2005 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? What does it matter? This unit focuses on nutrition during pregnancy, lactation, infancy, and childhood and is composed of seven 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)hormonal and molecular regulation of human milk production;. 4)utilization of dietary protein during early infancy;. 5)relationship between maternal nutritional status and pregnancy outcome;. 6)nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children; and. 7)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. Additionally, this research adheres to ARS Strategic Plan Goal 4 Improve the Nation's Nutrition and Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; Determine Food Consumption Patterns of Americans. 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 formulae can be appropriately modified to more optimally support the development of the formulae-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, the exact function of this protein in neonatal development and host defense remains unresolved. This was 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 are using 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 formulae to support intestinal iron balance and protection 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 dur;ing the maintenance of prolonged lactation. The orginal 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; and. 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: Hormonal and molecular regulation of human milk production The health benefits to infants in developing and developed countries are well established, and it is the recommendation of the American Academy of Pediatrics (AAP) that all infants be exclusively breast fed for at least 6 months and partially breast fed for an additional 6 months. Unfortunately fewer than 25% of infants are successful at breastfeeding for 6 months and even fewer continue to breast-feed partially for 1 year as is recommended by the AAP. Very little is known about the regulation of milk production in humans and the impact of breast-feeding on maternal metabolism. Milk volume is presumably determined by the rate of lactose synthesis, as the primary osmotic agent in milk. Thus, understanding the factors that regulate lactose synthesis and its impact on maternal metabolism may provide new strategies to facilitate milk production. Improved understanding of the regulation of milk synthesis and secretion will provide a scientific basis to the information provided women who wish to breast-feed and an evidence-based rationale for the development of new therapeutic strategies for women who struggle with or are not successful with breast-feeding. Such information will facilitate an increase in the number of infants who will be successfully breast-fed in whole or part over the first 6 months of life and provide evidence based strategies to help women lose weight following pregnancy. This work will benefit all women who attempt or are desirous of breast-feeding their infants, their infants, and all physicians, allied health professionals and nutritionist who provide information to such women. Project 4: Utilization of dietary protein during early infancy This research project addresses the problem of neonatal 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 these infants' 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 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 as measured by oxidation of 13C-leucine. 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 formula 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 and more appropriate nutritional management, which in turn should enhance rates of 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 should 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 5: 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 with very low birth weight, smaller head circumference, higher rates of illness. This is a big 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 mechanisms 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. 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 6: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children Two of the most important nutritional problems for young children worldwide are poor complementary feeding practices and zinc deficiency. The consequences are retarded physical and intellectual development, as well in increased child morbidity and mortality. The goal of this project is identifying and testing strategies to improve macronutrient nutrition and micronutrient nutrition in Malawian children less than 5 years of age. Our first research objective will develop a micronutrient-rich, energy-dense ready-to-use food and compare it with a fish-fortified porridge as a complementary food in 6- to 18-month-old children. Ready-to-use food is a lipid rich paste that requires no cooking and can be safely stored at ambient conditions for extended periods of time. The quantity and quality of consumed breast milk will be measured for the first objective, and children will be followed longitudinally. Additionally, the role of asymptomatic intestinal malabsorption in zinc homeostasis will be investigated in 3- to 5-year-old children, using site specific gastrointestinal sugar absorption tests and zinc stable isotope techniques. The effect of the probiotic agent Lactobacillus GG on intestinal function and zinc conservation will then be determined, once the relationship is elucidated. These objectives employ strategies that are feasible and can be provided at low cost, and thus have the potential for widespread applications in poor and compromised populations in the US and throughout the world. Project 7: Functional genomics of the casein gene cluster region The goal of this project is to understand the regulation of the casein genes (the major nutritional protein component in milk) and the region 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 the casein genes and nothing is known with regard to their regulation. Recent Children's Nutrition Research Center studies have indicated 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. 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 promoted as an important nutritional component supporting 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 one 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 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. The research results will lead to a fundamental understanding of the composition of the main milk source consumed by humans, 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 contribute to 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. 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 the milestones (indicators of progress) from your Project Plan. 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): We expected to complete the analysis of milk yield, milk composition, mammary gland development, and cell signaling in both the IRS-1 and IRS-2 knockout mice. We also expected to know if mammary gland or mammary tissue grafts will be required in the analysis of the double knockout mice. Year 2 (2006): We expected to 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 we will also study lactation, mammary development, and cell signaling in these mice. Year 3 (2007): We expected to complete the transplantation and cell culture studies on the IRS-1 and –2 knockouts and continue those on the double knockouts. Year 4 (2008): We expected to complete the transplantation studies on the double knockouts and continue the cell culture studies. Year 5 (2009): We will complete the cells culture studies on the double knockouts. Project 3: Hormonal and molecular regulation of human milk production Year 2 (2006): Recruitment complete 80% complete studies Develop primers and pilot data on other specific mRNA's Develop palatable drink Initiate pilot studies with 2 women Year 3 (2007): Completion of blood analyses 50% milk and protein Year 4 (2008): Complete clinical studies Initiate RT-PCR analyses Year 5 (2009): Complete analyses of alpha-lactalbumin and initiated other RNA analyses Analysis of stable isotopes Project 4: 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, 12m, and 18m 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 this age; Continue neurodevelopmental assessments at indicated ages. Year 4 (2008): Complete enrollment of all groups; 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 as indicated ages for these assessments are reached; Complete all 4 month and 12 month assessments of all groups; Begin analysis of data and preparation of manuscripts. Project 5: 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. 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 in 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 6: 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 is 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 analyses 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 to determine if tropical enteropathy as measured by dual sugar absorption test is correlated with endogenous fecal zinc losses from the intestine. Zinc homoestasis will be quantified in a limited number of these children. Complete data analyses, statistical consultations, summary reports and manuscripts for the macronutrient goals of the project, 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 will be performed. Year 5 (2009): Complete data analyses, statistical consultations, summary reports and manuscripts for the 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 7: 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 N-ECRs, 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 anti-sera 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.What was the single most significant accomplishment this past year? Project 1: Physiological role of lactoferrin in neonatal development and host defense NEUTROPHIL OXIDATIVE BURST IS IMPAIRED IN LACTOFERRIN KNOCKOUT MICE Lactoferrin (LF) is present at high quantities in neutrophils where it has been speculated to contribute to the cell's host defense response. To directly address the selective contribution of LF to neutrophil function, scientists at the Children's Nutrition Research Center in Houston, TX, have analyzed the host defense responses in lactoferrin knock-out mice. Interestingly, while neutrophil maturation, migration, phagocytosis, and antimicrobial response to acute Staphylococcus aureus and Pseudomonas aeruginosa experimental challenges were unaffected in adult lactoferrin knock-out mice, researchers showed that LF is required for the neutrophil's optimal oxidative burst activity. These findings provide researchers with greater knowledge of the beneficial impact lactoferrin has in neutrophils. 4b.List other significant accomplishments, if any. Project 2: Insulin and IGF signaling in lactogenesis and maintenance of lactation DECREASED LACTOSE CONCENTRATION NOT DUE TO IMPAIRED GLUCOSE TRANSPORT Understanding is lacking regarding the secretory cell glucose transport in humans, which has an impact on human lactation. Researchers at the Children's Nutrition Research Center (CNRC) in Houston, TX, have determined that decreased milk volume and lactose in knockout mice is not due to defects in secretory cell glucose transport. The knockout model has a reduced capacity to lactate and produces milk that has modest decreases in water and lactose. CNRC researchers have determined that this decreased milk lactose was not associated with decreased secretory cell glucose transport. This result suggests that the decreased lactose concentration in the milk of IRS protein deficient mice is not due to impaired glucose transport by the secretory cell and implicates some other aspect of glucose metabolism. MILK PROTEIN GENE EXPRESSION DOES NOT IMPACT MILK VOLUME Decreased milk volume and lactose in the IRS protein knockout mice is not due to decreased milk protein gene expression. Scientists at the Children's Nutrition Research Center in Houston, TX, have determined that this decreased milk lactose was not associated with decreases in the expression of a key gene necessary to lactose biosynthesis, alpha-lactalbumin. It was demonstrated that the expression of two other milk protein genes, beta-casein and whey acidic protein, were also unaffected by loss of IRS protein expression. Expression of the milk protein genes collected during lactation was compared among normal and IRS protein knockout mice. This result suggests the modest impairment of lactation in the IRS protein knockout mice is the result of factors that are unrelated to milk protein gene expression. GERMLINE MUTATION OF MOUSE GENE IMPAIRS SIGNALING IN MAMMARY TISSUE Decreased milk volume and lactose in the IRS protein knockout mice is associated with decreased insulin-dependent phosphorylation of the serine/threonine kinase AKT. Scientists at the Children's Nutrition Research Center in Houston, TX, have determined that the ability of the mammary glands of IRS protein knockout mice to respond to insulin through phosphorylation of a specific threonine residue on AKT was decreased. Normal and IRS protein knockout mice were given an intravenous insulin challenge at 3 days of lactation and mammary tissue collected from these insulin-injected mice was used to prepare extracts that were analyzed for AKT phosphorylation. This analysis demonstrated that the ability of insulin to induce AKT phosphorylation on threonine was decreased in IRS protein knockout mice. This result supports the conclusion that the germline mutation of IRS protein in mice impairs select aspects of insulin-dependent signaling in mammary tissue. UNDERSTANDING THE MECHANISMS THAT REGULATE LACTATION Development of transgenic vectors for use in mammary-specific, inducible, transgenic analysis of insulin and IGF signaling in lactation. Scientists at the Children's Nutrition Research Center in Houston, TX, developed the first of three transgenic vectors that will allow researchers to target the expression of specific gene products to the mammary glands of transgenic models in an inducible fashion. This first vector is designed to target the expression of a tetracycline-dependent transcriptional silence to the lactating mammary secretory cell. This vector, when used with a vector encoding a mammary specific tet-responsive CRE protein and a vector encoding a mammary specific FLOX-stopped gene of interest, will give us the unprecedented ability to inducibly regulate transgene expression in the lactating mammary gland. The use of the vectors in transgenic mice will create new models that will be important to understanding the signaling mechanisms that regulate lactation. Project 5: Relationship between maternal nutritional status and pregnancy outcome RELATIONSHIP BETWEEN LIVER FAT CONTENT AND VERY LOW DENSITY LIPOPOTEIN SYNTHESIS IN CHILDREN WITH PROTEIN ENERGY MALNUTRITION The reason why fat gets deposited in the livers of severely malnourished children is not known. Researchers at the Children's Nutrition Research Center in Houston, TX, found that the hepatic steatosis of severely malnourished children is not due to reduced hepatic synthesis of very low density lipoprotein as previously proposed and accepted in scientific literature. Actually, very low density lipoprotein synthesis rate is positively correlated to the degree of fat infiltration of the liver. In other words, children having a lot of fat in their livers were trying to remove the excess fat faster. This finding suggest that high-fat diets traditionally used to rehabilitate severely malnourished children should not be introduced early as it will accelerate fat deposition in the liver and worsen liver function. BREAKING DOWN KWASHIORKOR A lack of knowledge exists about kwashiorkor, a form of malnutrition caused by inadequate protein intake in the presence of fair to good energy (total calories) intake. Scientists at the Children's Nutrition Research Center in Houston, TX, found that malnourished children with kwashiorkor were breaking down body proteins at a slower rate than malnourished children with marasmus, a form of serious protein-energy malnutrition. This finding suggest that in kwashiorkor there may be a shortage of amino acids (derived from body proteins) to support the synthesis of proteins, peptides, and other important molecules critical for survival. These findings may explain why children with kwashiorkor are sicker and have a higher mortality rate than children with marasmus. Project 6: Nutritional pathogenesis, treatment and prevention of protein-energy malnutrition in young children GAINING INSIGHT ON TROPICAL ENTEROPATHY Increased knowledge is needed in the area of tropical enteropathy, the subclinical gastrointestinal condition that compromises nutrient absorption. Researchers at the Children's Nutrition Research Center in Houston, TX, have completed a controlled clinical trial of the probiotic Lactobacillus GG (maintains a healthy intestinal tract and strengthens the body's natural defenses) in 165 Malawian (African) children to determine if this has a beneficial impact on tropical enteropathy. Our findings indicate that 30 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. Project 7: Functional genomics of the casein gene cluster region LACTOGENIC HORMONE INDUCED TRANSCRIPTION-FACTOR BINDING AND HISTONE MODIFICATION There is a limited knowledge of factors and pathways involved in casein gene regulation. Scientists at the Children's Nutrition Research Center in Houston, TX, have determined changes in histone modifications and binding of transcription factors, promote one of the caseins and a nearby N-ECR in mammary epithelial cells. Changes in histone modifications and binding of transcription factors were determined at different time points after the addition of lactogenic hormones (prolactin and/or hydrocortisone) and were correlated to the expression of one of the casein genes. Hydrocortisone appears to be the mediator of histone modification changes and glucocorticoid receptor binding, whereas the cooperation of both hormones mediates the binding of a number of other transcription factors and casein gene expression. These findings point to the different roles lactogenic hormones have in milk protein gene regulation and will serve as a paradigm for analysis of other casein genes and milk protein genes. ESTABLISHMENT OF CHROMATIN IMMUNO-PRECIPTION (ChIP) ASSAYS ON MOUSE TISSUE Limited knowledge exists regarding the factors and pathways involved in casein gene regulation. Researchers at the Children's Nutrition Research Center in Houston, TX, have established methods to analyze DNA higher order structure in mouse tissues, both fresh and frozen. This will permit studies considering the changes in higher order DNA structure in the mammary gland at different developmental stages as well as non-mammary gland tissue to establish the role of DNA higher order structure in regulation of milk protein genes. Researchers optimized Chromatin Immuno-Precipitation assays using freshly harvested and frozen lactating mammary gland and liver tissues. These methods will enable us and other investigators to study histone modifications and transcription-factor binding in different animal tissues. MILK PROTEIN GENE PROMOTERS HAVE HISTONE MODIFICATION MARK INDICATIVE OF ACTIVE GENE TRANSCRIPTION IN LACTATING MAMMARY GLAND There is a limited knowledge of factors and pathways involved in casein gene regulation. Children's Nutrition Research Center scientists, in Houston, TX, have established that a histone modification associated with actively transcribed genes is over represented on milk protein gene promoters in the lactating mammary gland in comparison to the liver. Assays for histone modifications were performed on mammary gland tissue and liver tissue of lactating mice. Results indicated that higher order DNA structure remodeling occurs in association with gene expression. Such findings are important to other researchers and scientific investigators. 4c.List any significant activities that support special target populations. None. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. 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 show that the oxidative burst of LFKO-/- neutrophils is impaired. In addition, a slight increase in spontaneous abscess formation is observed in LFKO-/- mice as compared to WT control mice housed in the same SPF facility. We have successfully setup experimental conditions which establish a reproducible Citrobacter infection in suckling pups. This will enable us to determine the role of maternal milk-derived LF on suceptibility to enteropathogenic infection during the suckling period. The main impact of our research is the further elucidation of the physiological roles of LF which 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 nutritionalists 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 the fact that IRS1 is not entirely necessary for normal lactation and that loss of this protein only has modest negative effects on the capacity for milk synthesis. This result coupled with a reanalysis of the potential for other IRS proteins has led us to the conclusion that the focus of the project needs to be redirected to understanding the biological processes underlying the loss of milk synthesis with prolonged lactation. As a result of the refocusing, we have gained valuable preliminary data on some of the biological processes which occur during prolonged lactation, we have generated a sample collection that will be valuable in determining how changes in gene expression patterns both in the mammary gland and pituitary during prolonged lactation, and we have begun creating transgenic vector systems that will allow us to manipulation milk synthesis during prolonged lactation. 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 5: Relationship between maternal nutritional status and pregnancy outcome Scientists at the Children's Nutrition Research Center in Houston, TX, found that malnourished children with kwashiorkor were breaking down body proteins at a slower rate than malnourished children with marasmus, a form of serious protein-energy malnutrition. This finding suggest that in kwashiorkor there may be a shortage of amino acids (derived from body proteins) to support the synthesis of proteins, peptides and other important molecules critical for survival. These findings may explain why children with kwashiorkor are sicker and have a higher mortality rate than children with marasmus. Project 6: 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, the subclinical gastrointestinal condition that compromises nutrient absorption. The clinical aspects of the study are complete, as well as the laboratory studies. We have shown that 30 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 7: Functional genomics of the casein gene cluster region Using a mouse mammary epithelial cell line we show 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, we show that a particular change in the higher order DNA structure correlated with active gene expression is over represented in lactating mammary gland on milk protein gene promoters and some evolutionary conserved regions that might have a role in gene regulation. This work is 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. 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 milk protein gene expression. These results will serve as a paradigm for analysis of other casein genes and milk protein genes. 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. Additionally, this research adheres to ARS Strategic Plan Goal 4 Improve the Nation's Nutrition and Health, specifically Objective 4.1: Promote Healthier Individual Food Choices and Lifestyles and Prevent Obesity; Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods; Determine Food Consumption Patterns of Americans. 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 1: Physiological Role of Lactoferrin in Neonatal Development and Host Defense Conneely, O. 2004. Physiological role of lactoferrin in immuomodulation and host defense. Mead Johnson Site Visit, The Children's Nutrition Research Center, March 8, 2004, Houston, Texas. Project 5: Relationship between maternal nutritional status and pregnancy outcome Jahoor, F., Badaloo, A., Reid, M., Forrester, T. 2005. Protein metabolic differences between children with marasmus and kwashiorkor. Carribean Health Reseach Council, 50th Annual Council and Scientific Meetings, April 2005, Tobago. Badaloo, A., Reid, M., Forrester, T., Jahoor, F. 2005. Lipid kinetics in children with kwashiorkor and marasmus. Carribean Health Reseach Council, 50th Annual Council and Scientific Meetings, April 2005, Tobago. Project 7: Functional genomics of the casein gene cluster region Rijnkels, M. 2004. Of mammals and milk: the casein gene cluster region. Center for Comparative Genomics and Bioinformatics, Pennsylvania State University, October 20, 2004, College Park, Pennsylvania. Rijnkels, M. 2005. Milking the genome: Identification of Functional Elements in a Mammalian Specific Gene Cluster. Texas Medical Center Chromatin Interest Group, April 28, 2005, Houston, Texas. Review Publications Hadsell, D.L., Divisova, J., Cui, X., Lawrence, N.A., Lee, A.V. 2004. Mammary gland-specific overexpression of insulin receptor substrate-1 and -2 has minimal impact on downstream kinase activity and doesn't affect mammary involution [abstract]. In: Endocrine Society Meeting Proceedings, June 16-19, 2004, New Orleans, Louisiana. Abstract P3-37. p. 473. Hadsell, D.L., George, J., Lawrence, N., Torres, D., Fiorotto, M. 2004. Differential ability of insulin and long-r3-IGF-I to induce Akt phosphorylation in mammary gland of the virgin mouse is accounted for by cellular heterogeneity [abstract]. In: Endocrine Society Meeting Proceedings, June 16-19, 2004, New Orleans, Louisiana. Abstract P3-177. p. 507-508. Hadsell, D., Torres, D., George, J. 2005. Increased mammary gland oxidative damage and apoptosis during prolonged lactation in the mouse is little affected by overexpression of des(1-3)hIGF-I [abstract]. Journal of Dairy Science. 88(Suppl. 1):77. Rijnkels, M., Kabotyanski, E., Le, T., Rosen, J.M. 2005. Milking the genome: identification of functional elements in a mammalian specific gene cluster [abstract]. 24th Summer Symposium in Molecular Biology; Comparative and Functional Genomics, July 20-23, 2005, Pennsylvania State University, University Park, Pennsylvanai. p. 89. Rijnkels, M., Le, T., Thomas, J. 2005. Evolution and regulation of the casein gene cluster region: a genomics approach. Journal of Dairy Science. 88(Suppl. 1):76-77. Kabotyanski, E., Kemp, L., Rosen, J.M., Rijnkels, M. 2005. Understanding the regulation of a mammalian-specific genomic domain encoding secreted proteins [abstract]. Systems Biology: Genomic Approaches to Transcription Regulation. Cold Spring Harbor Meeting. p. 61. Kabotyanski, E., Le, T., Elnitski, L., Miller, W., Rosen, J.M., Rijnkels, M. 2004. Identification of functional elements in a mammalian specific gene cluster [abstract]. Cold Spring Harbor Meeting. p. 51. Carboni, J.M., Lee, A.V., Hadsell, D.L., Rowley, B.R., Lee, F.Y., Bol, D.K., Camuso, A.E., Gottardis, M., Ho, C., Hurlburt, W. 2005. Tumor development by transgenic expression of a constitutively active Insulin-Like Growth Factor I receptor. Cancer Research. 65:3781-3781. Kaplan, W., Rodriguez, L.M., Smith, O.E., Haymond, M., Heptulla, R.A. 2004. Effects of mixing glargine and short-acting insulin analogs on glucose control. Diabetes Care. 27:2739-2740. Hillier, L.W., Miller, W., Birney, E., Warren, W., Hardison, R.C., Ponting, C.P., Bork, P., Burt, D.W., Groenen, M.A., Rijnkels, M. 2004. Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature. 432:695-716. Heptulla, R.A., Rodriguez, L.M., Bomgaars, L., Haymond, M. 2005. The role of amylin and glucagon in the dampening of glycemic excursions in children with type 1 diabetes. Diabetes. 54:1100-1107. Jeha, G.S., Karaviti, L.P., Anderson, B., Smith, E.O., Donaldson, S., McGirk, T.S., Haymond, M.W. 2004. Continuous glucose monitoring and the reality of metabolic control in preschool children with type 1 diabetes. Diabetes Care. 27(12):2881-2886.