergo and it is linked to changes in risk of various diseases as compared to nulliparous women (those who have never given birth). The number of completed pregnancies changes the risk of autoimmune disease, Alzheimer's disease, cancers of the ovary, breast and liver, as well as asthma death later in life. However, it is unknown how these disease risks are altered by the child-bearing experience. DNA methylation (DNA-m) - the addition of a methyl group to cytosine in cytosine-phosphate-guanine (CpG) dinucleotides sequences in DNA - has been shown to be associated with multiple health outcomes later in life, such as atopy, eczema, hypertension, diabetes mellitus, and cancer. Our preliminary studies have shown that more CpGs (5.6% vs 0.7%) change levels of methylation during pregnancy (measured between age 18 years and again during pregnancy in women not yet pregnant at age 18) compared to pre-pregnancy (between ages 10 and 18 years), which may represent a plausible mechanism for altering post-pregnancy disease risks in parous women if these changes remain after pregnancy. To better understand the links between methylation changes during pregnancy and parous-related diseases later in life, we propose to identify pregnancy-related CpGs with significant pre- and post-pregnancy methylation changes through an epigenome-wide study. In Specific Aim 1, we will identify CpGs with significant methylation changes in parous women between ages 18 and 26-27 years compared to nulliparous women over the same time period. Then in Specific Aim 2, we will focus on the CpGs identified in Specific Aim 1 and test what proportion of them change from age 18 years to pregnancy and remain stable after parturition up to age 26-27 years. This proposed research has the potential to significantly contribute to 1) the discovery of biomarkers affected by a healthy pregnancy, 2) a better assessment of the long-term effects of parity on women in later life, and 3) the future development of epigenetic tools to detect these modified disease risks. Our collaborative research team is well-positioned to do this work with complementary expertise in genetics and epigenetics, reproductive epidemiology and (bio)statistics, and clinical medicine. We have a long track record of successful collaboration investigating DNA methylation changes during critical transition periods, such as puberty and pregnancy.