Christopher L. Coe, PhD - US grants

The proposed studies will assess the effect of psychological disturbance on several aspects of humoral and cellular immunity. Based on previous research showing altered immune responses in infant and juvenile primates, we propose to conduct a detailed characterization of macrophage function in young squirrel monkeys following two different types of separation from the mother. Phenotypic aspects of macrophages will be examined using: (1) surface morphology and nonspecific esterase staining, and (2) immunochemical techniques that utilize monoclonal antibodies directed against macrophage surface proteins. Functional capabilities of macrophages from control and stressed subjects will be compared by measuring (1) phagocytosis and intracellular killing, (2) antibody dependent cytolitic events, and (3) support and regulation of T lymphocyte functions. A second series of studies will compare the effect of acute psychological disturbance on T cell subsets in juvenile monkeys of two species, the squirrel monkey and rhesus macaque. The comparison is based on prior demonstrations of significant species differences in endocrine and immune responses, and will evaluate the influence of temporal factors, activity patterns, and the effect of providing social companions. Pharmacological manipulation of the pituitary-adrenal axis will also be employed to evaluate the role of cortisol in mediating the shifts in circulating levels of T lymphocytes. The third area of research is concerned with the impact of psychological disturbance during pregnancy on the ontogeny of infant immunity. The influence of female social status and relocation during pregnancy will be assessed by monitoring the differential transmission of IgG to newborn infants. IgG levels will be examined during the first 3 months of infant development, and the capacity of the infants to mount an antibody response to antigen challenge will be determined. The antigens we will use are the bacteriophage (OX174) and tetanus toxoid (TT) to assess the significance of psychoendocrine activity during pregnancy and the resulting alterations in the transmission of maternal antibody. We have already established the efficacy of both antigens and assay methods in previous research on nonhuman primates.

Prior research by our laboratory has demonstrated that stressful events experienced by the pregnant female monkey can affect her fetus and alter the development of several important immune responses in her young infant. The proposed studies will further investigate the relative vulnerability of the fetus by assessing the long-term effects of prenatal disturbance on immune responses and disease susceptibility during the first year of life. This research will also extend previous observations of a differential vulnerability of male and female infants. Immune assessment of the infant rhesus monkeys will focus on the development of lymphocyte cytotoxic responses, circulating levels of various cell subsets, and antibody responses. The potential clinical significance of the physiological alterations induced by prenatal disturbance will be evaluated by determining the infant's antibody responses to Haemophilus influenzae vaccination and, at an older age, by inoculation with Salmonella typhimurium. This attenuated strain of Salmonella is being used to deliver viral antigen to the mucosal immune system -- an approach that is currently being tested in vaccine studies to prevent transmucosal infection with simian immunodeficiency virus. In addition to assessing the immune effects of pregnancy disturbance in a reliable nonhuman primate model, the research will determine whether there are periods of particular vulnerability during pregnancy. One experiment will also assess whether the absence of breast milk and its soluble immune products exacerbates the effects of prenatal disturbance. The goal of the research program is to refine our understanding of how prenatal events influence the development of immune competence and facilitate the establishment of normal set points for certain physiological responses in the young infant.

posttraumatic stress disorder; female; neuroendocrine system; obesity; hormone regulation /control mechanism; psychoneuroimmunology; violence; body weight; hypothalamic pituitary axis; catecholamines; behavioral /social science research tag; human subject; clinical research;

virus; AIDS; immunology; communicable diseases; lymphatic system; Primates; Mammalia;

OBJECTIVE To evaluate the influence of prenatal conditions on the development of immune responses during infancy. RESULTS We continue to assess the way different pregnancy conditions affect the development of the nervous and immune systems of the infant monkey at birth and across the first year of life. We have shown that prenatal experiences can have long-lasting effects on many behavioral and lymphocyte responses during development, including the cellular ability to recognize and respond to nonself antigens. FUTURE DIRECTIONS To consider the relevance of these findings for understanding individual differences in postnatal immunity and response to infection. KEY WORDS infant development, prenatal, lymphocyte proliferation, natural killer cell activity FUNDING NIMH MH41659

virus; AIDS; immunology; endocrine gland /system; communicable diseases; lymphatic system; Primates; Mammalia;

virus; AIDS; immunology; communicable diseases; lymphatic system; Primates; Mammalia;

women's health; reproductive system; immunology; lymphatic system; Primates; Mammalia; aging;

OBJECTIVE: To evaluate the relative contribution of heritable and environmental factors to birthweight of the infant rhesus monkey. RESULTS A retrospective analysis of 25 years of breeding and births was conducted to develop a predictive model of factors that influence birthweight. Across this time period, there was a significant increase in infant birthweight, but the increases were relatively more evident in female than in male offspring. Moreover, there were distinct matrilines that showed this increase more than others. The relationship reflected a high correlation between maternal weight gain during pregnancy and infant birthweight. Specifically, those female matrilines that were prone to show larger weight gain during pregnancy were the ones to bear heavier offspring. We believe this association reflects that the transgenerational passage of a uterine constraint mechanism from mothers to their daughters. FUTURE DIRECTIONS This hypothesis will now be tested in prospective studies, and further statistical modelling of the existing data base. KEY WORDS birthweight, pregnancy, breeding

OBJECTIVE: To investigate to ability of a genetically altered bacterial vector to stimulate mucosal and humoral immunity against envelope proteins of simian immunodeficiency virus. RESULTS A recombinant strain of Salmonella typhimurium that expresses p27 was tested to determine if it would stimulate immunological memory and facilitate antibody responses against envelope proteins. Gut exposure to this strain of S.t. was sufficient to augment immunity, also demonstrating the important relationship beween the mucosal and humoral immune compartments. FUTURE DIRECTIONS Having established that this bacterial vector elicits immunological memory against envelope proteins it would be important to determine whether this vaccine strategy is sufficient to promote protective immunity. In addition, it will be important to verify that the attenuation process is stable and that the bacteria is not prone to mutate in the gut environment. KEY WORDS SAIDS, mucosal immunity, Salmonella typhimurium, vaccine

OBJECTIVES To examine the influence of cytokines on the release of soluble mediators in the peripheral blood and intrathecal compartments. RESULTS Cytokines, such as interleukin-1 (IL-1), are potent stimulators of endogenous cytokine release and the neuroendocrine axis, and thus play an important role in the communication between the immune system and the brain. We have been investigating the influence of IL-1 on other cytokines, such as interleukin-6, in the peripheral blood and intrathecal compartments. Following our demonstration that IL-6 is released endogenously from within the CNS, we have been investigating the primary cellular source. This research has focused on the endothelial cells of the blood-brain-barrier. In addition, we have been investigating the degree to which corticosteroids are involved in modulating these proinflammatory responses, which has bearing on the role of steroids in sepsis and anaphylactic shock. FUTURE DIRECTIONS Our studies are now focused on endothelial cells of the blood-brain-barrier. For this project, we have established endothelial cell cultures that will permit the research to go forward with in vitro models of the BBB. KEY WORDS cytokines, interleukin-1, interleukin-6 cerebrospinal fluid, psychoneuroimmunology FUNDING NIMH Dissertation Award, NSF Predoctoral fellowship, NIMH MH41659 PUBLICATIONS Reyes, T.M. and Coe, C.L. 1998. The proinflammatory cytokine network Interactions in the CNS and blood of rhesus monkeys. American Journal of Physiology 43 R139-149. Reyes, T.M. & Coe, C.L. 1998. Resistance of the central nervous system interleukin-6 to glucocorticoid inhibition in monkeys. American Journal of Physiology 44 R612-618.

OBJECTIVE: To evaluate changes in quinolinic acid production in response to viral infection and disease progression. RESULTS Previously we demonstrated that the neurotoxin, quinolinic acid, is increased in both systemic circulation and the intrathecal compartment following retroviral infection. We have now begun to investigate the potential sources of QUIN within the CNS. QUIN within the CNS may derive from diffusion across the blood-brain-barrier or may reflect endogenous production with the CNS by cells related to the monocyte/macrophage lineage. Previously collected, frozen postmortem tissue samples from infected animals are being analyzed to determine the regional distribution of QUIN within the brain. FUTURE DIRECTIONS One future direction would be to test the effectiveness of NMDA receptor antagonists in blocking the adverse effects of QUIN on neural tissue. KEY WORDS quinolinic acid, blood-brain-barrier, simian immunodeficiency virus, cerebrospinal fluid NIMH MH41659

DESCRIPTION (adapted form the investigator's abstract): This project proposes to evaluate psychological well-being, symptom expression, and endocrine/immune functioning in women with two musculoskeletal conditions, fibromyalgia (FMS), and rheumatoid arthritis (RA). Specifically, the studies will assess whether a capacity to sustain a sense of psychological wellbeing results in severity and self-reported experience of symptoms. The first study will compare women with FMS, RA, and healthy controls across two phases of the menstrual cycle for symptom expression, pain sensitivity, and physiological function. In a second intervention study, an attempt will be made to enhance psychological wellbeing and positive affect with an eight-week training program in women newly diagnosed with RA and FMS.

OBJECTIVE To evaluate the influence of prenatal conditions on the development of immune responses during infancy. RESULTS We continue to assess the way different pregnancy conditions affect the development of the nervous and immune systems of the infant monkey at birth and across the first year of life. We have shown that prenatal experiences can have long-lasting effects on many behavioral and lymphocyte responses during development, including the cellular ability to recognize and respond to nonself antigens. FUTURE DIRECTIONS To consider the relevance of these findings for understanding individual differences in postnatal immunity and response to infection. KEY WORDS infant development, prenatal, lymphocyte proliferation, natural killer cell activity FUNDING NIMH MH41659

The Biological Measures Core will provide the resources and expertise necessary to collect and analyze the hematological, neuroendocrine and cytokine indices for all research projects. This Core will create the requisite infrastructure and oversight to ensure the collection of reliable and high quality data through centralized testing and recordkeeping. Specifically, the Core will oversee the acquisition of specimens at the 3 regional General Clinical ResearchLaboratories, and direct the shipment of specimens to a central clinical laboratory in Madison, WI. It will also be the repository for the saliva samples from the diary studies to be used for assessing cortisol levels, and be the primary liason with the assay laboratory. Finally, the Core will maintain its own biochemical facility for the analysis of cytokine levels, and serve an archival function for the long-term storage of specimensfor the entire MIDUS program. In addition to assisting in the diagnostic testing, the Core will also be responsible for the creation of the data files of test results, and the dissemination of the information to primary investigators for each project. In this capacity, it will serve as the official repository for the paper records from all hematological and neuroendocrinetests, and will be responsible for all aspects of quality assurance, including assay reliability, data entry, and oversee any needed retesting of specimens. The Core will generate "reference norms" for all test results, both from the MIDUS as well as with respect to other studies and clinical laboratories, and thereby also serve a consulting role concerning the validity and meaning of the biological measures.

fertility; Primates; animal colony; animal puberty;

intestines; bacteria; Macaca mulatta; animal colony; infant animal;

[unreadable] DESCRIPTION (provided by applicant): This project will use a nonhuman primate model to investigate the impact of maternal influenza infection on infant behavior and brain functioning postpartum, building upon many studies showing that pregnancy conditions can significantly affect fetal development. In addition to being a cause of morbidity and mortality, concern has grown that flu infections during pregnancy may induce long-term changes in offspring even when maternal symptoms are seemingly benign. The effect of controlled flu infections with A/Sydney/5/97 [H3N2] in mid- and late- pregnancy will be prospectively assessed in a large cohort of 108 rhesus monkeys. After comparing gestational timing and severity of infection, a third experiment will determine the protective benefits of vaccination in the first trimester for ameliorating the deleterious effects on the baby. The phenotype and wellbeing of all infants will be characterized with standardized neurobehavioral and neuroendocrine tests. High resolution Magnetic Resonance Imaging (MRI) techniques will be employed to examine global and regional brain development at 8 and 12 months of age. White matter integrity and rate of myelinization will evaluated with Diffusion Tensor Imaging (DTI). These aims will be accomplished using the unique resources of the Harlow Primate Laboratory and Waisman 3-Telsa MR facility at the University of Wisconsin and the analytic expertise of Drs. J. Gilmore and G. Gerig at the University of North Carolina. Overall, the studies will test the central hypothesis that infection and immune activation can alter the natural progression of fetal development, be evinced by abnormal neuromotor responses in the neonate and still affect sensitive neural processes and the behavioral competence of the 1 year old infant. An important focus is the potential vulnerability of the hippocampus and its relationship to the regulatory set points of the hypothalamic-pituitary-adrenal axis. Primate studies of prenatal flu infection are critical at this juncture to bridge the findings in rodent models and concerns raised by retrospective analyses of human samples. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This R13 meeting proposal requests support to continue an annual training and mentoring program for 25 pre- and post-doctoral trainees at the upcoming 2008 meeting of the Psychoneuroimmunology Research Society (PNIRS). Opportunities for advanced, interdisciplinary training of younger scientists interested in the basic science aspects of behavioral and neuroimmune interactions and the translational relevance for prevention and intervention research are often limited at the university level. Since 1999 the PNIRS conference has received NIH support to provide formal didactic instruction and constructive career guidance for nearly 200 fellows. The primary goal is to create pedagogical opportunities and offer supportive mentoring at this pivotal, formative stage in a trainee's career in order to enhance their research potential and trajectory for success. [unreadable] [unreadable] PNI-related research has now definitively shown that psychological factors and life style can impact immune competence and influence disease susceptibility and progression. In addition to the relevance for infectious illness and autoimmunity, the findings are germane to neoplasia and help to account for variation in cancer morbidity and survival. PNI also offers unique assessment strategies and novel outcome measures for empirically evaluating the efficacy of nontraditional and complementary medicine approaches. The PNIRS is the primary professional organization representing the many diverse scientific disciplines involved in this type of inquiry. The society has approximately 300 regular members, including 40% at the early career stage. [unreadable] [unreadable] A Trainee Scholars Advisory Committee oversees the organization's commitment to this mentoring and ensures the quality of the program. It is comprised of society officers and program organizers, and benefits from 10 Senior Faculty Mentors who volunteer to participate in the training colloquium each year. Selection of the 25 Trainee Scholars is based on an open competition, and determined by the excellence of their recent research and promise. The impact of this program has been amply documented by the productivity of former awardees and their success in obtaining tenure track positions. In addition to the exposure to cutting edge science, plenary lectures, and symposia offered by leading scientists, the trainees participate in an Educational Short Course, a mentoring colloquium with Senior Faculty, and two workshops with Junior Faculty and NIH program staff, who provide advice on career building and funding opportunities. At the May 28-31, 2008 meeting in Madison, WI, there will also be luncheon roundtable discussion sessions and a trainee dinner to nurture the establishment of enduring, collaborative and professional relationships. This training and mentoring program is thus congruent with one of the primary aims of the NIH roadmap: to ensure the successful creation and productivity of the future scientific workforce of the United States. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chronic inflammatory states, steroid hormone exposure, and stress can affect iron-related biology, an essential micronutrient needed for infant growth and normal brain development. This project will investigate the novel hypothesis that some of the disabling effects of a stressful prenatal environment are mediated by undermining the placental transfer of maternal iron, which results in the subsequent depletion of iron stores in the growing infant. A nonhuman primate model will be used to determine how gestational stress impairs the placental transfer, sequestering, and use of iron by the young infant. One aim focuses on the timing of the gestational disturbance, comparing the effects of maternal stress during early, mid, or late pregnancy. To quantify placental transfer and bioavailability of iron in the neonate, an innovative approach with stable iron isotopes will be employed, contrasting absorption and transfer of 57Fe provided orally to 58Fe infused intravenously into the pregnant female. Iron-sensitive hematological measures will then be monitored in the developing infants from the stressed and undisturbed control pregnancies to prove that the postnatal iron deficiency is temporally associated with the occurrence of abnormal brain and renal functions. Based on previous findings in anemic monkeys, neural dysfunction will be indexed by the protein and metabolite profile of cerebrospinal fluid using Western blot and nuclear Magnetic Resonance Spectroscopy techniques. To further demonstrate the clinical significance of these deficits, the effects of prenatal stress and anemia on renal functioning observed in other species will be verified. When infants are 8 months of age, measures of glomerular filtration rate and renal sympathetic input will be obtained. A second study will support the veracity of iron mediation hypothesis by showing that oral iron supplementation during pregnancy can lessen the effects of prenatal stress on the developing infants. The two studies are comprised of 126 mother-infant pairs of rhesus monkeys, with the behavior and physiology of each infant evaluated prospectively from birth across the first year of life. This research will contribute to the growing awareness about the formative role of the fetal period in laying the foundation for postnatal health. Demonstrating that gestational stress affects iron homeostasis would reveal a deficit amenable to treatment. Iron deficiency has a broad public health significance because it is the most prevalent single nutrient deficiency worldwide. PUBLIC HEALTH RELEVANCE: Maternal stress, illness, and inflammatory processes during pregnancy can impair fetal growth, affect the likelihood of premature birth and obstetrical complications, and increase the risk for a number of postnatal behavioral and health problems in children. This project will investigate how gestational stress interferes with the placental transfer and utilization of maternal iron by the young baby, an important micronutrient needed for growth and normal brain development. Studies on iron deficiency are of particular relevance to the higher risk infants of low SES, anemic and stressed mothers, as well as of significance for babies from pregnancies complicated by diabetes and hypertension. The findings may reveal deficits that are amenable to prevention and treatment.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To evaluate factors associated with iron deficiency, sufficiency and excess. This project, initiated in December 2009, investigates maternal and dietary factors associated with iron deficiency anemia in infancy. One important factor is the amount of the iron in the diet, both for the mother during pregnancy and while lactating and nursing a young infant. Currently, many research facilities, including the WNPRC, use a commercial chow with more iron in it than previously fed to monkeys. Hence there was a compelling reason to compare the hematological status of infant monkeys gestated and reared on this data to preexisting norms. This ongoing study determines an iron panel on infant rhesus monkeys at 6 and 12 months age to evaluate factors associated with iron deficiency, sufficiency and excess. The offspring are from primiparous and multiparous females because younger, first time mothers are at higher risk for generating iron deficient offspring. Previous research has demonstrated that iron deficiency is associated with a number of significant effect on brain development, including on the essential monoamine neurotransmitter pathways. This research used WNRPC Animal Services. PUBLICATION: Coe CL, Lubach GR, Bianco L, Beard JL. A history of iron deficiency anemia during infancy alters brain monoamine activity later in juvenile monkeys. Dev Psychobiol. 2009 Apr;51(3):301-9. PMID: 19194962.

DESCRIPTION (provided by applicant): Iron deficiency is the most common micronutrient deficiency worldwide, and is particularly significant for women of child-bearing age and rapidly growing infants. Conventional methods of treating iron deficiency orally are inadequate as evidenced by poor compliance and the periodic need for iron injections. Our primary goal is to investigate the benefits of a new medical food supplement for treating anemia. We will establish the utility and safety of providing iron in yeast biotechnically modified to express human ferritin One advantage of this modification is that yeast can acquire therapeutic levels of iron in a bioavailable form without significant change in texture or palatability. In addition, ferritin is a highly conserved protein enabling us to provide a natural tissue storage form of iron using yeast as the delivery vehicle. To test its efficacy, we take advantage of a well-characterized nonhuman primate model of infant anemia. Three studies will be conducted in young rhesus monkeys under controlled laboratory conditions, empirically verifying the value of this yeast-ferritin complex in infants, a likely target population in humans. We will directly compare its benefits to a common standard of care: oral treatment with ferrous sulfate. Beyond traditional hematological tests and iron- related measures in serum, several novel endpoints will be determined in cerebrospinal fluid, including quantitation of iron management proteins and two important protein indices previously identified by proteomic analysis to be abnormal in anemic infants. Prior proteome and metabolome analyses revealed that when infant anemia is not resolved, it impacts functional proteins in the developing CNS, including prostaglandin D2 synthase and amyloid beta A4 protein-like, and impairs brain energetics. In addition to verifying the effectiveness of this innovative treatment, we will determine the safety if an iron-sufficient infant were to consume yeast containing iron, a critical prerequisite for human clinical trials. Measures of the heme and intrathecal compartments will be determined before, during, and after treatment. Based on previously found behavioral differences in anemic monkeys, emotional reactivity, motor activity, and cognitive performance will be assessed after supplementation. The core hypothesis is that this yeast-ferritin complex will provide iron in a readily absorbed form, and most significantly, that direct provision of ferritin will replenish the iron-deficient CNS more effectively. Using a multi-tiered nutritional and developmental neuroscience approach, several novel aspects of iron delivery and utilization will be investigated. The research has a clear translational relevance with the potential for establishing a new therapeutic modality. It will set the stage for a Phase I/II clinical trial and, at the same time, validate new biomarkers for tracking how anemia and iron supplementation affect the developing brain. Our capacity to carry out this unique inter-disciplinary project is based on a history of collaboration between two laboratories with the essential resources and expertise.

DESCRIPTION (provided by applicant): The gut microbiota of the infant, acquired by exposure to the mother and the early rearing environment, plays a critical role in establishing a functional gastrointestinal tract and promoting health via stimulating the maturation of the immune, endocrine, and enteric nervous systems. Our project uses a nonhuman primate model to specifically determine the mechanistic pathways through which the infant's gut microbiome affects brain development, as well as emotional and behavioral responsiveness during the first year of life. The mother's microbiome will be experimentally controlled during the pregnancy and nursing periods, enabling us to generate infants with two distinctive microbiome profiles. Microbial community analyses will be run on Illumina MiSeq to acquire high quality 16S rRNA data in order to track the development of the infant's microbiota while it is with the mother, and subsequently after weaning when the gut bacterial community is reorganized, due to the consumption of solid foods and exposure to peers. Based on preliminary results, we focus on two sets of signaling mediators: 1) the production of neurochemicals by gut bacteria, including monoamine neurotransmitters and GABA, and 2) inflammatory cytokine activity in the blood and intrathecal compartments. At one year of age, state-of-the-art neuroimaging will be used for structural analyses of cortical brain regions (gray and white matter volumes), and for quantifying the pace of myelination with diffusion tensor imaging. During the initial R21 phase, we will verify that 20 infant monkeys with distinct microbiome profiles can be reliably generated, and that population differences persist, allowing us to create peer social groups comprised of infants with comparable microbiota. During the R33 phase, a larger cohort of 40 infants will be generated, enabling us to also use fecal transplant methods to systematically change the composition of the gut microbiota and to test the hypothesis that behavioral and neural trajectories will shift to that of the donor animal. This project takes advantage of the unique resources of a major primate breeding facility, permitting us to evaluate a large number of infant monkeys under controlled environmental conditions, and the multi-disciplinary expertise and resources of the collaborating research team at 4 institutions. Innovative methods are employed, including the biopsy collection of gut tissue specimens to validate sequencing and neurochemical conclusions from directed metabolomic panels derived from the more routinely collected rectal swabs. Our project directly addresses a central tenet of the RFA, which is to investigate the influence of the maternal and infant microbiome on behavioral and brain maturation, and to determine if a dysregulated microbiome is associated with atypical development. Multi-tiered, molecular biology and developmental neuroscience approaches are employed. This research has a clear translational relevance for child health, contributing to extant findings that indicate an abnormal microbiome is evident in several pediatric disorders. We are poised to validate new biomarkers for tracking the influence of the gut microbiota on systemic physiology and to make novel discoveries on how the gut and enteric nervous system interface with the microbiome to influence the normal functioning and development of the central nervous system.

? DESCRIPTION (provided by applicant): There is a dearth of evidence in humans definitively showing that stress confined to the prenatal-infancy period has programming-like effects on the immune system. In addition, human studies of immune correlates of adverse childhood conditions have not focused specifically T-cell phenotypes involved in immune regulation that may compromise the ability to respond to infection. Both of these gaps in the literature are highly significant in that they limit our ability to understand the mechanisms through which early life stress (ELS) impairs health across the life span. This R21 addresses these lacunae by conducting sophisticated immunophenotyping and functional immune assays in a unique population of youth (14 through 17 years of age) who as infants (< 36 months) were adopted from orphanages/institutions in Russia/Eastern Europe into well-resourced and generally stable, low-life-stress families in the US. The early life stress (ELS) for these youth, thus, was confined to the prenatal-infancy period. There are two aims. Aim 1: Determine the impact of ELS on immune responses of these youth now that they are adolescents. Blood samples will be obtained from 30 post-institutionalized (PI) and 30 comparison adolescents. Sophisticated multi-color immunophenotyping will be used to characterize lymphocyte subsets and mononuclear cell responses will be probed in vitro with stimulants, and cytokine levels in supernatants determined with multi-cytokine arrays. Complete blood counts and C-reactive protein levels also will be determined. Saliva cortisol collected at testing will assess whether transient cortisol elevations in response to blood sampling contribute to the immune differences between groups. Aim 2. Delineate factors that account for differential vulnerability among ELS youth, including distorted hormone biorhythms and severity of the ELS. The second phase of this project will expand the sample size to include 30 additional PI's (total=60), providing sufficient diversity to determine the influence of the severity and duration of institutional care and the mediating influence of dysregulated HPA diurnal rhythms to predict variation in the T cell repertoire and cellular responses in stimulated cultures. This work is highly significant because, by focusing on youth who experience radical improvements in care post-infancy, it will provide the strongest evidence to date of prenatal-infancy programming-like effects on the human immune system and underscores the importance of preventing early deprivation and neglect. It also has potential significance for improving the preventive care of individuals with a history of early adverse care, by delineating specific immune alterations that may underlie potential health risks later in adulthood. It is highly innovative in being the first study of ELS in humans to employ such a sophisticated immune panel and to combine measures that are integral to a pro-inflammatory bias and immune competence. This R21 project will lay the groundwork for a new avenue of ELS-immune research that is focused on lingering sequelae of childhood experiences.

Abstract - Core B - BioCore Core B provides the resources and expertise to process specimens for the hematological, neuroendocrine, glucoregulatory, metabolic and cytokine indices needed for the research projects. The Core has the requisite infrastructure and oversight capabilities to ensure reliable and high quality data are generated in a high throughput manner with centralized testing at single sites. Specifically, the Core works with the 3 clinician- researchers who supervise nursing staff acquiring blood, urine and saliva specimens at the 3 regional Clinical Research Units. The BioCore then coordinates many tests at two CAP and CLIA-certified clinical laboratories for evaluating glucoregulation and lipid metabolism, as well as neuroendocrine assays, including catecholamines via LC/MS at the Endocrine Services unit of the UW-CTR. Some analytes are determined at labs that specialize in specific methods, such as dried blood spots (DBS) for the Retention-Early Warning project, run at the CLIA-CAP accredited Clinical Laboratories of the University of Washington School of Medicine. Core B also conducts assays, including multi-cytokine arrays using an electrochemiluminescence platform. Core B receives the saliva specimens for the Daily Stress project, monitors shipment integrity of the specimens upon receipt, and oversees the assay of salivary cortisol. To fulfill these functions, the Core is the primary liaison with project investigators and with medical directors at the testing labs, including Meriter Labs in Madison, WI and ARUP in Salt Lake City, UT. It has its own biochemical facilities for safe processing, including certified biosafety cabinets and fume hoods, follows Biosafety Level-2 protocols, and has the requisite number of ultracold freezers for long-term storage of archival aliquots. The scientific expertise of the Core staff positions MIDUS to be able to add innovative indices, such as ones employed in the Gene Expression project. It has enabled us to refine and validate more sensitive assays with Lower Limits of Detection (e.g., reducing the LLOD for urinary epinephrine by 1000-fold). In addition to assays and diagnostic testing, the Core is responsible for creating the data files of initial test results, which employ a distinct study code to protect participant confidentiality. In keeping with this record-keeping role, the Core serves as the repository for all original paper records of test results. It is responsible for all aspects of quality assurance, including monitoring the integrity of shipped specimens, quality control testing of assay reliability over time, and determines the need for any retesting of specimens, as well as oversees the 24/7 surveillance of our archival specimen repository in ultracold freezers. Finally, the Core has a pedagogical role. It generates norms for MIDUS test results to serve as a reference for investigators. In this educational capacity, the Core Director also provides instruction, giving workshops on specific biomarkers at the MIDUS meetings and consults with investigators on interpreting the meaning of biomarkers. The effectiveness and past success of the Core are evinced by 122 publications using MIDUS biomarker data, 47% of which were published in the last two years.

DESCRIPTION (provided by applicant): The gut microbiota of the infant, acquired by exposure to the mother and the early rearing environment, plays a critical role in establishing a functional gastrointestinal tract and promoting health via stimulating the maturation of the immune, endocrine, and enteric nervous systems. Our project uses a nonhuman primate model to specifically determine the mechanistic pathways through which the infant's gut microbiome affects brain development, as well as emotional and behavioral responsiveness during the first year of life. The mother's microbiome will be experimentally controlled during the pregnancy and nursing periods, enabling us to generate infants with two distinctive microbiome profiles. Microbial community analyses will be run on Illumina MiSeq to acquire high quality 16S rRNA data in order to track the development of the infant's microbiota while it is with the mother, and subsequently after weaning when the gut bacterial community is reorganized, due to the consumption of solid foods and exposure to peers. Based on preliminary results, we focus on two sets of signaling mediators: 1) the production of neurochemicals by gut bacteria, including monoamine neurotransmitters and GABA, and 2) inflammatory cytokine activity in the blood and intrathecal compartments. At one year of age, state-of-the-art neuroimaging will be used for structural analyses of cortical brain regions (gray and white matter volumes), and for quantifying the pace of myelination with diffusion tensor imaging. During the initial R21 phase, we will verify that 20 infant monkeys with distinct microbiome profiles can be reliably generated, and that population differences persist, allowing us to create peer social groups comprised of infants with comparable microbiota. During the R33 phase, a larger cohort of 40 infants will be generated, enabling us to also use fecal transplant methods to systematically change the composition of the gut microbiota and to test the hypothesis that behavioral and neural trajectories will shift to that of the donor animal. This project takes advantage of the unique resources of a major primate breeding facility, permitting us to evaluate a large number of infant monkeys under controlled environmental conditions, and the multi-disciplinary expertise and resources of the collaborating research team at 4 institutions. Innovative methods are employed, including the biopsy collection of gut tissue specimens to validate sequencing and neurochemical conclusions from directed metabolomic panels derived from the more routinely collected rectal swabs. Our project directly addresses a central tenet of the RFA, which is to investigate the influence of the maternal and infant microbiome on behavioral and brain maturation, and to determine if a dysregulated microbiome is associated with atypical development. Multi-tiered, molecular biology and developmental neuroscience approaches are employed. This research has a clear translational relevance for child health, contributing to extant findings that indicate an abnormal microbiome is evident in several pediatric disorders. We are poised to validate new biomarkers for tracking the influence of the gut microbiota on systemic physiology and to make novel discoveries on how the gut and enteric nervous system interface with the microbiome to influence the normal functioning and development of the central nervous system.

PROJECT SUMMARY/ABSTRACT Long-term cognitive and behavioral deficits are the sequelae of iron deficiency (ID) prior to 3 years of age in children. A causal relationship between early-life ID and brain dysfunction has been established in rodent models, but not as clearly in humans due to a lack of peripheral biomarkers of brain iron status and function. Using a nonhuman primate model that closely mimics human iron biology, brain development and metabolism, we propose to discover novel biomarkers that index brain dysfunction in the pre-anemic stage of ID and evaluate the efficacy of early iron treatment for mitigating ID-induced brain dysfunction. We will serially measure from birth until 12 months, conventional hematological and iron-related indices, and novel proteomic- and metabolomic-based biomarkers in the blood (serum) and intrathecal (CSF) compartments of ID infants and iron sufficient control infants, concluding with neuroanatomical (MRI) and functional (behavior) assessments. Aim 1 will determine how best to employ serum proteomics and metabolomics to detect impending ID- induced brain dysfunction by delineating which analytes and when in the course of ID, the serum proteome and metabolome accurately reflect the brain metabolic, structural and functional impairments. We predict that specific protein and metabolite changes reflecting distinct iron-regulated pathways will be detected in the serum in the pre-anemic period and provide biomarkers of impending brain dysfunction. Aim 2 will quantify and model the sensitivity of conventional hematological and serum iron parameters for detecting brain dysfunction by serially monitoring these parameters relative to the metabolomic and proteomic indices of brain dysfunction in concurrently obtained CSF. We predict that brain ID and dysfunction will be evident prior to the appearance of anemia, indicating that hematological parameters used in clinical practice are insensitive as biomarkers of brain iron and metabolic status. Aim 3 will test the hypothesis that iron treatment prior to anemia is essential to mitigate the adverse neurological effects of ID. ID infants will be randomized to iron treatment either in the pre-anemic stage of ID or after the development of anemia. The efficacy of the two therapies for restoring hematological indices and brain iron status, metabolism, structure and function will be determined. We predict that both treatments will normalize hematological indices, but only iron treatment begun in the pre-anemic stage will fully restore brain iron status, metabolism, structure and function. This project is significant, because it focuses on the benefits of early screening and interventions for improving the neurodevelopment of children at risk for early-life ID. It is innovative because it will employ novel proteomic and metabolomic analyses to simultaneously probe the blood and intrathecal compartments in a primate model that uniquely mimics the iron and metabolic demands of the human infant. The discovery of functional biomarkers will achieve our ultimate translational goal of optimizing screening and treatment strategies in children at risk for early-life brain ID.

Negative emotions happen every day and those thoughts and feelings can undermine a person's success and health. Research shows that the harmful effects of negative emotions are greater among Americans compared to East Asians. This project focuses on understanding how negative emotions assert their negative influence. It considers whether the way a person views and interprets negative emotions influences the effects of those emotions. One theory is that unfavorable beliefs about negative emotions make those negative emotions more harmful. The primary hypothesis is that negative emotions per se are not negative. How people respond to those negative emotions, however, produces the negative outcomes. Research suggests that Americans, compared to East Asians, view negative emotions as more harmful. Such differences in how people think about negative emotions produce the negative outcomes. This project lays the foundation for the design of interventions that promote more effective ways to respond to negative emotions. The research will help teachers improve the learning and success of their students and help counselors who work with patients who have mood and anxiety disorders.

This project examines the cognitive, physiological, mental health, and performance outcomes of cultural differences in beliefs about negative emotions. Multiple behavioral experiments examine how people respond to stressful events. Measures of cognitive, behavioral, and hormonal responses are used to compare American with East Asian samples. Stress responses to emotional experiences and arousal are measured via cortisol. Participants respond to different demanding tasks (such as giving a speech or doing difficult math problems) to test for performance and physiological reaction differences to the stressors. Interpretations of one's negative emotions are measured to test for subsequent outcome differences in stress and performance. This project helps to develop more general principles about the causes and effects of negative emotions. In addition to advancing the fields of psychology and affective neuroscience, this research offers many practical and applied implications for improving student success and developing interventions for patients with mood and anxiety disorders.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.