Project description:Investigating alterations the intestinal microbiome in a diet induced obesity (DIO) rat model after fecal transplant from rats, which underwent Roux-Y-Gastric-Bypass surgery (RYGB). The microbiomes of the RYGB-donor rats, the DIO rats, and DIO rats after receiving the fecal transplant from the RYGB rats. As controls lean rats as well as lean, RYGB and DIO rats after antibiotics treatment were used.
Project description:<p>This project follows a cohort of 78 Very Low Birth Weight (VLBW) previously enrolled infants in a R21 grant plus additional 25 infants through their Neonatal Intensive Care Unit (NICU) stay until they reach the age of 4 years. The data, gathered over 6 weeks of the NICU stay, includes multiple factors, such as prenatal and postnatal events and illnesses, received human milk amount, weekly means of cytokines, chemokines, growth factors, and secretory Immunoglobulin A in the milk, and weekly levels of fecal calprotectin. These factors could potentially alter the gut microbiome. Microbiome species and diversities will be measured in the laboratory of Dr. Jack Gilbert at Argonne National Laboratory using state of the science deep sequencing and amplification of microbial sRNA genes. The microbiome will again be measured in stool samples from those children at the ages of 2 and 4 years. Relationship between the prenatal and postnatal factors, human milk volume and immunobiology, fecal calprotectin levels, and the very early microbiome will be analyzed. The predictive power of the VLBW infant gut microbiome for determining later childhood microbiomes will be analyzed prospectively. The relationships between microbiomes across time and later growth, development and health will be determined. VLBW infants are at risk for both early and later health effects, and the role of the microbiome in these effects will be measured in this prospective study. </p>
Project description:Over the course of milk digestion, native milk proteases and infant digestive proteases fragment intact proteins into peptides with potential bioactivity. This study investigated the release of peptides over three hours of gastric digestion in 14 preterm infant sample sets. The peptide content was extracted and analyzed from milk and gastric samples via Orbitrap tandem mass spectrometry. The relative ion intensity (abundance) and count of peptides in each sample were compared over time and between infants fed milk fortified with bovine milk fortifier and infants fed unfortified milk. Bioactivity of the identified peptides was predicted by sequence homology to known bioactive milk peptides. Both total and bioactive peptide abundance and count continuously increased over three hours of gastric digestion. After accounting for infant weight, length, and post-conceptual age, fortification of milk limited the release of peptides from human milk proteins. Peptides that survived further gastric digestion after their initial release were structurally more similar to bioactive peptides than non-surviving peptides. This work is the first to provide a comprehensive profile of milk peptides released during gastric digestion over time, which is an essential step in determining which peptides are most likely to be biologically relevant in the infant.
Project description:There is little information regarding the allergen content of milk feeds in the preterm population. Previous studies have evaluated specific proteins/peptides via ELISA, but no studies have performed a broad analysis of the allergenic peptide content and protease activity of milk feeds in this population. Preterm infants spend a critical window of time for immune development in the Newborn Intensive Care Unit (NICU), and may receive fortified donor milk, maternal milk or formula feeds via nasogastric tube or bottle instead of fresh breastmilk via breastfeeding.
Project description:Human milk, the best enteral selection for a preterm infant, becomes altered during freezing and soluble free fatty acid is generated over time. Free fatty acids may form complexes, such as the oleic acid-bound protein called HAMLET (human α‐lactalbumin made lethal to tumor cells). We determined the in vitro biological activity of preterm human milk protein‐oleic complexes (HAMLET-like complexes) and tested the hypothesis that laboratory-synthesized HAMLET exhibits cytotoxicity in human immature epithelial intestinal cell culture. Thirty-four milk samples from 15 mothers of hospitalized preterm infants were donated over time. Milk fractions were tested repeatedly for FHs 74 Int and HIEC-6 fetal cell cytotoxicity, using a sensitive viability assay. Protein and fatty acid identities were confirmed by Western blot, high performance liquid chromatography, and mass spectrometry. Cytotoxicity of intestinal cells exposed to milk increased respective to milk storage time (p<0.001) and was associated with free oleic acid (p=0.009). Synthesized HAMLET was cytotoxic in cultures of both lines. Preterm milk samples killed most cells in culture after an average 54 days in frozen storage (95% C.I. 34-72 days). After prolonged storage time, preterm milk and HAMLET showed a degree of cytotoxicity to immature intestinal cells in culture. Protein was reduced 221 in 1 mM dithiothreitol and alkylated in 5.5 mM
Project description:Injury occurring during critical periods of development may have long-term effects on inflammatory responses. Periventricular leukomalacia (PVL) is the most common cause of cerebral palsy (CP) in preterm infant. Activated leukocytes are the main source of inflammatory cytokines that give rise to white matter damage and CP in preterm infant. Here, we tested the hypothesis that inflammation profiles as pathogenic mediators for the occurrence of PVL in the neonatal period may persist in preterm children with CP at school age. Five preterm children with PVL-induced CP and gestational age-matched five preterm children with normal neurodevelopment were recruited from follow up clinics. Proinflammatory gene expression in the PBMCs from preterm children were determined by Superarray PCR study.
Project description:Hundreds of naturally occurring milk peptides have been found in term human milk. Whether there are differences between the levels of these peptides between term and preterm milk remains unknown. Premature milk is produced before complete maturation of the mammary gland and is under the influence of a different hormonal milieu, which could change enzymatic activity and protein expression within the mammary gland and result in an altered peptide profile. We employed nano-liquid chromatography tandem mass spectrometry to identify naturally occurring peptides in term and premature milks at multiple time-points across lactation and compare the abundances of these peptides. We found that preterm milks produced more unique peptide sequences than term milks on average (359 vs. 286). The peptides identified were searched for overlapping sequences with an in-house database of known functional peptides. Specifically, we found that both term and preterm milks contain peptides overlapping with known sequences with antimicrobial, opioid antagonist and immunomodulatory actions. We also compared the enzymes involved in degradation of both milks via bioinformatic analysis. This analysis reveals that plasmin is more active in preterm milk than term milk.
Project description:Necrotizing enterocolitis (NEC) is a severe gastrointestinal complication of prematurity. Using small intestinal organoids derived from fetal tissue of a gestational age similar to an extremely preterm infant, this study aims to assess the effect of diet on intestinal epithelial growth and differentiation to elucidate the role nutrition type plays in intestinal development and modifies the risk for NEC. Organoids were cultured for 5 days in growth media and 5 days in differentiation media supplemented 1:40 with four different diets: maternal milk (MM), donor human milk (DHM), standard formula, or extensively hydrolyzed formula. Images were captured daily and organoids were quantified. Organoids were preserved for RNA sequencing and immunofluorescence staining with Ki67, cleaved caspase 3, and chromogranin-A. Media was saved for cytokine/chemokine and growth factor analysis.Human milk supplementation improved growth and differentiation of intestinal organoids generating larger organoids during the growth phase and organoids with longer and wider buds during differentiation compared to formula. Ki67 staining confirmed the proliferative nature of milk-supplemented organoids and chromogranin A staining proved that MM-supplemented organoids induced highest enteroendocrine differentiation. Human milk supplementation also upregulated genes involved in proliferation and promoted a homeostatic immune landscape while those supplemented with formula had a downregulation of cell-cycle-promoting genes and a more inflammatory immune signature. Our results show that MM, and to a lesser extent DHM, support robust intestinal epithelial proliferation and differentiation, suggesting a critical role for factors enriched in human milk in intestinal epithelial health.
Project description:Very little is known about miRNAs found in breastmilk cells, which also reflect the cells of the lactating mammary epithelium. Our hypothesis is that breastmilk cells are richer in miRNA compared to other milk fractions, such as skim milk. Further, the effects of milk removal by the infant on milk cell miRNA content and/or composition have not been investigated. Breastmilk cells conserved higher miRNA content than previously published lipid and skim fractions of breastmilk as well as other known sources of miRNA in humans. Specifically, 1,467 known mature miRNAs were identified and a further 1996 novel miRNAs, of which 89 were highly expressed. As previously shown, post-feed milk contained more cells than pre-feed milk, and the same was observed for miRNA content. However, no statistically significant difference was found in the expression of the total known and novel miRNAs between pre- and post-feed milk (p=0.76), although 27 known miRNAs and 1 novel miRNA were higher expressed in post-feed milk. As expected, samples richer in viable cells contained more known miRNAs (p = 0.01). Functional analysis of the top 10 most highly expressed known miRNAs showed that they may be potentially involved in crucial roles for the infant, including body fluid balance, thirst, appetite, immune responses, and development. In conclusion, breastmilk is highly rich in miRNA which may play important functions in the breastfed infant and the lactating breast. Milk removal by the infant can influence the total miRNA content of breastmilk, similar to its cell and fat content, but the miRNA composition remains constant