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Title:Impact of formula additives on immune and gastrointestinal development in the piglet
Author(s):Hester, Shelly
Director of Research:Donovan, Sharon M.
Doctoral Committee Chair(s):Gaskins, H. Rex
Doctoral Committee Member(s):Donovan, Sharon M.; Woods, Jeffrey A.; Miller, Michael J.
Department / Program:Nutritional Sciences
Discipline:Nutritional Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):infant formula
beta glucan
nucleotides
human milk oligosaccharides
rotavirus
influenza vaccination
Abstract:Infections are a major cause of morbidity and mortality in infants and children world-wide. However, the severity of response of neonates to rotavirus (RV) infection or their ability to generate sufficient antibodies following influenza vaccination can vary due to immune immaturity and dietary differences. Manipulating the composition of infant formula, by adding different ingredients, such as β-(1,3)-glucans (βG), nucleotides (NT), and human milk oligosaccharides (HMO) could stimulate the development of the neonatal immune and gastrointestinal systems. Dietary βG are able to boost the natural defense mechanism by stimulating both innate (Descroix et al., 2006) and adaptive immune responses (Akramiene et al., 2007). NT are conditionally essential for infants, particularly under stressful conditions, such as during disease and rapid growth. Clinical studies demonstrate that supplemental NT improve immune function, modify the intestinal microbiota and reduce diarrhea in formula fed infants. HMO contribute to the immunomodulatory and immunoprotective actions of human milk. Thus, the objective of this dissertation research was to investigate how early nutrition influences immune and gastrointestinal development. βG have been shown to potentiate immune system responses, and it was hypothesized that orally administered βG would boost the neonatal immune system. Therefore we evaluated the influence of purified yeast (1,3/1,6)-β-D-glucan [WGP®] on the development of the intestinal and systemic immune systems in neonatal piglets. Piglets were fed formula containing 0 (control), 1.8, 18 or 90 mg WGP/kg body weight and vaccinated with human influenza vaccine (Fluzone). Piglets were euthanized at 7 or 21 days of age. Piglet weight and formula intake were similar for piglets in each dietary treatment group. Small intestinal length and weight were unaffected by dietary WGP. In addition, WGP did not affect ileal crypt depth, ileal villus height or ascending colon cuff depth. Immune parameters analyzed included T cell phenotypes, cytokine gene expression, and cell proliferation. These were not affected by WGP® supplementation. Overall, the doses of 1.8, 18, and 90 mg (per kg body weight) of dietary WGP had no effect on intestinal development or intestinal and systemic immune development in neonatal piglets. Additionally, dietary WGP did not improve the antibody response to Fluzone vaccination. We concluded that dietary supplementation with WGP is not beneficial for intestinal or immune development in colostrum-fed, full-term neonates. NT and HMO have been shown to impact epithelial cell growth, however their combined effects had not previously been examined. Herein, a fetal epithelial cell line (FHs-74 Int) was used to determine the impact of NT and HMO on intestinal cell proliferation, apoptosis, necrosis and cell cycle. Cells were incubated with media containing 2.5% FBS and no epidermal growth factor (EGF) (Control); Fucosyl-lactose (FL) mix [85% 2'FL/15% 3'FL], Sialyl-lactose (SL) mix [40% 6'SL/10% 3'SL/50% sialic acid (SA)] or LNnT at 125, 250, 500 or 1000 μg/mL with and without 250 μg/mL NT (43% CMP, 18.5% UMP, 16.4% AMP, and 22.0% GMP) for 24 or 72h. NT alone significantly increased proliferation, but did not affect cell cycle or apoptosis/necrosis. All HMO treatments at 1000 μg/mL significantly decreased proliferation and some were also inhibitory at 250 or 500 μg/mL. Inhibition of proliferation increased with longer HMO exposure (72h vs. 24h). When NT and HMO were simultaneously added, NT reduced the anti-proliferative effect of HMO. FL mix treatments significantly increased cells S phase and SL mix and LNnT treatments significantly increased cells in G2/M and S phases which concomitantly decreased cells in G0/G1 phase. HMO with NT decreased the amount of cells in the G2/M phase compared to HMO treatments alone. Higher HMO doses significantly increased the percentage of apoptotic and necrotic cells compared to control. The percentage of necrotic and apoptotic cells were higher after 24h vs. 72h exposure to HMO. In conclusion, HMO reduced cell proliferation in vitro and this effect is partially ameliorated by the presence of NT. It appears that HMO initially induced apoptosis/necrosis, which is later evidenced by cell cycle arrest and decreased proliferation. Further studies are needed to determine whether HMO influence intestinal cell growth in vivo. Thus, we evaluated the interaction between chronic NT feeding and acute HMO treatment within the ileal lumen of both healthy and acutely rotavirus (RV)-infected piglets. Piglets (n=9 group) were fed formula containing 64 mg NT/L (FF) or 314 mg NT/L (FF+NT) from d2 to d21 of age. At d21, a midline laparotomy was performed and six 10-cm loops of ileum were isolated in situ. The following treatments were injected into the loops: media, 2 mg/mL neutral HMO (LNnT), 2 mg/mL acidic HMO mixture (aHMO: sialic acid (SA), 6'sialyllactose (SL), 3'SL, SA) or each treatment + SA-dependent OSU strain RV (1 x 107 FFU). After 6h, samples were collected. Body weight, formula intake, jejunum villus length/crypt depth, ileal proliferation, ileal macrophages and T cell phenotypes were unaffected by dietary NT. Loops treated with either HMO treatments + RV had lower RV replication, as assessed by NSP4 mRNA expression, than RV-treated loop alone. Ileal loops not treated with RV showed no NSP4 expression. There was no difference in the mRNA expression of NF-κβ, TNF-α, CCL2 or IL-1 in ileal mucosa as analyzed by loop or dietary treatment. However, NT fed piglets had significantly greater IL-8 ileal mucosa mRNA expression than formula piglets and aHMO treatment had significantly greater IL-8 mRNA expression than control loops. Thus, chronic dietary NT only had minor effects on the parameters measured, but HMO decreased NSP4 replication during acute RV infection. The dissertation comes to a close with a discussion of overall conclusions and future directions for this work. Future studies are warranted to determine the effect of these ingredients on the development of the neonatal immune and gastrointestinal systems.
Issue Date:2013-05-24
URI:http://hdl.handle.net/2142/45693
Rights Information:Copyright 2012 Shelly N Hester
Date Available in IDEALS:2013-05-24
2013-08-30
2015-08-30
Date Deposited:2012-05


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