The immunopotentiating effects of shark-derived protein hydrolysate.

OBJECTIVE: Peptides derived from natural sources can act as immunomodulating agents and prevent infections. The aim of this study was to investigate the immunopotentiating and protective effects of a shark-derived protein hydrolysate (SPH) against an enterotoxigenic Escherichia coli H10407 infection in a murine model. METHODS: Mice were fed an aqueous solution of SPH for 7 days before being inoculated with an experimental enterotoxigenic Escherichia coli H10407 infection. After euthanasia, small intestines were removed for histological study and the number of IgA and IgG producing cells was determined by direct immunofluorescence. Cytokines were measured in the serum and the intestinal fluid. RESULTS: The oral administration of SPH enhanced the gut barrier function via up-regulation of immunoglobulin A-producing cells and intestinal cytokines production, including interleukin-6 and tumor necrosis factor-α. The increase of transforming growth factor-ß and interleukin-10 contribute to the down-regulation of uncontrolled-inflammatory reaction induced by E. coli infection. From these results, the anti-inflammatory properties of SPH may be caused by regulation and priming mechanisms of the immune system. CONCLUSION: Enzymatic protein degradation confers immunomodulating and protective potentials to shark proteins and the resulted peptides could be used as an alternative therapy to reduce the risk of bacterial infections and inflammatory-related diseases.

Bovine colostrum enhances natural killer cell activity and immune response in a mouse model of influenza infection and mediates intestinal immunity through toll-like receptors 2 and 4.

Oral administration of bovine colostrum affects intestinal immunity, including an increased percentage of natural killer (NK) cells. However, effects on NK cell cytotoxic activity and resistance to infection as well as a potential mechanism remain unclear. Therefore, we investigated the effects of bovine colostrum (La Belle, Inc, Bellingham, WA) on the NK cytotoxic response to influenza infection and on toll-like receptor (TLR) activity in a primary intestinal epithelial cell culture. We hypothesized that colostrum would increase NK cell activity and that TLR-2 and TLR-4 blocking would reduce interleukin 6 production by epithelial cells in response to contact stimulation with colostrum. Four-month-old female C57BL/6 mice were supplemented with 1 g of colostrum per kilogram of body weight before and after infection with influenza A virus (H1N1). Animals were assessed for weight loss, splenic NK cell activity, and lung virus titers. Colostrum-supplemented mice demonstrated less reduction in body weight after influenza infection, indicating a less severe infection, increased NK cell cytotoxicity, and less virus burden in the lungs compared with controls. Colostrum supplementation enhanced NK cell cytotoxicity and improved the immune response to primary influenza virus infection in mice. To investigate a potential mechanism, a primary culture of small intestine epithelial cells was then stimulated with colostrum. Direct activation of epithelial cells resulted in increased interleukin 6 production, which was inhibited with TLR-2 and TLR-4 blocking antibodies. The interaction between colostrum and immunity may be dependent, in part, on the interaction of colostrum components with innate receptors at the intestinal epithelium, including TLR-2 and TLR-4.

Anti-oxidant, anti-inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds.

PURPOSE: Enzymatic protein hydrolysates of yellow pea seed have been shown to possess high anti-oxidant and anti-bacterial activities. The aim of this work was to confirm the anti-oxidant, anti-inflammatory and immunomodulating activities of an enzymatic protein hydrolysate of yellow field pea seeds. METHODS: The anti-oxidant and anti-inflammatory properties of peptides from yellow field pea proteins (Pisum sativum L.) were investigated in LPS/IFN-γ-activated RAW 264.7 NO⁻ macrophages. The immunomodulating potential of pea protein hydrolysate (PPH) was then studied in a murine model. RESULTS: Pea protein hydrolysate, after a 12 h pre-treatment, showed significant inhibition of NO production by activated macrophages up to 20%. Moreover, PPH significantly inhibited their secretion of pro-inflammatory cytokines, TNF-α- and IL-6, up to 35 and 80%, respectively. Oral administration of PPH in mice enhanced the phagocytic activity of their peritoneal macrophages and stimulated the gut mucosa immune response. The number of IgA+ cells was elevated in the small intestine lamina propria, accompanied by an increase in the number of IL-4+, IL-10+ and IFN-γ+ cells. This was correlated to up-regulation of IL-6 secretion by small intestine epithelial cells (IEC), probably responsible for B-cell terminal differentiation to IgA-secreting cells. Moreover, PPH might have increased IL-6 production in IECs via the stimulation of toll-like receptors (TLRs) family, especially TLR2 and TLR4 since either anti-TLR2 or anti-TLR4 was able to completely abolish PPH-induced IL-6 secretion. CONCLUSIONS: Enzymatic protein degradation confers anti-oxidant, anti-inflammatory and immunomodulating potentials to pea proteins, and the resulted peptides could be used as an alternative therapy for the prevention of inflammatory-related diseases.

Effects of the oral administration of the products derived from milk fermentation by kefir microflora on immune stimulation.

Nutritional status has a major impact on the immune system. Probiotic effects ascribed to fermented dairy products arise not only from whole microorganisms but also from metabolites (peptides, exopolysaccharides) produced during the fermentation. We recently demonstrated the immunomodulating capacity of kefir in a murine model. We now aimed at studying the immunomodulating capacity in vivo of the products derived from milk fermentation by kefir microflora (PMFKM) on the gut. BALB/c mice received the PMFKM for 2, 5 or 7 consecutive days. IgA+ and IgG+ cells were determined on histological slices of the small and large intestine. IL-4, IL-6, IL-10, IL-12, IFNgamma and TNFalpha were determined in the gut, intestinal fluid and blood serum. IL-6 was also determined in the supernatant of a primary culture of small intestine epithelial cells challenged with PMFKM. PMFKM up-regulated IL-6 secretion, necessary for B-cell terminal differentiation to IgA secreting cells in the gut lamina propria. There was an increase in the number of IgA+ cells in the small and large intestine. The increase in the number of IgA+ cells was accompanied by an increase in the number of IL-4+, IL-10+ and IL-6+ cells in the small intestine. Effects of PMFKM in the large intestine were less widely apparent than the ones observed at the small intestine lamina propria. All cytokines that increased in the small intestine lamina propria, also did so in blood serum, reflecting here the immunostimulation achieved in the gut mucosa. We observed that the PMFKM induced a mucosal response and it was able to up and down regulate it for protective immunity, maintaining the intestinal homeostasis, enhancing the IgA production at both the small and large intestine level. The opportunity exists then to manipulate the constituents of the lumen of the intestine through dietary means, thereby enhancing the health status of the host.

Effect of milk fermented with a Lactobacillus helveticus R389(+) proteolytic strain on the immune system and on the growth of 4T1 breast cancer cells in mice.

Previous studies on a murine model have demonstrated that the administration of Lactobacillus helveticus and Lactobacillus casei inhibits the development of fibrosarcoma and colon carcinoma, respectively. The aim of this work was to study the beneficial effects of the consumption of milk fermented by L. helveticus on a murine model for mammary carcinoma. Female BALB/c mice were challenged by a single subcutaneous injection of tumoral cells (American Type Culture Collection 4T1) in the left mammary gland. Prior to tumour injection, mice were fed for two, five or seven consecutive days with fermented milk. The following factors were monitored for 2 months: rate of tumour development, histological studies, apoptosis, phagocytic index, peritoneal macrophages, determination of beta-glucuronidase enzyme in peritoneal macrophages, determination of gamma-interferon (INFgamma) and tumour necrosis factor-alpha (TNF-alpha) in blood serum, determination of CD4+, CD8+, interleukin-6 (IL-6), IL-10, TNF-alpha and INFgamma by immunoperoxidase, and measurement of beta-glucuronidase activity in intestinal fluid. The administration of L. helveticus delayed the development of the tumour in all cases, a 2- or 7-day feeding period being most effective. This work demonstrates that milk fermented with L. helveticus decreases the growth rate of mammary tumours. The effect was mediated by increased apoptosis and decreased production of pro-inflammatory cytokines, in particular IL-6, implicated in oestrogen synthesis.

Immunomodulating capacity of commercial fish protein hydrolysate for diet supplementation.

Dietary proteins harbour bioactive peptides that can be released by a fermentation process. Fish proteins are a valuable and little-exploited source of potentially active biopeptides. The aim of this research was to evaluate the effects of a commercially available fermented fish protein concentrate (Seacure) (FPC) derived from a fermentation process, on the mucosal immune response in a murine model. BALB/c mice received the FPC or the non-fermented powder at different concentrations (0.20, 0.25 or 0.30mg/ml) for 2, 5 or 7 consecutive days. At the end of each feeding period, histological studies of the gut were carried out and the phagocytic activity of peritoneal macrophages, the number of IgA+ cells in the small intestine lamina propria and bronchial tissue and the number of IL-4+, IL-6+, IL-10+, IFNgamma+ and TNFalpha+ cells in the small intestine lamina propria were determined. Different accumulative doses of FPC did not induce any inflammatory immune response and the normal morphology of the small intestine was not affected. Phagocytic activity of peritoneal macrophages was enhanced following FPC administration at 0.3mg/ml for 7 consecutive days. The number of IgA+ cells increased in the small intestine lamina propria but not in the bronchial tissue. IL-4, IL-6 and IL-10 were all significantly increased in the lamina propria of the small intestine of animals that received FPC. At the same time, some pro-inflammatory cytokines such as IFNgamma and TNFalpha also increased, but the intestinal homoeostasis was maintained and no tissue damage was observed. We conclude that FPC is an immunomodulating food with a demonstrated capacity to enhance non-specific host defense mechanisms.

Effects of kefir fractions on innate immunity.

Innate immunity that protects against pathogens in the tissues and circulation is the first line of defense in the immune reaction, where macrophages have a critical role in directing the fate of the infection. We recently demonstrated that kefir modulates the immune response in mice, increasing the number of IgA+ cells in the intestinal and bronchial mucosa and the phagocytic activity of peritoneal and pulmonary macrophages. The aim of this study was to further characterize the immunomodulating capacity of the two fractions of kefir (F1: solids including bacteria and F2: liquid supernatant), by studying the cytokines produced by cells from the innate immune system: peritoneal macrophages and the adherent cells from Peyer's patches. BALB/c mice were fed either kefir solid fraction (F1) or kefir supernatant (F2) for 2, 5 or 7 consecutive days. The number of cytokine (IL-1alpha, IFNgamma, TNFalpha, IL-6 and IL-10) producing cells was determined on peritoneal macrophages and adherent cells from Peyer's patches. Both kefir fractions (F1 and F2) induced similar cytokine profiles on peritoneal macrophages (only TNFalpha and IL-6 were up-regulated). All cytokines studied on adherent cells from Peyer's patches were enhanced after F1 and F2 feeding, except for IFNgamma after F2 administration. Moreover, the percentage of IL-10+cells induced by fraction F2 on adherent cells from Peyer's patches was significantly higher than the one induced by fraction F1. Different components of kefir have an in vivo role as oral biotherapeutic substances capable of stimulating immune cells of the innate immune system, to down-regulate the Th2 immune phenotype or to promote cell-mediated immune responses against tumours and also against intracellular pathogenic infections.

Effects of the oral administration of the exopolysaccharide produced by Lactobacillus kefiranofaciens on the gut mucosal immunity.

The probiotic effects ascribed to lactic acid bacteria (LAB) and their fermented dairy products arise not only from whole microorganisms and cell wall components but also from peptides and extracellular polysaccharides (exopolysaccharides) produced during the fermentation of milk. There is a lack of knowledge concerning the immune mechanisms induced by exopolysaccharides produced by lactic acid bacteria, which would allow a better understanding of the functional effects described to them. The aim of this study was to investigate the in vivo immunomodulating capacity of the exopolysaccharide produced by Lactobacillus kefiranofaciens by analyzing the profile of cytokines and immunoglobulins induced at the intestinal mucosa level, in the intestinal fluid and blood serum. BALB/c mice received the exopolysaccharide produced by L. kefiranofaciens for 2, 5 or 7 consecutive days. At the end of each period of administration, control and treated mice were sacrificed and the numbers of IgA+ and IgG+ cells were determined on histological slices of the small and large intestine by immunofluorescence. Cytokines (IL-4, IL-6, IL-10, IL-12, IFNgamma and TNFalpha) were also determined in the gut lamina propria as well as in the intestinal fluid and blood serum. There was an increase of IgA+ cells in the small and large intestine lamina propria, without change in the number of IgG+ cells in the small intestine. This study reports the effects of the oral administration of the exopolysaccharide produced by L. kefiranofaciens in the number of IgA+ cells in the small and large intestine, comparing simultaneously the production of cytokines by cells of the lamina propria and in the intestinal fluid and blood serum. The increase in the number of IgA+ cells was not simultaneously accompanied by an enhance of the number of IL-4+ cells in the small intestine. This finding would be in accordance with the fact that, in general, polysaccharide antigens elicit a T-independent immune response. For IL-10+, IL-6+ and IL-12+ cells, the values found were slightly increased compared to control values, while IFNgamma+ and TNFalpha+ cells did not change compared to control values. The effects observed on immunoglobulins and in all the cytokines assayed in the large intestine after kefiran administration were of greater magnitude than the ones observed in the small intestine lamina propria, which may be due to the saccharolytic action of the colonic microflora. In the intestinal fluid, only IL-4 and IL-12 increased compared to control values. In blood serum, all the cytokines assayed followed a pattern of production quite similar to the one found for them in the small intestine lamina propria. We observed that the exopolysaccharide induced a gut mucosal response and it was able to up and down regulate it for protective immunity, maintaining intestinal homeostasis, enhancing the IgA production at both the small and large intestine level and influencing the systemic immunity through the cytokines released to the circulating blood.

Immunomodulating capacity of kefir.

Kefir is a fermented milk produced by the action of lactic acid bacteria, yeasts and acetic acid bacteria, trapped in a complex matrix of polysaccharides and proteins. Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a long tradition of being regarded as good for health in countries where it is a staple in the diet. However, published human or animal feeding trials to substantiate this view are not numerous. The aim of this work was to determine the immunomodulating capacity of kefir on the intestinal mucosal immune response in mice and to demonstrate the importance of dose and cell viability on this response. BALB/c mice were fed with commercial kefir ad libitum (diluted 1/10, 1/50, 1/100 or 1/200) or pasteurized kefir (diluted 1/6, 1/10, 1/50, 1/100) for 2, 5 or 7 consecutive days. At the end of each feeding period, the bacterial translocation assay was performed in the liver. Small intestine structure was studied by haematoxilin-eosin staining and light microscopy. The number of IgA+ and IgG+ cells was also determined. For the functional doses chosen, cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha and IFN-gamma) were determined. Kefir and pasteurized kefir were able to modulate the mucosal immune system in a dose-dependent manner. Kefir was administred 10-times more diluted than pasteurized kefir, but it induced an immunomodulation of similar magnitude, indicating the importance of cell viabilty. The results suggest that a Th1 response was controlled by Th2 cytokines induced by kefir feeding. Pasteurized kefir would induce both Th2 and Th1 responses. This is the first study in vivo regarding the mechanisms involved in the immunomodulating capacity of the oral administration of kefir containing viable or heat-inactivated bacteria at different doses.