Mechanism of Action of Probiotics: Two Concepts

Two concepts have been proposed to under­stand the beneficial effects of probiotics (26). The first was based on the effects observed when only a particular strain was added as a food supplement (e.g., L. plantarum and L. casei Shirota). The validity of this concept was demonstrated in several models and will be discussed below. A more recent concept has been derived from the use of a combination of lactic acid bacteria and bifidobacteria such as those present in VSL#3. This product con­tains 3 x 1011 cells/g of three strains of Bifidobacterium (B. longum, B. infantis, B. breve), four strains of Lactobacillus (L. acidophilus, L. casei, L. plantarum, L. delbrueckii subsp. bulgaricus) and one strain of Streptococcus (S. salivarius subsp. thermophilus). The stability and importance of this cGmbination for regulating gastrointestinal inflammation has been demonstrated and will also be reviewed below (27).

Probiotics - The Classical Concept

The classical explanation of the effects of probiotics in the gastrointestinal tract is through the direct or indirect modulation of the endogenous flora or the immune system (28). In this explanation, direct contact with intestinal epithelial cells is a prerequisite. Evidence of cross-talk between lactobacilli and antigen-presenting cells of the intestine, such as M-cells, was observed in electron microscopic studies (Fig. 3). More recent microarray studies have shown that lactobacilli in contact with epithelial cells induce mRNA of several genes. The colonization of the intestine or the contact of probiotics with the intestinal wall might be achieved by different mechanisms, such as adherence, aggregation or high growth to avoid washing out, or continued consumption of probiotics (25).
Cross-talk between bacteria and between bacteria and epithelial cells

According to Bengmark (7), probiotics seem to be effective in controlling overgrowth of potentially pathogenic microorganisms with a bacterial, viral, and fungal origin (29). In addition to the effects of probiotics in antagonizing noxious or unwanted microorganisms, eliminating toxins and stimulating the intestinal immune defense, some data suggest that probiotics also participate in the regulation of intestinal function (23). Thus, the mechanisms by which the indigenous intestinal bacteria inhibit pathogens include competition for colonization sites and nutrients, production of toxic compounds, and stimulation of the immune system. These mechanisms are not mutually exclusive, and inhibition may include one, several, or all of these mechanisms (30). Probiotics counteracted various enteric pathogens in experimental models such as Salmonella typhimurium in mice (31), Shigella (32), Clostridium difficile (33), Campylobacter jejuni (34) and Escherichia coli (35). Studies currently aim to understand the cross-talk mechanisms between different bacteria and between bacteria and intestinal epithelial cells. The use of new technologies such as microarray provides a new view of the richness of these relationships (Kevin Collins, personal communication).
Modulation at the endogenous flora

The modulation of the endogenous flora is beli.eved to occur through the antagoriistic interaction between several bacterial strains that colonize the intestine. In order to colonize the intestine and exert their beneficial effects, probiotic bacteria must be able to adhere to mucosal epithelial cells. Since colonization is species-specific, the microorganisms should be of human origin, as mentioned above, and survive in the intestine while ingested (36).
Adherence and colonization

Adherence and colonization are crucial for the competitive exclusion of pathogens. Colonization also leads to immune modulation. Therefore, direct contact with intestinal epithelial cells is a prerequisite for some probiotic effects on the immune system. This helps to enhance leukocyte phagocytic activity against enterobacteria, an effect that was detected following administration of probiotic strains to healthy individuals. Lactic acid bacteria in food can transiently colonize the intestine and induce beneficial effects. Survival during intestinal transit and adhesion to the epithelium seem to be important for modifying the host's immune reactivity. Since L. acidophilus strain La1 adheres to enterocytes in vitro, it was hypothesized that contact with immune cells may also occur in vivo. Bifidobacterium bifidium strain Bb12, which shows high fecal colonization, is another potential immunomodulator (37). The most successful studies examining this idea involved the use of Lactobacillus GG at a dose of 1 x 1010 viable organisms per day and Saccharomyces boulardii yeast at a dose of 1 g/day (38).
Enhancement of the intestinal barrier

Another mechanism by which probiotic bacteria may induce protection is through the enhancement of intestinal barrier function (39). Isolauri et al. studied the effect of lactobacilli on the gut mucosal barrier. In an experimental model, rat pups were divided into three feeding groups at the age of 14 days. In addition to normal mother's milk the "milk" group received a daily gavage of cow's milk, the "milk-GG" group received L. casei strain GG with cow's milk, whereas controls received the same vol­ume of water. At 21 days, the absorption of horse­radish peroxidase across patch-free jejunal segments and segments containing Peyer's patches was studied in Ussing chambers. Results showed that the mean absorption of intact horseradish peroxidase was significantly different in the study groups both in patch-free segments and in segments containing peyer's patches. There was a significant increase in the frequency of antibody secretor cells to B-Iactoglobulin (enzyme-linked im­munospot assay) in the milk-GG group. They concluded that prolonged cow's milk challenge in suckling rats increases gut permeability to intact proteins, whereas Lactobacillus GG counteracts this permeability disorder. These results suggest a link between the intensity of the antigen-specific immune response and the stabilization of the mucosal barrier (39).
Immunomodulatory effects

Although probiotics may promote nonspecific stimulation of the host immune system, such as immune cell proliferation, enhanced phagocytic activity and increased production of secretory immunoglobulin A (lgA), some experimental evidence suggest that lactobacilli decrease the host's immune response (6).

The presence of bacteria in the lumen and its epithelial cell adherence produce a variety of chemo­attractants and cytokines that can pass on luminal signals to mucosal immune cells (40). On the hu­moral side, Lactobacillus GG significantly increas­es in IgG, IgA, and IgM secretion from circulating lymphocytes (22).

There is some evidence suggesting that probiotics nonspecifically modulate the host's immune system. The state of the immune system is characterized by the number of T- or B-cells, the levels of cytokines, the levels of antibodies and the phago­cytic activity as a measure of cellular function. Activated T-cells can be divided into two major sub­sets based on their cytokine profile. In mice, T-helper 1 (Th1) cells produce principally IL-2, interferon-y (IFN-y) and Iymphotoxin, now called tumor necrosis factor-~ (TNF-~), whereas T-helper 2 (Th2) cells produce IL-4, IL-5, IL-6, IL-10 and IL-13. Human Th1 and Th2 cells not only produce a different set of cytokines but also present distinct functional properties and preferential expression of some activation markers (41) IL-1a and IL-1~ has a prominent involvement in both systemic and local inflammato­ry reactions. IL-1 ~ has numerous biological effects which are of relevance to inflammatory bowel disease such as induction of fever, arthralgia, hypotension, neutrophilia, increased acute phase response proteins, and hypoalbuminemia. A specific antago­nist, the IL-1 receptor antagonist (IL-1 RN) can block the effect of IL-1 by binding to IL-1 receptors with­out agonistic effects. It has been suggested that an imbalance in the IL-1~/IL-1ra rati9·may lead to a dysregulated immune response: A mucosal imbalance of IL-1 WIL-1 ra has been observed in inflammatory bowel disease (42). The molecular mecha­nisms underlying this imbalance are unknown, but evidence suggests that polymorph isms in these genes also affect the number of IL-1 and IL-1ra (43). In this respect, McGeehan et al. (44) described the capacity of a metalloproteinase inhibitor, G1129471, to block TNF-a secretion both in vitro and in vivo. This inhibition occurs at a post­translational label. They suggest that TNF-a processing is mediated by a unique Zn2+ endopeptidase, which is inhibited by GI 129471 and represents a new target for therapeutic intervention in TNF-a associated pathologies. As described below, some probiotics help to control the inflammatory response and indirectly inhibit metalloproteinase activity. Fabia et al. (45) showed a significant decrease in lactobacilli concentrations in colonic biopsy specimens from patients with active ulcerative colitis. This may also be of importance in the host with a heightened abnormal immune response.

In patients with Crohn's disease there is a de­crease in fecal Bifidobacteria concentration and the administration of Lactobacillus GG increased the intestinal IgA immune response and thereby promoted the gut immunological barrier (46). Similarly, in patients with active pouchitis there is a reduced fecal concentration of lactobacilli and bifi­dobacteria (47).

Ivanova et al. have examined the protective role of lyophilic dairy products (bulgaricum and bio­stim) with an active bacteria lactic acid strain. The protective effect was determined by the phagocytic activity and the activity of major immunoglobulins. These products were taken for one month in doses of 50 g/day. There were positive responses between the immune system and paraspecific resistance. The immunologic response was significantly higher after a lyophilic dairy diet (48).



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