Pre-clinical in vitro and in vivo safety evaluation of bovine whey derived osteopontin, Lacprodan® OPN-10.

Lacprodan® OPN-10 is a proprietary whey-based protein product that contains bovine-derived osteopontin (OPN), found in human milk and other bodily tissues. In vitro genotoxicity tests conducted according to accepted guidelines at up to 5000µg/plate OPN failed to induce genetic mutations in Salmonella typhimurium strains and Escherichia coli strain and did not induce chromosomal aberrations or cytotoxicity in human lymphocytes. Administration of an acute dose of Lacprodan® OPN-10 (2300mg/kg body weight) to male and female mice did not induce chromosomal damage or mitotic apparatus damage to erythroblasts from bone marrow. Lacprodan® OPN-10 was evaluated in a 13-week oral toxicity study in which rats were fed diets containing 0.5%, 1.0% and 2.0% Lacprodan® OPN-10. No test-article-related clinical observations or toxicological effects on body or organ weights, food consumption, ophthalmic effects, locomotor activity, hematology, clinical chemistry, urinalysis, or pathology were identified. In a teratogenicity study, administration of Lacprodan® OPN-10 up to 2500mg/kgbw/day via gavage to pregnant rats had no effect on dams or pups. The No Observed Adverse Effect Level (NOAEL) for Lacprodan® OPN-10 in the 13-week toxicity study was 2.0% of the diet (equivalent to 1208mg/kgbw/day in male rats and 1272mg/kgbw/day in female rats).

Effects of term infant formulas containing high sn-2 palmitate with and without oligofructose on stool composition, stool characteristics, and bifidogenicity.

OBJECTIVES: Levels of stool fatty acid soaps and beneficial bacteria differ between formula-fed and breast-fed infants; addition of specific formula ingredients may reduce these differences. This study evaluated the effects of a term infant formula containing high sn-2 palmitate term infant formula (sn-2) or an identical formula supplemented with oligofructose (OF) at 2 concentrations (sn-2+3 g/L OF, sn-2+5 g/L OF) on stool composition, stool characteristics, and fecal bifidobacteria. METHODS: Healthy, term formula-fed infants 7 to 14 days old (n = 300) were randomized in a double-blind manner to receive standard formula (control), sn-2, sn-2+3 g/L OF, or sn-2+5 g/L OF for 8 weeks. Human milk (HM)-fed infants (n = 75) were studied in parallel. Stool samples were collected from all subjects at week 8 for fatty acid soaps and mineral content, and from a subset at baseline and week 8 for bifidobacteria. Stool characteristics were assessed via 3-day diary. RESULTS: The sn-2 group had 46% less stool soap palmitate (P < 0.001) and softer stools than control (20% more mushy soft stools, P = 0.026; 50% fewer formed stools, P = 0.003). Addition of OF resulted in even fewer formed stools versus control (65% fewer for sn-2+3 g/L OF, 79% fewer for sn-2+5 g/L OF), with 5 g/L OF more closely resembling that of HM-fed infants. Both sn-2 (P < 0.05) and sn-2 with OF groups (P < 0.01) had significantly higher fecal bifidobacteria concentrations than control at week 8, not differing from HM-fed infants. CONCLUSIONS: High sn-2-palmitate formulas led to reduced stool soaps, softer stools, and increased bifidobacteria, whereas addition of OF further improved stool consistency. Those modifications brought outcomes in formula-fed infants closer to that in HM-fed infants.

Postnatal dietary supplementation with either gangliosides or choline: effects on spatial short-term memory in artificially-reared rats.

This study addressed the hypothesis that dietary supplementation with either gangliosides or choline during the brain growth spurt would enhance short-term spatial memory. Male Long-Evans rats were reared artificially from postnatal days (PD) 5-18 and were fed diets containing either (i) choline chloride 1250 mg/l (CHL), (ii) choline chloride 250 mg/l and GD3 24 mg/l (GNG) or (iii) choline chloride 250 mg/l (STD). A fourth group (SCK) was reared normally. Rats were weaned onto AIN 93G diet and on PD 35 were trained on a cued delayed- matching-to-place version of the Morris water maze. All groups learned to swim to the beacon that indicated the platform position on the first trial; similarly, on the second un-cued trial, the distance swam to reach the platform decreased to the same extent in all groups over the five days of training. The groups also responded in the same way to an increase in delay between the first and second trial from 1 min to 1 h, showing an increase in the distance swam, accompanied by a decrease in the number of direct swims to the platform. Thus, all rats were equally proficient at using spatial short-term memory, regardless of the choline or ganglioside content of the preweaning diet.

Dietary ganglioside inhibits acute inflammatory signals in intestinal mucosa and blood induced by systemic inflammation of Escherichia coli lipopolysaccharide.

Our previous study demonstrated that feeding ganglioside increased total ganglioside content while decreasing cholesterol and caveolin-1 content in developing rat intestinal lipid microdomains. Cholesterol or caveolin depletion in membranes inhibits inflammatory signaling by disrupting microdomain structure. We hypothesized that dietary ganglioside-induced reduction in cholesterol content will reduce proinflammatory mediators in the intestinal mucosa after acute exposure to bacterial endotoxin. Weanling rats were fed semipurified diets with 0.1% (wt/wt of total fat) gangliosides (treatment) or without ganglioside (control). After 2 weeks of feeding, half of animals from each diet group were injected with saline or lipopolysaccharide (LPS) endotoxin (Escherichia coli serotype O111:B4, intraperitoneal, 3 mg/kg body weight) to induce acute gut inflammation. Intestinal mucosa and blood were collected after 6 h. The effect of dietary ganglioside on proinflammatory mediators including cholesterol, platelet-activating factor, prostaglandin E2, leukotriene B4 (LTB4), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNF-alpha) was determined in inflamed mucosa and blood. Feeding animals the control diet increased cholesterol content in intestinal lipid microdomains by 92% after LPS injection compared with saline injection. Animals fed the ganglioside diet significantly decreased cholesterol content in lipid microdomains by 60% compared with animals fed the control diet. Feeding animals the ganglioside diet increased total ganglioside content by 90% while decreasing platelet-activating factor content by 45% in the inflamed mucosa by acute systemic exposure to LPS compared with animals fed the control diet. When animals were fed the ganglioside diet, the levels of prostaglandin E2, LTB4, IL-1beta, and TNF-alpha were lower in inflamed mucosa, and LTB4, IL-1beta, and TNF-alpha were decreased in plasma by 41%, 58%, and 55% compared with control animals, respectively. The present study demonstrates that dietary gangliosides inhibit proinflammatory signals in the intestine and blood induced by acute inflammation of LPS and suggests therapeutic potential in the treatment and management of acute local and systemic inflammatory diseases.

Dietary gangliosides increase the content and molecular percentage of ether phospholipids containing 20:4n-6 and 22:6n-3 in weanling rat intestine.

This study was conducted to determine whether dietary ganglioside (GG) increases the content of ether phospholipids (EPL) in intestinal mucosa. Weanling Sprague-Dawley rats were fed a semipurified diet consisting of 20% fat as a control diet. Two experimental diets were formulated by adding either 0.1% (w/w fat) GGs (GG diet) or 1.0% (w/w fat) sphingomyelin (SM diet) to the control diet. Fatty acid methyl esters from the alkenylacyl, alkylacyl and diacyl subclasses of phospholipids were measured to determine total and molecular percentage of EPL comprising the choline phosphoglyceride (CPG) and ethanolamine phosphoglyceride (EPG) fraction. Animals fed the GG diet significantly increased total EPL content both in CPG (by 36%) and in EPG (by 66%), and the molecular percentage of EPL in CPG (by 76%) and in EPG (by 59%) compared to animals fed the control diet. Dietary GG-induced increase in EPL resulted in a higher level of polyunsaturated fatty acids (PUFA) specifically in 20:4n-6 and 22:6n-3 compared to control animals, leading to a decrease in the ratio of saturated fatty acids (SFA) to PUFA both in CPG and in EPG. Feeding animals the SM diet showed a higher level of EPL than control animals with a concomitant increase in 22:6n-3 in EPL. The present data demonstrate that dietary GG increases the content and composition of EPL containing PUFA in the weanling rat intestine.

Dietary ganglioside decreases cholesterol content, caveolin expression and inflammatory mediators in rat intestinal microdomains.

Membrane microdomains rich in cholesterol and sphingolipids, including gangliosides (GGs), are known to be important regions for cell signaling and binding sites for various pathogens. Cholesterol depletion inhibits the cellular entry of pathogens and also reduces inflammatory signals by disrupting microdomain structure. Our previous study showed that dietary gangliosides increased total ganglioside incorporation while decreasing cholesterol in the intestinal mucosa. We hypothesized that diet-induced reduction in cholesterol content in the intestinal mucosa disrupts microdomain structure resulting in reduced pro-inflammatory signals. Male weanling Sprague-Dawley rats were fed semipurified diets for 2 weeks. Experimental diets were formulated to include either ganglioside-enriched lipid (GG diet, 0.02% gangliosides [w/w of diet] ) or polyunsaturated fatty acid (PUFA diet, 1% arachidonic acid and 0.5% docosahexaenoic acid, w/w of total fat), in a control diet containing 20% fat. Levels of cholesterol, GG, caveolin, platelet activating factor (PAF), and diglyceride (DG) were measured in the microdomain isolated from the intestinal brush border. The GG diet increased total gangliosides by 50% with a relative increase in GD3 and a relative decrease in GM3. Cholesterol content was also reduced by 23% in the intestinal microdomain. These changes resulted in a significant decrease in the ratio of cholesterol to ganglioside. The GG diet and the PUFA diet were both associated with reduction in caveolin, PAF, and DG content in microdomains, whereas no change occurred in the ganglioside profile of animals fed the PUFA diet. Dietary gangliosides decrease the cholesterol/ganglioside ratio, caveolin, PAF and DG content in microdomains thus exerting a potential anti-inflammatory effect during gut development.

Cutting edge: cyclooxygenase-2 activation suppresses Th1 polarization in response to Helicobacter pylori.

Helicobacter pylori infection causes a Th1-driven mucosal immune response. Cyclooxygenase (COX)-2 is up-regulated in lamina propria mononuclear cells in H. pylori gastritis. Because COX-2 can modulate Th1/Th2 balance, we determined whether H. pylori activates COX-2 in human PBMCs, and the effect on cytokine and proliferative responses. There was significant up-regulation of COX-2 mRNA and PGE(2) release in response to H. pylori preparations. Addition of COX-2 inhibitors or an anti-PGE(2) Ab resulted in a marked increase in H. pylori-stimulated IL-12 and IFN-gamma production, and a decrease in IL-10 levels. Addition of PGE(2) or cAMP, the second messenger activated by PGE(2), had the opposite effect. Similarly, stimulated cell proliferation was increased by COX-2 inhibitors or anti-PGE(2) Ab, and was decreased by PGE(2). Our findings indicate that COX-2 has an immunosuppressive role in H. pylori gastritis, which may protect the mucosa from severe injury, but may also contribute to the persistence of the infection.