Claudin-2 and occludin protein expressions.

<p>Western blot was used to measure claudin-2 (Fig 6A) and occludin (Fig 6B) protein expressions in intestine. The levels of claudin-2 and occludin in FF and BO groups were significantly decreased compared to the FO group. Claudin-2 and occludin levels were also lower in the FF group compared to the BO group. The data are presented as mean ± SE (* P<0.05, **p<0.01, N = 5–8 / group).</p

p65 NF-κB and TLR4 activation and expression.

<p>Intestinal p65 NF-κB activation in each group was assayed by EMSA. NF-κB mRNA was determined by qRT-PCR. P65 NF-κB and TLR4 proteins were measured by Western blot. Fig 3A: Image and bar graphs showed TLR4 protein expressions. Significant increase in TLR4 protein were seen in FF and BO groups. Fig 3B: Intestinal NF-κB mRNA expression levels. NF-κB mRNA levels in FF group were higher than BO and FO groups. Fig 3C: Intestinal p65 NF-κB protein levels. The levels of p65 NF-κB protein from FF group were higher than BO and FO groups. Fig 3D: Bar graphs, obtained by densitometric analysis of EMSA (the images not shown). The DNA binding activities in FF and BO groups were significantly increased compared to FO group. The data are represented as mean ± SE (* P<0.05, **P<0.01, N = 5–8 / group).</p

Data analysis.

<p>Data analysis.</p

The activation of NF-κB in FHs 74 Int cells.

<p>FHS 74 int cells were transfected with pNF-κB-Luc vector, then treated with filtered fermented formula (100 μl/ml) or propionic acid (2.5 mM) in serum-free media for 18 h. Cells were harvested for luciferase activity. NF-κB luciferase activity data are expressed as fold induction of normalized luciferase activity. The levels of NF-κB luciferase activity in FF and propionic acid groups were significantly increased compared to control group. The data are represented as mean ± SE (* P<0.05, **P<0.01, N = 4 / group).</p

Gross pathology from fermented formula (FF), bacteria only (BO) and formula only (FO) groups.

<p>Fig 1A. Gross pathology from FF group: 1a-i. Small intestinal inflammation. Representative sample of small intestine from FF group animal fed with a mixture of nontoxigenic <i>E</i>. <i>coli</i> and standard infant formula. The bowel shows evidence of severe inflammation, transmural necrosis, and bowel wall edema, discontinuous “skip lesions. 1a-ii. Pneumatosis intestinalis. Representative sample of small intestine from FF group animal. Clear demonstration of gas bubbles in the sub-serosa of the small intestine confirming pneumatosis intestinalis. 1a- iii. Portal venous gas. Representative dissection of pancreas and portal venous tributary in FF group animal. Pancreatic parenchyma was retracted to demonstrate gas bubbles in the portal venous system confirming portal venous gas. Fig 1B. Gross pathology from BO group. Representative image of small intestine of an animal fed only E. coli suspended in normal saline. This mixture has an equivalent PH as the formula + bacteria mixture (PH = 5.5). The bowels are pink, healthy, and show no evidence of inflammation. Fig 1C: Gross pathology from FO group. Representative image of small intestine of an animal fed only infant formula, the bowels are pink, healthy, and show no evidence of inflammation. <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201172#pone.0201172.g002" target="_blank">Fig 2</a>.</b> Histology from Fermented Formula (FF), Bacteria only (BO) and Formula only (FO) groups.</p