2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This CRIS unit focuses on nutrition during pregnancy, lactation, infancy, and childhood and is composed of six individual research projects:. 1)physiological role of lactoferrin in neonatal development and host defense;. 2)insulin and IGF signaling in lactogenesis and maintenance of lactation;. 3)utilization of dietary protein during early infancy;. 4)relationship between maternal nutritional status and pregnancy outcome;. 5)nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children; and. 6)functional genomics of the casein gene cluster region. This research falls within ARS National Program 107 - Human Nutrition and addresses components 1 (Nutrient Requirements), 2 (Diet, Genetics, Lifestyle and the Prevention of Obesity and Disease), 5 (Health Promoting Intervention Strategies for Targeted Populations) and 7 (Bioavailability of Nutrients and Food Components) of the Human Nutrition Action Plan. Project 1: Physiological role of lactoferrin in neonatal development and host defense It is well known that breast-feeding confers multiple benefits to the developing infant. Therefore, a major need for research is to determine which milk components are responsible for these beneficial effects so that formulas can be appropriately modified to more optimally support the development of the formula-fed infant. Lactoferrin (LF) is the second most abundant protein in human breast milk. While the iron binding and host defense properties of LF predict that it may be one of the more critically important beneficial milk components, its exact function in neonatal development and host defense remains unresolved, largely due to lack of a relevant model to adequately test the function of this protein in vivo. Development of suitable model systems are therefore required to rigorously test the functions of LF in a physiologically relevant in vivo environment to help elucidate the potential benefits of this protein for neonatal health and well being. Our overall hypothesis is that LF functions as a critical bioactive milk component that is responsible, in part, for proper iron homeostasis and host defense protection in the intestinal tract of the neonate. We will use mouse models of LF deficiency and overexpression to address the physiological role of lactoferrin. The main impact of our research will be to help elucidate the physiological roles of the major milk protein LF and to determine the potential use of this protein as a nutritional and/or bioactive supplement for infant formulas to support intestinal iron balance and to protect against microbial infection and inflammation in the gastrointestinal tract of the developing infant. These studies will provide invaluable information to researchers, clinicians, and nutritionists on the bioactivities of LF relevant to infant development and host protection. Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation This research project aims to understand how insulin and IGF1 regulate normal biological processes that occur in mammary cells both during the onset of lactation and during the maintenance of prolonged lactation. The original aims of this project were:. 1)determine the impact of germline mutations in the IRS1 and IRS2 genes on mammary gland development, lactation, secretory cell differentiation and mammary gland-specific IGF-I/insulin-dependent signaling;. 2)determine if lactation defects attributed to loss of IRS-1 and/or -2 are due to mammary gland-specific effects;. 3)compare the impact of germline mutations in the IRS1 and IRS2 genes on biosynthetic and metabolic capacity of mammary cells in primary culture. An understanding of the genetic, biochemical, and nutritional mechanisms that regulate milk synthesis and composition is important to human lactation and, ultimately, the overall wellbeing of the breastfeeding newborn. In addition, knowledge gained from studies on mammary cell metabolism and the regulation thereof will enhance productivity and sustainability of the US dairy industry through the development of novel strategies to enhance lactation. Project 3: Utilization of dietary protein during early infancy This research project addresses the problem of newborn growth failure, which is experienced by the majority of infants who weigh less than 1500 grams at birth, and is thought to be at least partially responsible for these infants remaining small, even into early adulthood, and to contribute to their higher prevalence of developmental deficits. The project addresses the primary hypothesis that there is a finite period in early infancy during which utilization of protein for growth is maximal and the corollary hypothesis that maximizing protein intake during this period will improve early growth and reduce subsequent growth and neurodevelopmental deficits. The goals of the project are: (1) To confirm that there is a finite period during which the human neonate maximally utilizes protein intake for growth; (2) To define this period; (3) To determine if a higher protein intake during this period improves early growth; (4) To determine if the greater rate of early growth is reflected by a greater length and weight at 18 months of age; (5) To determine if a higher protein intake during this early period and improved growth reduce subsequent neurodevelopmental deficits. The period of maximal protein utilization will be defined by an increase in amino acid oxidation. Differences in short- and long-term growth and neurodevelopmental indices will be determined by comparing these outcomes in infants fed conventional vs. higher protein intakes for the first 4 months of life. This research is particularly relevant for infants who weigh <1500 g at birth (~1.5% of all live births in the U.S., or ~60,000 infants annually), medical personnel who care for these infants, and manufacturers of infant formulas and food. The research will provide a better understanding of protein metabolism during the neonatal period and the protein needs of low birth weight infants during early infancy. This better understanding should result in more appropriate feeding recommendations for this vulnerable group of infants and more appropriate nutritional management which, in turn, should enhance growth. These impacts will have the potential of reducing hospital stay and the cost of caring for these infants (currently estimated to be ~ $5,000/day of hospitalization). Better nutritional management also is expected to improve long-term growth and neurodevelopmental outcome, with obvious benefits for the surviving infant and his/her family as well as for society, which assumes the bulk of the cost of caring for those with compromised function. Project 4: Relationship between maternal nutritional status and pregnancy outcome In obese and underweight women and young adolescent girls, pregnancy causes increased health risks for both the mother and fetus. Obesity in pregnancy is increasing just as fast as in the general population, and it increases hypertension, diabetes, cesarean delivery, infections, the risk of delivering a larger than normal baby, who is in turn at an increased risk of childhood obesity. Underweight women and teenagers on the other hand are at increased risk of premature delivery, developing hypertension or dying during pregnancy. They have more babies with malformations, short stature, very low birth weight, smaller head circumference, higher rates of illness. This is a severe problem in the USA because about 31% of all women are obese and 13% of all births are from teenagers. Although the contribution of these two extremely malnourished conditions to poor pregnancy outcomes are well-established the mechanism through which they cause complications are not known. This research project aims to provide a better understanding of how over nutrition (obesity) and under nutrition (wasted) alter normal pregnancy processes in the body to produce a bad outcome and will test nutritional therapies aimed at reducing or correcting the adverse outcomes. In experiments that will be performed in obese pregnant women and underweight teenagers in Houston and in underweight and normal weight adult women in India we aim to determine whether: 1. Pregnant obese women develop hypertension because of inflammation and harmful compounds called oxidants produced by cells during inflammation; 2. Dietary supplements of antioxidant vitamins and a substance called cysteine that boosts production of an anti-oxidant called glutathione will neutralize the oxidants and reduce the risk of hypertension in pregnant obese women; 3. Moderate exercise plus an adequate balanced diet will improve blood sugar levels in obese and overweight pregnant women who develop diabetes during pregnancy; 4. Underweight pregnant women and teenagers give birth to short low and very low birth weight infants because they cannot provide sufficient glucose (sugar) and amino acids (protein building blocks) for growth of the fetus; 5. The inability of underweight pregnant women and teenagers to produce adequate amounts of glucose and amino acids is related to their deficient body tissues; and 6. A balanced dietary supplement of energy, protein and micronutrients (vitamins and minerals) to underweight pregnant women and teenagers will reduce the risk of having short low and very low birth weight infants. This research is relevant to obese pregnant women, undernourished pregnant women, and pregnant underweight teenagers. The potential benefits of this research project are that obese women may have a reduced risk of developing hypertension, diabetes, and infections during pregnancy. They will also have a reduced risk of having a cesarean delivery and delivering a larger than normal baby. Additionally, under weight pregnant adult women and teenage girls may have a reduced risk of having a premature delivery and a short low birth weight or very low birth weight baby. They may also have a reduced risk of premature delivery, developing hypertension, or dying during pregnancy. Project 5: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children The goal of this project is to identify and test strategies to improve macronutrient nutrition and micronutrient nutrition of Malawian children under 5 years of age. We have developed 2 strategies to improve macronutrient nutrition, and a major clinical trial is underway. The subject enrollment and follow-up for the trial comparing the 2 strategies to improve macronutrient nutrition is nearing completion, but the data have not been analyzed. Once we assess the effectiveness of the 2 interventions, a plan for further implementation can be made. An intervention to improve micronutrient nutrition, our first and best hypothesis, has been tested and found to be ineffective. Specifically this intervention was the administration of a probiotic, Lactobacillus GG. Probiotics are bacteria that can colonize the human intestinal tract and displace harmful bacteria. Although the problem is not major, other possibilities exist to eliminate the harmful bacteria in the intestine, such as antibiotic treatment, and a small trial to determine if antibiotics will eliminate this bacteria is planned. I am now planning a small trial with antibiotics to see if this will eliminate the harmful bacteria in the intestine. Together with other CNRC researchers, we are assessing what the best course should be to find an effective method to improve micronutrient nutrition. The work is relevant to nutritionists and aid programs (US AID) throughout the developing world, and the impact of the work could be to provide the scientific basis for more effective interventions to improve child health in the least developed countries. Project 6: Functional genomics of the casein gene cluster region The primary goal of this project is to understand the regulation of the casein genes (the major nutritional protein component in milk) and the region in the genome in which these genes reside. The knowledge of the factors and pathways involved in casein gene regulation is limited to the proximal promoter regions of a subset of the casein genes and nothing is known with regard to their coordinate regulation. Recent studies in our laboratory indicate that the casein genes are located in a region in the genome encoding a number of secreted proteins (caseins and non-caseins) with similar spatial expression patterns and comparable physiological properties. We do not know the role of the non-casein gene products in nutrition, health, and development of the breast tissue and suckling infants. To achieve the stated goal we will determine the function of non-coding evolutionary conserved regions (N-ECR) with potential regulatory function in the Casein (CSN)-gene cluster by analyzing the higher order DNA structure, determining the functional role in gene regulation of N-ECRs in the CSN-gene cluster by expression of large transgenes harboring portions of the casein gene cluster with or without N-ECR, performing computational analysis of N-ECR and promoter regions to identify sequence elements that are involved in regulation of the CSN region and studying the function of the gene products encoded by the non-casein genes in the CSN region as proteins involved in immuno-modulation, mineral homeostasis and nutrition. We also will determine the relationships (functional and evolutionary) of casein and non-casein genes in this region. Milk is a critical nutrient for the early development of the neonate providing pivotal factors for bone development, growth, gastrointestinal-tract maturation, early immunity and host defense. In addition, cow's milk is currently considered an important nutritional component promoting increased calcium intake in children for healthy bone and teeth development. An understanding of the genetic and nutritional mechanisms that regulate milk synthesis and composition is important to the overall well being of the breastfeeding newborn, with respect to human milk, and for consumers of dairy-based products with regards to cow's milk. Also, as not all women can breast feed their newborns for 1 year, the development of synthetic formulas based on the quality of human milk is important. This work is relevant to other scientists in the field of lactation biology, researchers in the area of infant nutrition, scientists working in the area of breast cancer, clinicians working with breastfeeding mothers, formula producers, breastfeeding mothers and their infants, dairy scientists, and dairy farmers. Research results will lead to a fundamental understanding of the composition of the main milk source consumed by humans (of human and bovine origin), and the organization and regulation of the genes encoding the major milk proteins. Additionally, these studies will provide insight into the genetic factors regulating milk composition, and growth promoting properties and host defense mediated by milk consumption. This information should improve our understanding of normal lactation and aid in improving the composition of synthetic formulas, milk production in livestock, and the production of biologically important proteins in the milk of transgenic animals. Furthermore, this research should lead to the development of improved vector systems for expression of milk proteins and biopharmaceuticals in transgenic livestock and mouse models with modified milk protein content. 2.List by year the currently approved milestones (indicators of research progress) Project 1: Physiological role of lactoferrin in neonatal development and host defense Year 1 (2005) Complete postnatal iron diet studies in LFKO versus WT mice Complete analysis of SI-LF and LI-LF founder lines Setup Pilot Citrobacter infections in mice Year 2 (2006) Complete iron studies in SI-LF versus WT mice Complete the analysis on microflora in LFKO versus WT mice Complete the analysis of Citrobacter infections in SI-LF versus WT mice and LFKO mice Setup pilot studies of NEC in mice Year 3 (2007) Complete iron analysis in double SI-LF/HFE mice versus single SI-LF or HFE mice Complete the analysis of microflora in SI-LF versus WT mice Setup pilot Salmonella bacterial infections in mice Complete the analysis of NEC in LI-LF versus WT mice Year 4 (2008) Complete the analysis of microflora in LI-LF versus WT mice Complete the analysis of Salmonella infections in LI-LF versus WT and LFKO mice Setup pilot Listeria bacterial infections in mice Complete the analysis of CD in LI-LF versus WT mice Year 5 (2009) Complete the analysis of Listeria infections in SI-LF versus WT and LFKO mice Complete the analysis of UC in LI-LF versus WT mice Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation Year 1 (2005) Complete the analysis of milk yield, milk composition, mammary gland development and cell signaling in both the IRS-1 and IRS-2 knockout mice. To determine if mammary gland or mammary tissue grafts will be required in the analysis of the double knockout mice. Year 2 (2006) Complete the cell turnover and lactogenesis studies on the IRS-1 and –2 knockout mice and begin the transplantation and cell culture studies. If the double knockouts are fertile, lactation, mammary development, and cell signaling will be studied in these mice. Year 3 (2007) complete the transplantation and cell culture studies on the IRS-1 and -2 knockouts and continue those on the double knockouts. Year 4 (2008) Complete the transplantation studies on the double knockouts and continue the cell culture studies. Year 5 (2009) Complete the cell culture studies on the double knockouts. Project 3: Utilization of dietary protein during early infancy Year 1 (2005) Enroll 20 breast-fed and 40 formula-fed infants Perform gas spectrometric analyses of breath and urine as enrolled infants reach the designated ages Perform growth and body composition measurements as infants reach the designated ages Perform neurodevelopmental assessments at indicated ages (4m, 9m, 2m and 18 m post menstrual age (PMA)) Year 2 (2006) Continue enrollment of formula-fed (n=40) and breast-fed infants (n=20) Complete required mass spectrometric analyses in 15 breast-fed infants and 15 infants fed each formula Continue growth and body composition assessments at indicated ages Continue neurodevelopmental assessments at indicated ages Year 3 (2007) Continue enrollment of formula-fed (n=40) and breast-fed (n=20) infants Continue growth and body composition assessments of formula-fed and breast-fed infants who have reached the indicated ages Continue neurodevelopmental assessments at indicated ages Year 4 (2008) Complete enrollment of all groups (120 formula-fed and 60 breast-fed infants) Continue indicated growth, body composition, and neurodevelopmental assessments Year 5 (2009) Continue to follow all groups until 18 months PMA performing growth, body composition and neurodevelopmental assessments at indicated ages for these assessments Complete all 4-month and 12-month assessments of all groups Begin analysis of data and preparation of manuscripts Project 4: Relationship between maternal nutritional status and pregnancy outcome Year 1 (2005) Start screening and enrolling 10 normal weight and 40 obese pregnant subjects in Houston for both experimental protocols Start screening and enrolling 20 obese pregnant subjects in Houston for experiment; Start baseline isotope infusions and subject assignment to different diet and exercise treatment groups Start screening and enrolling 30 underweight and 30 normal weight pregnant women in Bangalore Start baseline isotope infusions to measure glucose kinetics and gluconeogenesis, protein kinetics and amino acid fluxes. Start laboratory analyses of samples Start screening and enrolling 60 underweight pregnant women in Bangalore for supplementation intervention Start baseline isotope infusions to measure glucose kinetics, gluconeogenesis and protein kinetics Year 2 (2006) Start analyzing samples for TNF(alpha), CRP, oxidant damage (8-isoprostane, hydroperoxides), total antioxidant potential, urinary protein and GSH and GSSG Start supplementing 10 subjects with dietary supplements of vitamins C, E and Beta-carotene plus N-acetylcysteine Start screening and enrolling 30 underweight and 30 normal weight pregnant teenagers in Houston Start laboratory analyses of samples Continue enrollment and start mid-pregnancy isotope infusions in women in Bangalore Continue enrollment and start post-treatment isotope infusions in women in Bangalore Year 3 (2007) Complete laboratory analyses of samples and complete enrollment and start post-treatment isotope infusions Continue and complete experimental protocol in Bangalore and start analyzing data Continue enrollment and start mid-pregnancy isotope infusions of teenagers in Houston Start screening and enrolling 60 underweight and pregnant teenagers in Houston for supplementation intervention Start baseline isotope infusions to measure glucose kinetics, gluconeogenesis and protein kinetics Complete experimental protocol in Bangalore and start analyzing data Year 4 (2008) Complete experimental protocol and start analyzing data and writing manuscript Year 5 (2009) Continue and complete experimental protocol in Houston and start analyzing data and writing manuscript Project 5: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children Year 1 (2005) Prepare the prototype RTUF that is to be used as the complementary food Field test the prototype for acceptability and taste in small groups of children Prepare deuterated water for human administration, work with village mothers and leaders to secure understanding and cooperation with the trial Initiate enrollment of 100 children in RTUF complementary feeding group Initiate enrollment of 100 children in fish-fortified porridge complementary feeding group Enroll 150 Malawian village children in trial of Lactobacillus GG Perform analyses of quality and quantity of breast milk in 20 mother-child pairs before and after initiation of complementary feeding Data analyses, statistical consultations, preparation of summary reports and manuscripts from the trial of Lactobacillus GG Year 2 (2006) Follow these 200 children for 12 months Collect and analyze samples for iron, zinc and selenium Plan a clinical trial to determine if antibiotic therapy improves the intestinal dysfunction of tropical enteropathy Year 3 (2007) Enroll 12 Malawian children in trial of to determine if tropical enteropathy as measured by dual sugar absorption test is correlated with endogenous fecal zinc losses from the intestine Quantify zinc homoestasis will be quantified in a limited number of these children. Complete data analyses, statistical consultations, summary reports and manuscripts for the complementary feeding study conducted in Years 1 and 2 Conduct a clinical trial to determine if antibiotic therapy improves the intestinal dysfunction of tropical enteropathy Year 4 (2008) Zinc stable isotope analyses from the 12 Malawian children treated with Lactobacillus GG Year 5 (2009) Complete data analyses, statistical consultations, summary reports and manuscripts for all objectives After data is reviewed, enroll any additional children needed to achieve the objective. Sample analyses from additional children. Complete data analyses, statistical consultations, preparation of summary reports and manuscripts for the project. Project 6: Functional genomics of the casein gene cluster region Year 1 (2005) Initiate analysis of higher order DNA structure of the non-coding evolutionary conserved regions (N-ECR) and promoters in the CSN-region in a mouse mammary gland cell line, mouse mammary gland tissue at different developmental stages as well as non-mammary gland tissue Computational analysis of N-ECR and promoter regions to identify common sequence elements that are involved in regulation of genes in the CSN-region with available comparative and analytical computational genomics tools Determine in which tissue and cell type the genes in the CSN-region are expressed Year 2 (2006) Continue analysis of higher order DNA structure of the N-ECRs and promoters in the CSN region in a mouse mammary gland cell line, mouse mammary gland tissue at different developmental stages as well as non-mammary gland tissue Modify Bacterial Artificial Chromosomes (BACs) to be used as transgenes in functional analysis of N-ECRs Continued computational analyses, as new tools will become available Year 3 (2007) Complete higher order DNA structure analysis, and expand to analysis of binding of transcription factors. Generate transgenic mice with modified BAC transgenes (from #2 2006) and establish lines to analyze N-ECR function Conduct computational analyses using knowledge generated by (#1 2007) Extend the analysis of the spatial expression of the genes in the CSN-region (#3 2005) to determine expression levels using (semi-) quantitative methods Year 4 (2008) Continue analysis of binding of transcription factors, and initiate higher order DNA structure analysis of transgenic animals (#2 2007) Analyze transgenic animals harboring BAC transgenes with modified or deleted N-ECRs (#2 2007), for effects on expression of CSN-region resident genes in mammary gland and other tissues Continue conducting computational analyses Year 5 (2009) Continue higher order DNA structure analysis of transgenic animals, and expand to transcription factors Continue analysis of transgenic animals harboring BAC transgenes with modified NECRs, for effects on expression of CSN-region resident genes in mammary gland and other tissues Complete computational analyses Determine the presence of gene products in milk and or saliva (availability of antisera permitting) Using information provided by the initial analysis of spatial expression and expression levels of the genes in the CNS-region determine potential function of gene-products (computational analysis) 4a.List the single most significant research accomplishment during FY 2006. Project 1: Physiological role of lactoferrin in neonatal development and host defense LF is not required to protect against high dose Citrobacter infections in suckling mice. Lactoferrin is present at high levels in human milk and in the milk of other mammals and serves as the first line of defense in protection against pathogens; yet, a major concern in the field of pediatrics is infantile diarrhea. Children's Nutrition Research Center researchers have used a Citrobacter rodentium infection model in mice to examine the consequences of lactoferrin on susceptibility to Enteropathogenic (EPEC) bacterial infections, a major cause of infantile diarrhea disease worldwide. Control and lactoferrin knockout mice pups were given different doses of Citrobacter rodentium (106-108) orally and the infection was allowed to proceed for 2 - 7 days. There was no increased susceptibility to infection in the lactoferrin knockout mice versus controls as measured by colonic Citrobacter colony forming unit counts. These results indicate that lactoferrin ablation does not increase susceptibility of pups to this infection at such doses. Future studies will determine whether lactoferrin is required to protect against low dose Citrobacter infection. [ARS National Program 107 - Component 2 (Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease)] 4b.List other significant research accomplishment(s), if any. Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation Lactation supported by hexokinase I Children's Nutrition Research Center Scientists have discovered a molecular basis within the mammary cell for the negative impact of loss of IRS-1 on lactation. The subcellular localization of the enzyme hexokinase I in mammary tissue samples prepared from lactating wild-type and IRS-1 null mice models was determined. This analysis demonstrated that loss of IRS-1 caused a shift in hexokinase I localization from the mitochondrial to the cytoplasmic compartment of the mammary cell. These findings are important as it demonstrates that IRS-1 supports the process of lactation through regulation of hexokinase I localization and activity within the mammary cell. Furthermore, knowledge gained from studies on mammary cell metabolism and the regulation thereof will enhance productivity and sustainability of the US dairy industry through the development of novel strategies to enhance lactation. [ARS National Program 107 - Human Nutrition and addresses components 1 Nutrient Requirements] IRS-2 reduction reduces lactation Understanding mammary cell metabolism and regulation is important in order to enhance milk productivity and sustainability of the US dairy industry. Scientists at the Children's Nutrition Research Center in Houston, TX, demonstrated that IRS-2 loss causes a modest reduction in lactation capacity in mice. This was accomplished by comparing litter growth in mice that were either wild-type or null for IRS-2. This result supports the conclusion that IRS-2 as well as IRS-1, plays some role in supporting normal lactation. [ARS National Program 107 - Human Nutrition and addresses components 1 Nutrient Requirements] Understanding mammary cell mitochondrial Understanding mammary cell metabolism and the regulation is important in order to enhance milk productivity and sustainability of the US dairy industry. Scientists at the Children's Nutrition Research Center in Houston, TX, demonstrated that significant changes occur in the mammary gland mitochondrial population during a single lactation cycle. These changes were examined by measuring the expression of mitochondrial markers in mammary tissue during both early and prolonged lactation. Both mitochondrial genome number and the expression of mitochondrial proteins increase significantly during early lactation, followed by significant decreases during prolonged lactation. These results support the conclusion that significant variations in mitochondrial number and function occur within the mammary gland in support of the changes that occur in milk synthesis capacity of the organ. [ARS National Program 107 - Component 2 Diet, Genetics, Lifestyle, and the Prevention of Obesity and Disease] Project 4: Relationship between maternal nutritional status and pregnancy outcome Glucose production in pregnant women In 2006, Children's Nutrition Research Center researchers measured glucose production and rate of gluconeogenesis in underweight and normal weight pregnant women during the first trimester and found that underweight women produced glucose at the same rate as normal weight women. This was the first study of its kind. The aim of the study was to test the hypothesis that underweight pregnant women produce less glucose than normal weight pregnant women because they have less body protein that can be used to make glucose by gluconeogenesis. If true, this new finding means that underweight pregnant women will not be able to provide sufficient glucose to sustain growth of their fetus. This aim was accomplished by a 6-hour intravenous infusion of glucose in overnight fasted pregnant women and collection of timed blood samples. The results of this study suggest that at least in the first trimester there was no deficit in the availability of glucose in underweight pregnant women. [ARS National Program 107 - Components 7 Bioavailability of Nutrients and Food Components] Project 5: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children Improved complementary feeding intervention conducted Poor child nutrition in children aged 6-18 months accounts for over half of the childhood mortality worldwide. Children's Nutrition Research Center researchers devised 2 novel foods designed to improve complementary feeding (food that young children receive in addition to breast milk) in Malawi, a soy/peanut micronutrient-fortified paste and a moist corn dough with fish powder mixture. 240 children aged 6 months and their mothers were recruited in 8 villages and randomly assigned to feed their children one of these foods daily for a period of 12 months. The children were monitored biweekly, and measured monthly. All subjects successfully completed the 12-month intervention. If these novel strategies are shown to improve childhood growth and health, it could be used in a broader intervention to increase child survival. [ARS National Program 107 - Component 5 Health Promoting Intervention Strategies for Targeted Populations] Project 6: Functional genomics of the casein gene cluster region Identification of casein genes in the Laboratory opossum (Monodelphis domestica). There is a limited knowledge of factors and pathways involved in the regulation of casein genes that impact mammalian lactation. Comparative genomics using species with different lactation systems will allow researchers to identify the most basic mechanisms regulating lactation and milk protein gene expression shared by all mammals. Children's Nutrition Research Center researchers established the presence of casein and other major milk protein gene transcripts in lactating mammary gland tissue of the laboratory opossum. Using computational approaches to predict the presence of milk protein genes in the opossum genomic sequence, our lab determined the genomic localization and gene structure of the corresponding genes and identified the promoting regions and their conservation when compared to other mammals. Further computational analyses were used to determine gene structure and align the opossum casein promoter regions with those of other mammals, showing conservation of a number of presumed transacting factor binding sites. These research findings indicate that despite a vastly different lactation strategy in opossum, some basic lactogenic signaling pathways are the same and enable researchers to focus their studies of casein gene regulation to the pathways and transacting factors that mediate those signals. [ARS National Program 107 - Component 7 Bioavailability of Nutrients and Food Components] Milk protein gene promoter have epigenetic marks correlating with gene transcription in mammary gland. There is a limited knowledge of factors and pathways involved in the regulation of casein genes, thus scientists desire to identify changes in higher order DNA structure associated with casein gene expression. Children's Nutrition Research Center researchers established that epigenetic marks associated with actively transcribed genes are present at the promoter and a nearby non-coding evolutionary conserved region (N-ECR) of one of the casein genes as well as other n-ECRs in lactating and late pregnant mammary tissue. Assays were performed on mammary and non-mammary gland tissues of lactating and pregnant mice. Combined with the previously established presence of histone modification marks indicative of active gene transcription, our research results confirm that an open higher order DNA structure is established in association with gene expression in the mammary gland. These results suggests a regulatory role for these nECRs, and these will be subject of more detailed studies. (ARS National Program 107 - Component 2 Diet, Genetics, Lifestyle and the Prevention of Obesity and Disease) 4c.List significant activities that support special target populations. None. 5.Describe the major accomplishments to date and their predicted or actual impact. This research falls within ARS National Program 107 - Human Nutrition and addresses components 1 (Nutrient Requirements), 2 (Diet, Genetics, Lifestyle and the Prevention of Obesity and Disease), 5 (Health Promoting Intervention Strategies for Targeted Populations) and 7 (Bioavailability of Nutrients and Food Components) of the Human Nutrition Action Plan. Project 1: Physiological role of lactoferrin in neonatal development and host defense We have used lactoferrin knockout mice (LFKO) to examine the role of LF in the host defense response. We have shown that neutrophil maturation, migration, phagocytosis and antimicrobial response to acute Staphylococcus aureus and Pseudomonas aeruginosa experimental challenges were unaffected in adult LFKO mice. However, we showed that the oxidative burst of LFKO neutrophils is impaired using a dihydrorhodamine assay. Further, we have shown that there is a slight decrease in superoxide production in LFKO neutrophils. Studies examining the cell surface expression of CD11b under resting and activating conditions indicate that the impairment of the oxidative burst response of the LFKO neutrophil is not due to an overall difference in the activation status of these cells. We also have shown a slight increase in spontaneous abscess formation in LFKO mice as compared to WT control mice housed in the same SPF facility. We have successfully established experimental conditions for a reproducible Citrobacter infection in suckling pups (a mouse model of EPEC infections) and have shown that LF ablation does not increase the susceptibility of pups to high dose infection with Citrobacter. The main impact of our research is the further elucidation of the physiological roles of LF that will help determine the potential use of this protein as a nutritional and or bioactive supplement for infant formulae. These studies will benefit researchers, clinicians, and nutritionists by further defining the bioactivities of LF relevant to infant development and host protection. Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation Since the beginning of 2005, our work on this project has illustrated that both IRS-1 and -2 play some role in supporting mammary cell function during lactation. This conclusion is based on the fact that targeted mutations of these genes in mice results in reduced capacity to support litter growth coupled with altered milk composition and, in the case of IRS-1, diminished insulin-dependent signaling and altered sub cellular localization of hexokinase I. We have also gained valuable preliminary data on some of the biological processes which occur during prolonged lactation and have generated a sample collection that will be valuable in determining changes in gene expression patterns both in the mammary gland and during prolonged lactation. In particular, we have generated microarray data on RNA expression in the mammary gland over a prolonged lactation cycle and we have collected exciting observations of the regulation of the mammary energy metabolism through the mitochondria. Potential customers or users of the knowledge obtained from these studies include but may not be limited to; other scientists in the field of lactation biology, researchers in the area of breast cancer, clinicians working with breastfeeding mothers, dairy scientists, and dairy farmers. Project 3: Utilization of dietary protein during early infancy Because of the long-term nature of this project, a significant accomplishment is not expected until sometime after the end of the second year. This is because the requisite number of infants to detect statistically significant differences in 1-13C-leucine oxidation will not be enrolled until approximately mid-2006 and the studies necessary to determine differences in 1-13C-leucine oxidation will not be finished until at least 4 months after the requisite number of infants is enrolled. Even more infants are required to detect expected differences in growth, body composition and neurodevelopmental outcome. On the other hand, considering the difficulties of enrolling and maintaining this vulnerable population, the fact that our enrollment goals have been substantially met is considered a major accomplishment. Project 4: Relationship between maternal nutritional status and pregnancy outcome Pregnant women have to produce extra glucose to support their increased energy needs plus the needs of the growing fetus. Most of this extra glucose is made by gluconeogenesis from amino acids released from protein breakdown. Because underweight women (BMI<19) have less lean body mass, hence less body protein, we hypothesized that underweight pregnant women will produce less glucose than normal weight (BMI>19) pregnant women. This, in turn, will cause them to deliver small for gestational age babies. Our results so far do not support this hypothesis. The underweight pregnant women made glucose at the same rate as the normal weight women. In fact, both the rate of gluconeogenesis and the production of glucose trended to be higher in the underweight women. These findings suggest that the small for gestational age babies delivered by underweight women is not related to a shortage of glucose as a source of energy for fetal growth. Project 5: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children Researchers have completed a double-blind, placebo controlled clinical trial of the probiotic Lactobacillus GG in 165 Malawian children to determine whether this heals tropical enteropathy, a subclinical gastrointestinal condition that compromises nutrient absorption. The clinical aspects of the study are complete, as well as the laboratory studies. Thirty days of Lactobacillus GG does not improve intestinal function or integrity in Malawian children aged 3-5 years. This is a hypothesis that has been discussed for over a decade by nutrition scientists, and we tested it using a rigorous study design that is likely to be accepted as solid evidence by the scientific community. The primary customers of this research are children and those agencies that provide nutrition and health services to these children. The secondary customers are the scientific nutrition community that have received the results from this work, and can use these results to direct their own work. Project 6: Functional genomics of the casein gene cluster region Using a mouse mammary epithelial cell line our lab indicates that lactogenic hormones stimulate changes in higher order DNA structure and transcription factor binding, correlating to gene expression. After establishing an assay to study higher order DNA structure in animal tissue, our team identified several hallmarks of the higher order DNA structure correlating with active gene expression in lactating mammary gland on milk protein gene promoters and some evolutionary conserved regions that might have a role in gene regulation. In addition, we have shown that hallmarks of the higher order DNA structure correlating with inactive DNA (no-gene expression) are absent on at least one of the milk protein promoters in the lactating mammary gland and present in non-mammary gland tissues (that do not express the gene). These results point to the different roles lactogenic hormones have in milk protein gene regulation and the involvement of changes in DNA higher order structure in regulation of milk protein gene expression. These results will serve as a paradigm for analysis of other casein genes and milk protein genes. Such research experiments are relevant to other scientists in the field of lactation biology, researchers in the area of infant and specialty formulas, scientists working in the area of breast cancer, clinicians working with breastfeeding mothers, formula producers, breastfeeding mothers and their infants, dairy scientists, and dairy farmers. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? None. 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Project 3: utilization of dietary protein during early infancy Invited Speaker: Heird, W.C. "Effect of Maternal Docosahexaenoic Acid (DHA) Supplementation on Neuropsychological and Visual Status of Former Breast-fed Infants at Five Years of Age". PERILIP Consensus Conference on "Dietary fat intakes during the perinatal period in health and disease", Wildbad Kreuth, Bavaria, Germany, September 11-14, 2005. Heird, W.C. "Current Electrolyte Intakes of Infants and Toddlers". American Dietetic Association FITS II Symposium, St. Louis, MO, October 22, 2005. Heird, W.C. "Start Healthy Feeding Guidelines for Infants and Toddlers". Russian Federation for Infant Nutrition. Moscow, Russia, February 6, 2006. Heird, W.C. "Evidence Based Method for Development of the Start Healthy Feeding Guidelines for Infants and Toddlers". Russian Federation for Infant Nutrition. Moscow, Russia, February 6, 2006. Lecture/Co-Program Director: Heird, W.C. "Concept of Catch-Up Growth". Neonatal Nutrition Conference, Baylor College of Medicine and Texas Children's Hospital, Houston, TX. March 5-8, 2006. Moderator, Symposium: Heird, W.C. "Advances in Meeting the Nutritional Needs of infants Worldwide". Annual American Society of Nutrition Meeting, San Francisco, CA, April 5, 2006. Butte N.F., Heird, W.C. Knowing what's best for baby to eat. Agriculture Research Magazine 2005;53:7. Project 6: Functional genomics of the casein gene cluster region Invited Speaker: Rijnkels, M. "Milking the Genome: Regulation of Mammary Gland Specific Gene Expression." At "Functional Development of the Mammary Gland" Program Project Retreat, University of Colorado Health Science Center, Denver, Colorado. January 2006. Rijnkels, M. "Milking the Genome: Evolution and Regulation of the Casein Gene Cluster Region" For the Interdisciplinary Faculty of Toxicology, Texas A&M University, College Station, Texas. April 2006.