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|Title: ||Influence of Beta-lactoglobulin on the uptake and passage of IgG in the intestinal cell in vivo and in vitro|
|Authors: ||Sutton, Leonard Franklin Jr.|
|Advisors: ||Brenda Alston-Mills, Committee Chair|
|Issue Date: ||2-Dec-2002|
|Abstract: ||Studies were conducted to determine the effects of the whey protein Beta-lactoglobulin (BLG) on the delivery and transport of a particular immunoglobulin, IgG. The presence of the two proteins in vivo in certain animals prompted an investigation into the possibility that BLG might have some effect on the passage of IGG not only to the intestinal cell but also through it.
The first experiment consisted of a serum analysis of blood from neonatal piglets. Two trials were conducted in this experiment, the first with a duration of 5 days (trial 1) and the second 28 days (trial 2). The influence of BLG on IgG uptake, endogenous production of various immunoglobulins (IgG, IgM, IgA), and sera levels of BLG were investigated. Eighteen piglets from three sows were taken immediately following parturition and divided into three experimental groups. In the first group, piglets received a milk replacer diet consisting of commercial bovine colostrum supplemented with BLG (treatment 1, n = 6). Piglets in group 2 (n = 6) received the same bovine colostrum powder with no supplementation (treatment 2) while the remaining piglets (n = 6) served as controls by suckling the sow (treatment 3). In trial 1, groups 1 and 2 received the treatment diets for 36 hours and were then provided a liquid, gravity-fed diet. The control group remained with the sow for the duration of the trial. For the second trial, the same dietary regimens were imposed with all piglets weaned at day 20 and placed onto a solid commercial piglet diet. In both trials, IgG uptake was highest when BLG was added as a supplement while piglets from the sow reared control group had the highest endogenous porcine IgG and BLG uptake.
The second part of the initial investigation examined intestinal growth, morphology and function. Enzymatic activity (maltase, lactase, alkaline phosphatase), total DNA synthesis, crypt depth and villus height were all parameters used to evaluate the effect of BLG on intestinal development of the neonatal piglet. Piglets from the sow reared control group had the greatest villi height after 28 days as well as total DNA. Piglets fed colostrum supplemented with BLG had the greatest total DNA after 5 days with no differences observed in enzymatic activity among all treatments.
As a result of the in vivo results, the second experiment was performed to determine whether BLG served as a facilitator of transcellular transport of IgG in a human intestinal cell line (Caco-2). The specificity of IgG ligand receptor binding, the presence and type of Fc receptors and overall passage of IgG from apical to basolateral surfaces of the intestinal cell were investigated.
Cells were grown in a porous transwell system, seeded at 4.6 x 10 cells/well, in a total of 36 wells. Media were added to both compartments in the transwell system with 2.5 mL of media on the basolateral side and 1.5 mL on the apical side. Media were changed every other day. Cells were cultured as a monolayer until confluency was reached (day 14) and the tightness of this monolayer was determined by transepithelial electrical resistance (TEER). After apical projections and villi differentiations were observed, approximately day 21, several protocols were used to identify uptake and passage of IgG.
Fluorescently labeled IgG was added with unlabeled IgG to determine specificity of binding to the apical receptor. Labeled IgG was added alone and with BLG in varying concentrations to determine levels of uptake and passage through the cell. IgG uptake and transcellular transport were evaluated by fluorimetry, flow cytometry and fluorescent microscopy. Unlabeled IgG and labeled IgG competitively bound to the polymeric Ig receptor. Uptake of IgG was evident after incubation with the Caco-2 cells but passage through the monolayer was highest in cells in which BLG was also added. These results suggest that BLG may, in fact, enhance and protect IgG in passage of Caco-2 cells. Results were best when IgG and BLG were added to Caco-2 cells separately rather than as a complex.|
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