Yuan Kun Lee

Microflora of the gastrointestinal tract: a review.

The mucosal surface of the human gastrointestinal (GI) tract is about 200-300 m2 and is colonized by 1013-14 bacteria of 400 different species and subspecies. Savage has defined and categorized the gastrointestinal microflora into two types, autochthonous flora (indigenous flora) and allochthonous flora (transient flora). Autochthonous microorganisms colonize particular habitats, i.e., physical spaces in the GI tract, whereas allochthonous microorganisms cannot colonize particular habitats except under abnormal conditions. Most pathogens are allochthonous microorganisms; nevertheless, some pathogens can be autochthonous to the ecosystem and normally live in harmony with the host, except when the system is disturbed. The prevalence of bacteria in different parts of the GI tract appears to be dependent on several factors, such as pH, peristalsis, redox potential, bacterial adhesion, bacterial cooperation, mucin secretion, nutrient availability, diet, and bacterial antagonism. Because of the low pH of the stomach and the relatively swift peristalsis through the stomach and the small bowel, the stomach and the upper two-thirds of the small intestine (duodenum and jejunum) contain only low numbers of microorganisms, which range from 103 to 104 bacteria/mL of the gastric or intestinal contents, mainly acid-tolerant lactobacilli and streptococci. In the distal small intestine (ileum), the microflora begin to resemble those of the colon, with around 107-108 bacteria/mL of the intestinal contents. With decreased peristalsis, acidity, and lower oxidation-reduction potentials, the ileum maintains a more diverse microflora and a higher bacterial population. Probably because of slow intestinal motility and very low oxidation-reduction potentials, the colon is the primary site of microbial colonization in humans. The colon harbors tremendous numbers and species of bacteria. However, 99.9% of colonic microflora are obligate anaerobes.

Competition for adhesion between probiotics and human gastrointestinal pathogens in the presence of carbohydrate

The adhesion of Lactobacillus rhamnosus GG to human enterocyte-like Caco-2 cells was not inhibited by eight carbohydrates tested, namely N-acetyl-glucosamine, galactose, glucose, fructose, fucose, mannose, methyl-alpha-D-mannopyranoside and sucrose. The degree of hydrophobicity predicted the adhesion of L. rhamnosus GG to Caco-2 cells. L. rhamnosus GG, however, was able to compete with Escherichia coli and Salmonella spp. of low hydrophobicity and high adhesin-receptor interaction for adhesion to Caco-2 cells. The interference of adhesion of these gastrointestinal (GI) bacteria by L. rhamnosus GG was probably through steric hindrance, and the degree of inhibition was related to the distribution of the adhesin receptors and hydrophobins on the Caco-2 surface. A Carbohydrate Index for Adhesion (CIA) was used to depict the binding property of adhesins on bacteria surfaces. CIA was defined as the sum of the fraction of adhesion in the presence of carbohydrates, with reference to the adhesion measured in the absence of any carbohydrate. The degree of competition for receptor sites between Lactobacillus casei Shirota and GI bacteria is a function of their CIA distance. There were at least two types of adhesins on the surface of L. casei Shirota. The study provides a scientific basis for the screening and selection of probiotics that compete with selective groups of pathogens for adhesion to intestinal surfaces. It also provides a model for the characterisation of adhesins and adhesin-receptor interactions.

Handbook of Probiotics and Prebiotics

PART I: PROBIOTICS. Chapter 1: Probiotic microorganisms. 1.1 Definition. 1.2 Screening, identification and characterization of Lactobacillus and Bifidobacillus strains. 1.3 Detection and enumeration of gastrointestinal microorganisms. 1.4 Enteric microbial community profiling in gastrointestinal tract by Terminal-Restriction Fragment Length Polymorphism (T-RFLP). 1.5 Effective dosage for probiotic effects. 1.6 Incorporating probiotics into foods. 1.7 Safety of probiotic microorganisms. 1.8 Legal status and regulatory issues. Chapter 2: Selection and maintenance of probiotic microorganisms. 2.1 Isolation of probiotic microorganisms. 2.2 Selection of probiotic mircroorganisms. 2.3 Maintenance of Probiotic Microorganisms. Chapter 3: Genetic modification of probiotic microorganisms. 3.1 Mutants obtained from probiotic microorganisms by random mutagenesis. 3.2 Plasmids. 3.3 Vectors for Lactobacilli and Bifidobacteria. 3.4 Genetic recombination. Chapter 4: Role of probiotics in health and diseases. 4.1 Cell line models in research. 4.2 Laboratory animal models in research. 4.3 Effects on human health and diseases. 4.4 Effects on farm animals. Chapter 5: Mechanisms of probiotics. 5.1 Adhesion to intestinal mucus & epithelium by probiotics. 5.2 Combined probiotics and pathogen adhesion and aggregation. 5.3 Production of antimicrobial substances. 5.4 Immune effects of probiotic bacteria. 5.5 Alteration of microecology in human intestine. Chapter 6: Commercially available human probiotic microorganisms. 6.1 Lactobacillus acidophilus, LA-5?. 6.2 Lactobacillus acidophilus NCDO1748. 6.3 Lactobacillus acidophilus NFCM. 6.4 Lactobacillus casei Shirota. 6.5 Lactobacillus gasseri OLL2716 (LG21). 6.6 Lactobacillus paracasei ssp paracasei, F19(R). 6.7 Lactobacillus paracasei ssp paracasei, L. CASEI 431(R). 6.8 Lactobacillus rhamnosus GG, LGG(R). 6.9 Lactobacillus rhamnosus, GR-1(R) and Lactobacillus reuteri , RC- 14(R). 6.10 Lactobacillus rhamnosus HN001 and. 6.11 LGG(R)MAX, a multispecies probiotic combination. 6.12 Bifidobacterium animalis ssp. lactis, BB-12(R). 6.13 Bifidobacterium breve strain Yakult. 6.14 Bifidobacterium longum BB536. 6.15 Bifidobacterium longum strains BL46 and BL2C - Probiotics for adults and ageing consumers. PART II: PREBIOTICS. Chapter 7: Prebiotics. 7.1 The prebiotic concept. 7.2 A brief history of prebiotics. 7.3 Advantages and disadvantages of the prebiotic strategy. 7.4 Types of prebiotics. 7.5 Production of prebiotics. 7.6 Prebiotic mechanisms. 7.7 Modulating the intestinal microbiota in infants. 7.8 Modulating the intestinal microbiota in adults. 7.9 Modifying the intestinal microbiota in the elderly. 7.10 Health effects and applications of prebiotics. 7.11 Functional foods for animals. 7.12 Safety of prebiotics. 7.13 Regulation of prebiotics. 7.14 Conclusion.

Chemopreventive Effect of Lactobacillus rhamnosus on Growth of a Subcutaneously Implanted Bladder Cancer Cell Line in the Mouse

Lactic acid bacteria are known to have beneficial effects on the host, such as preventing carcinogenesis. The present study was designed to evaluate the chemopreventive effects of Lactobacttlus rhamnosus strain GG (LGG) in suppressing bladder cancer formation in a murine subcutaneous model of bladder cancer involving the inoculation of MB49 cells in C57B/L6 mice. After tumor implantation, one group of mice (n=8) was fed LGG immediately. The remaining mice that had tumors between 0.03–0.1 cm3 were divided into two groups: those fed LGG after 7 days (n=7) and those fed saline (n=7). A second group of mice without any inoculation of MB49 cells was fed either LGG (n=10) or saline (n=10) and served as non‐tumor‐bearing controls. LGG was administered orally at 1.6×l08 colony‐forming units daily. Mice fed LGG immediately after tumor cell implantation formed smaller tumors and some did not develop tumors (2 out of 8 mice), when the tumor burden was small. The level of spleen CD3, CD4 and CD8a T lymphocytes, as well as natural killer cells in mice fed immediately with LGG was also higher than that in control tumor‐bearing mice. There was an increase in lymphocytes and granulocytes in tumor sections, especially from the immediately fed group as compared to the controls. Our results suggest that oral consumption of LGG may prevent tumor growth via modulation of the immune system. The potential of LGG as an adjunct therapy in the treatment of bladder cancer could be further explored.

Lactobacillus Species is More Cytotoxic to Human Bladder Cancer Cells Than Mycobacterium Bovis (Bacillus Calmette-Guerin)

PURPOSEnWe determined if Lactobacillus species has growth inhibitory effects in human bladder cancer cell lines and how this effect compares with the known effects of Mycobacterium bovis, that is bacillus Calmette-Guerin (BCG).nnnMATERIALS AND METHODSnThe growth of MGH and RT112 cells were determined by cell counts after 24, 48 and 72 hours of exposure to L. casei strain Shirota (Yakult, Singapore) or L. rhamnosus strain GG (National Collection of Industrial and Marine Bacteria, Ltd., Aberdeen, Scotland) (1 x 10 and 1 x 10 cfu) or BCG (1 x 10 cfu) in the presence and absence of streptomycin. Annexin-V was used to monitor the presence of pre-apoptotic cells.nnnRESULTSnL. rhamnosus GG inhibited MGH proliferation and it was cytotoxic to RT112 cells (p <0.05). L. casei Shirota was cytotoxic to the 2 cell lines (p <0.05). BCG had a similar cytotoxic effect in MGH cells as Lactobacillus species but was not as effective in RT112 cells. Streptomycin abrogated the cytotoxic effect of Lactobacillus species but not that of BCG. Cytotoxic activity was not found in Lactobacilli culture supernates but it was induced in the presence of mammalian cells. L. rhamnosus GG induced apoptosis in RT112 but not in MGH cells. No apoptotic cells were detected after treatment with L. casei Shirota.nnnCONCLUSIONSnLactobacillus species induced cytotoxic effects in bladder cancer cells. Unlike BCG, it requires bacterial protein synthesis. Like BCG, L. casei Shirota induces cell death primarily via necrosis. The cytoxicity of these lactobacilli in bladder cancer cells raises the possibility of using this species of bacteria as intravesical agents for treating bladder cancer.

Free fucose is a danger signal to human intestinal epithelial cells

Fucose is present in foods, and it is a major component of human mucin glycoproteins and glycolipids. l-Fucose can also be found at the terminal position of many cell-surface oligosaccharide ligands that mediate cell-recognition and adhesion-signalling pathways. Mucin fucose can be released through the hydrolytic activity of pathogens and indigenous bacteria, leading to the release of free fucose into the intestinal lumen. The immunomodulating effects of free fucose on intestinal epithelial cells (enterocyte-like Caco-2) were investigated. It was found that the presence of l-fucose up regulated genes and secretion of their encoded proteins that are involved in both the innate and adaptive immune responses, possibly via the toll-like receptor-2 signalling pathway. These include TNFSF5, TNFSF7, TNF-alpha, IL12, IL17 and IL18. Besides modulating immune reactions in differentiated Caco-2 cells, fucose induced a set of cytokine genes that are involved in the development and proliferation of immune cells. These include the bone morphogenetic proteins (BMP) BMP2, BMP4, IL5, thrombopoietin and erythropoietin. In addition, the up regulated gene expression of fibroblast growth factor-2 may help to promote epithelial cell restitution in conjunction with the enhanced expression of transforming growth factor-beta mRNA. Since the exogenous fucose was not metabolised by the differentiated Caco-2 cells as a carbon source, the reactions elicited were suggested to be a result of the direct interaction of fucose and differentiated Caco-2 cells. The presence of free fucose may signal the invasion of mucin-hydrolysing microbial cells and breakage of the mucosal barrier. The intestinal epithelial cells respond by up regulation and secretion of cytokines, pre-empting the actual invasion of pathogens.

Lipid peroxidative stress and antioxidative enzymes in brains of milk-supplemented rats.

Skim milk cultured with lactic acid bacteria has been previously reported to reduce lipid peroxidation in rat livers. In this study, the effects of skim milk and cultured milk supplementation on peroxidative stress in brains of weanling rats were investigated. We observed a reduction of brain thiobarbituric acid reacting substances (TBARS) concentration in milk-supplemented animals as compared with controls. In brains of control rats, the superoxide dismutase (SOD) enzyme levels were significantly higher than those from the milk-supplemented animals. In addition, SOD activity in control animal brains had a positive correlation with the TBARS concentration. There was no significant differences in the brain glutathione-S-transferase (GST) levels of all the three groups of animals. The results suggest that milk supplementation may be beneficial in reducing peroxidative stress in the developing rat brain.

Expression of chemokine/cytokine genes and immune cell recruitment following the instillation of Mycobacterium bovis, bacillus Calmette–Guérin or Lactobacillus rhamnosus strain GG in the healthy murine bladder

Mycobacterium bovis, bacillus Calmette–Guérin (BCG) is the current gold standard for bladder cancer therapy. In this study a profile of the gene expression changes that occur after BCG instillation in the bladders of healthy mice was produced and compared to the type of immune cells recruited into the bladder. A similar comparison was made for Lactobacillus rhamnosus strain GG (LGG) instillations in healthy mice to determine its potential in the immunotherapy of bladder cancer. Mice were given six weekly instillations and were killed after the fourth, fifth and sixth instillations of BCG or LGG. Their bladders were harvested for chemokine/cytokine messenger RNA analysis using an array as well as semi‐quantitative reverse transcription–polymerase chain reaction. In a second set of mice both the bladder and draining lymph nodes were harvested for the analysis of immune cells. BCG significantly upregulated genes for T helper type 1 (Th1) chemokines: Cxcl2, Cxcl9, Cxcl10, Xcl1; and increased the expression of Th1/Th2 chemokines: RANTES, Ccl6 and Ccl7; Th1 polarizing cytokines: Il1β and Tnfa; and Fcγr1 and iNOS as early as after four weekly instillations. Most of these genes remained highly expressed after 6u2003weeks. In contrast, LGG transiently induced Cxcl10, Il16, Fcεr1 and Il1r2. Despite these findings, LGG instillation induced the recruitment of natural killer cells into the bladder and draining lymph nodes, as was observed for BCG instillation.