Harsharnjit S. Gill

Enhancement of natural and acquired immunity by Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019)

Consumption of lactic acid bacteria (LAB) has been suggested to confer a range of health benefits including stimulation of the immune system and increased resistance to malignancy and infectious illness. In the present study, the effects of feeding Lactobacillus rhamnosus (HN001, DR20), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019, DR10) on in vivo and in vitro indices of natural and acquired immunity in healthy mice were examined. Mice were fed daily with L. rhamnosus, L. acidophilus or B. lactis (10(9) colony forming units) and their immune function was assessed on day 10 or day 28. Supplementation with L. rhamnosus, L. acidophilus or B. lactis resulted in a significant increase in the phagocytic activity of peripheral blood leucocytes and peritoneal macrophages compared with the control mice. The proliferative responses of spleen cells to concanavalin A (a T-cell mitogen) and lipopolysaccharide (a B-cell mitogen) were also significantly enhanced in mice given different LAB. Spleen cells from mice given L. rhamnosus, L. acidophilus or B. lactis also produced significantly higher amounts of interferon-gamma in response to stimulation with concanavalin A than cells from the control mice. LAB feeding had no significant effect on interleukin-4 production by spleen cells or on the percentages of CD4+, CD8+ and CD40+ cells in the blood. The serum antibody responses to orally and systemically administered antigens were also significantly enhanced by supplementation with L. rhamnosus, L. acidophilus or B. lactis. Together, these results suggest that supplementation of the diet with L. rhamnosus (HN001), L. acidophilus (HN017) or B. lactis (HN019) is able to enhance several indices of natural and acquired immunity in healthy mice.

Dietary Probiotic Supplementation Enhances Natural Killer Cell Activity in the Elderly: An Investigation of Age-Related Immunological Changes

Many elderly subjects are at increased risk of infectious and noninfectious diseases due to an age-related decline in lymphoid cell activity (immunosenescence). Noninvasive means of enhancing cellular immunity are therefore desirable in the elderly. Previous reports have suggested that dietary supplementation could represent an effective means of enhancing the activity of circulating natural killer (NK) cells in the elderly. In the present study, we have conducted a pre–post intervention trial to determine the impact of dietary supplementation with probiotic lactic acid bacteria (LAB) on peripheral blood NK cell activity in healthy elderly subjects. Twenty-seven volunteers consumed low-fat/low-lactose milk supplemented with known immunostimulatory LAB strains (Lactobacillus rhamnosus HN001 or Bifidobacterium lactis HN019) for a period of 3 weeks. A dietary run-in of milk alone was shown to have no significant effect on NK cells. In contrast, the proportion of CD56-positive lymphocytes in peripheral circulation was higher following consumption of either LAB strain, and ex vivo PBMC tumoricidal activity against K562 cells was also increased. Supplementation with HN001 or HN019 increased tumoricidal activity by an average of 101 and 62%, respectively; these increases were significantly correlated with age, with subjects older than 70 years experiencing significantly greater improvements than those under 70 years. These results demonstrate that dietary consumption of probiotic LAB in a milk-based diet may offer benefit to elderly consumers to combat some of the deleterious effects of immunosenescence on cellular immunity.

Systemic Immunity-Enhancing Effects in Healthy Subjects Following Dietary Consumption of the Lactic Acid Bacterium Lactobacillus rhamnosus HN001

Objective: To determine the effects of the probiotic lactic acid bacterium, Lactobacillus rhamnosus HN001, on natural cellular immunity when delivered orally in normal low-fat milk (LFM) or lactose-hydrolyzed low-fat milk (LFM-LH). Design: A three stage, pre-post intervention trial, spanning nine weeks. Setting: Taipei Medical College Hospital, Taipei, Taiwan. Subjects: Fifty-two healthy middle-aged and elderly volunteers (17 males, 35 females; median age 63.5, range 44–80). Interventions: Stage 1 (run-in diet): 25g/200 mL reconstituted LFM powder, twice daily for 3 weeks. Stage 2 (probiotic intervention): LFM or LFM-LH, supplemented with 109 CFUs/g L. rhamnosus HN001 in each case, for 3 weeks. Stage 3 (wash-out): LFM for 3 weeks. Measures of Outcome: In vitro phagocytic capacity of peripheral blood polymorphonuclear (PMN) leukocytes; in vitro tumoricidal activity of natural killer (NK) leukocytes. Results: Immunological responses were unaffected by the run-in diet of LFM alone. In contrast, the relative proportion of PMN cells showing phagocytic activity increased by 19% and 15%, respectively, following consumption of HN001 in either LFM or LFM-LH; the relative level of NK cell tumor killing activity increased by 71% and 147%. In most cases these levels declined following cessation, but remained above baseline. Conclusions: Dietary consumption of L. rhamnosus HN001, in a base of low-fat milk or lactose-hydrolyzed low-fat milk, appears to enhance systemic cellular immune responses and may be useful as a dietary supplement to boost natural immunity.

Oligosaccharides and glycoconjugates in bovine milk and colostrum

Oligosaccharides and glycoconjugates are some of the most important bioactive components in milk. A great deal of information is available on the biological function of the components from human milk. Their primary role seems to be in providing protection against pathogens by acting as competitive inhibitors for the binding sites on the epithelial surfaces of the intestine. Evidence is also available to support the role of some of these components as growth promoters for genera of beneficial microflora in the colon. Compared with human milk, levels of oligosaccharides in bovine milk are very low. Nevertheless, a number of neutral and acidic oligosaccharides have been isolated from bovine milk and characterised. The highest concentration of these molecules is found in early postparturition milk (colostrum). The chemical structure of the oligosaccharides and many of the glycoconjugates from bovine milk are similar to those in human milk. It is likely that bovine oligosaccharides and glycoconjugates can be used in milk products as bioactive components in human nutrition.

Milk immunoglobulins and complement factors

The importance of colostrum for the growth and health of newborn offspring is well known. In bovine colostrum, the antibody (immunoglobulin) complement system provides a major antimicrobial effect against a wide range of microbes and confers passive immunity until the calfs own immune system has matured. Bovine serum and lacteal secretions contain three major classes of immunoglobulins: IgG, IgM and IgA. The immunoglobulins are selectively transported from the serum into the mammary gland, as a result of which the first colostrum contains very high concentrations of immunoglobulins (40-200 mg/ml). IgG1 accounts for over 75 % of the immunoglobulins in colostral whey, followed by IgM, IgA and IgG2. All these immunoglobulins decrease within a few days to a total immunoglobulin concentration of 0.7-1.0 mg/ml, with IgG1 representing the major Ig class in milk throughout the lactation period. Together with the antibodies absorbed from colostrum after birth, the complement system plays a crucial role in the passive immunisation of the newborn calf. The occurrence of haemolytic or bactericidal complement activity in bovine colostrum and milk has been demonstrated in several studies. This review deals with the characteristics of bovine Igs and the complement system to be exploited as potential ingredients for health-promoting functional foods.

Probiotics to enhance anti-infective defences in the gastrointestinal tract

Several clinical studies have demonstrated the therapeutic and/or prophylactic efficacy of specific probiotics against acute viral gastroenteritis and antibiotic-associated diarrhoea (including Clostridium difficile infection). Emerging evidence also suggests beneficial effects against Helicobacter pylori infection. The evidence of efficacy against travellers diarrhoea remains, however, inconclusive. The precise mechanisms by which probiotics potentiate host gastrointestinal defences and mediate protection are not fully known. There is evidence to suggest, however, that probiotics might contribute to host defence by reinforcing non-immunological defences and stimulating both specific and non-specific host immune responses. Little is known about the relative importance of the probiotic-stimulated mechanisms in host protection. This review summarises the evidence for the anti-infective effects of probiotics and discusses the effect of orally delivered probiotics on non-immunological and immunological defence mechanisms in the host, especially in the gastrointestinal tract.

Stimulation of the immune system by lactic cultures

An optimally functioning immune system is essential for protection against infectious diseases and cancers. Deficiency in any component of the immune system can predispose an individual to a greater risk of infection or may enhance the severity of disease. Several studies have shown that dietary supplementation with lactic acid bacteria (LAB) can be used to promote health and well-being; LAB are normal components of the human intestinal flora and are commonly used as starter cultures in dairy products. Consumption of LAB has been associated with a variety of health benefits including enhanced immune performance, increased resistance to infectious diseases, alleviation of food allergies and the suppression of cancer development. The precise mechanisms by which LAB act on the immune system are not fully understood. However, there is sufficient evidence to suggest that LAB exert their immunity enhancing effects by augmenting both non-specific (e.g. phagocyte function, NK-cell activity) and specific (e.g. antibody production, cytokine production, lymphocyte proliferation, delayed-type hypersensitivity) host immune responses. It is important to note that most of the evidence supporting these immunoenhancing effects is derived from in vitro or animal studies and there is a scarcity of carefully designed and properly controlled clinical studies demonstrating immune health benefits for humans, especially healthy human subjects. This article discusses the impact of LAB on different immune functions, the role of LAB-induced immunoenhancement in disease resistance, and highlights gaps in our knowledge that need further research.

Immunoregulatory peptides in bovine milk.

Bovine milk is known to contain a number of peptide fractions that can affect immune function. The vast majority of immunoregulatory peptides that have been characterised are hydrolysate derivatives of major milk proteins. Recent research has also indicated that the metabolic activity of probiotic lactic acid bacteria can generate de novo immunoregulatory peptides from milk, via enzymatic degradation of parent milk protein molecules. In contrast, relatively little is known of endogenous, preformed immunoregulatory peptides in milk that may be relevant to modulating human health. The natural in vivo role of preformed and enzymatically derived peptides is likely to be one of regulation of the neonatal (bovine) gastrointestinal tract immune system, in order to modulate immune function with respect to the development of immunocompetence and avoidance of undesirable immunological responses (e.g. tolerance, and hypersensitivity to nutrients). There is scope for the further characterisation of both the origin and function of milk-derived immunoregulatory peptides, so that their potential to influence human health can be fully appraised. This review highlights our current knowledge of milk-derived immunoregulatory peptides, and outlines areas that are of relevance for further research.

Bovine milk antibodies for health

The immunoglobulins of bovine colostrum provide the major antimicrobial protection against microbial infections and confer a passive immunity to the newborn calf until its own immune system matures. The concentration in colostrum of specific antibodies against pathogens can be raised by immunising cows with these pathogens or their antigens. Immune milk products are preparations made of such hyperimmune colostrum or antibodies enriched from it. These preparations can be used to give effective specific protection against different enteric diseases in calves and suckling pigs. Colostral immunoglobulin supplements designed for farm animals are commercially available in many countries. Also, some immune milk products containing specific antibodies against certain pathogens have been launched on the market. A number of clinical studies are currently in progress to evaluate the efficacy of immune milks in the prevention and treatment of various human infections, including those caused by antibiotic resistant bacteria. Bovine colostrum-based immune milk products have proven effective in prophylaxis against various infectious diseases in humans. Good results have been obtained with products targeted against rotavirus, Shigella flexneri, Escherichia coli, Clostridium difficile, Streptococcus mutans, Cryptosporidium parvum and Helicobacter pylori. Some successful attempts have been made to use immune milk in balancing gastrointestinal microbial flora. Immune milk products are promising examples of health-promoting functional foods, or nutraceuticals. This review summarises the recent progress in the development of these products and evaluates their potential as dietary supplements and in clinical nutrition.

Potential probiotic lactic acid bacteria Lactobacillus rhamnosus (HN001), Lactobacillus acidophilus (HN017) and Bifidobacterium lactis (HN019) do not degrade gastric mucin in vitro

The mucus layer (mucin) coating the surface of the gastrointestinal tract (GIT) plays an important role in the mucosal barrier system. Any damage or disturbance of this mucin layer will compromise the hosts mucosal defence function. In the present study, the ability of three potential probiotic lactic acid bacteria (LAB) strains (Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, Bifidobacterium lactis HN019) to degrade mucin in vitro was evaluated, in order to assess their potential pathogenicity and local toxicity. The LAB strains were incubated in medium containing hog gastric mucin (HGM, 0.3%) at 37 degrees C for 48 h, following which any decrease in carbohydrate and protein concentration in the ethanol-precipitated portion of the culture medium was determined, using phenol-sulphuric acid and bicinchonic acid (BCA) protein assays, respectively. The change in molecular weight of mucin glycoproteins, following incubation with the test strains, was monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In order to expose any ability of the test strains to degrade mucin visually and more directly, the test strains were also cultured on agarose containing 0.3% HGM and incubated anaerobically for 72 h at 37 degrees C. No significant change in the carbohydrate or protein concentration in mucin substrates was found following incubation with the test strains. No mucin fragments were derived from the mucin suspension incubated with test strains, and no mucinolysis zone was identified on agarose. These results demonstrate that the potential probiotic LAB strains tested here were unable to degrade gastrointestinal mucin in vitro, which suggests that these novel probiotic candidates are likely to be non-invasive and non-toxic at the mucosal interface.