Preuves scientifiques

Abstract. The objective of this study was to determine if supplementation with milk fermented by yogurt cultures and Lactobacillus casei (strain DN-114 001) could lessen acute diarrhoea in healthy children. The study was conducted over six months, with 287 children aged 18.9 (SD 6.0) months, comprising three periods of one month supplementation, each month being followed by one month without supplementation. Subjects were supplemented daily with either 125 g or 250 g (according to age) of one of three tested dairy products : standard yogurt, milk fermented by yogurt cultures and Lactobacillus casei (10⁸ cfu/ml), or a jellied milk (control product). A daily record was kept of the number and type of stools. Although the incidence of diarrhoea was not shown to be different between the groups, the severity of diarrhoea over the six-month study was significantly decreased (4.3 days) with the supplementation of L.casei fermented milk compared with the jellied milk (8.0 days) (p=0.009).

Abstract. The aim of this study was to determine if supplementation of healthy children with milk fermented by yogurt cultures and Lactobacillus casei strain DN-114 001 could affect the incidence of acute diarrhoea when compared with traditional yogurt. The study was a multicentre, randomised, double-blind trial, conducted over four months, on 928 children aged, at inclusion, 6-24 months. The study consisted of two periods: supplementation and observation. Subjects were supplemented daily with 100 g of one of the two dairy products being tested: standard yogurt and milk fermented by yogurt cultures and Lactobacillus casei (10⁸ cfu/ml). Frequency or duration of any diarrhoea episode was evaluated. As far as frequency was concerned there was a statistically significant difference between the groups, the incidence of diarrhoea being significantly reduced by supplementation with L.casei fermented milk (15.9%) compared with yogurt (22%) (p=0.03). These results suggest an additional benefit of L.casei in acute diarrhoea in children compared with standard yogurt.

Abstract : The aim of this work was to study feasibility of diarrhoea control in children (6 months to 5 y of age) by feeding fermented milk preparations. The design used was a randomized controlled clinical trial and the study was carried out at the Delhi University College Hospital providing tertiary care, and a nearby community centre Nand Nagri, a resettlement colony in East Delhi. Children suffering from acute diarrhoea (75 patients from the hospital and 75 from the community) were allocated to three groups by double-blind technique. Group 1 was given a fermented milk, Actimel, containing 10⁸ of each Lactobacillus casei DN-114001, Lactobacillus bulgaricus and Streptococcus thermophilus per gram. Group 2 was given Indian Dahi (Lf 40) containing 108 of each Lactococcus lactis, Lactococcus lactis cremoris and Leuconostac mesenteroides cremoris per gram. Group 3 was given ultra-heat-treated yoghurt preparation (no live bacteria). Actimel was also used as a starter to prepare the curd in order to study the preventive effect of diarrhoea in children in a community. In the hospital study Indian Dahi and Actimel administration reduced mean duration of diarrhoea by 0.3 and 0.6 day (P<0.001), respectively. The corresponding figures in the community study were 0.2 and 0.5 day (P<0.05), respectively. The families using Actimel as a starter showed a reduction in diarrhoeal morbidity episodes by 40% of the children tested in a 3 month follow-up. In conclusion, Actimel, fermented milk containing Lactobacillus casei DN-114001, and Indian Dahi can significantly reduce the duration of diarrhoea in children; the former preparation being superior.

Abstract: Probiotics are being increasingly studied for their ability to enhance host resistance to, and recovery from, infection. The probiotic strain Lactobacillus casei DN-114 001 has previously been shown to reduce the incidence and duration of episodes of diarrhoea in children. Our controlled pilot study aimed to evaluate the effect of supplementation for 3 weeks with milk fermented with yoghurt cultures and L. casei DN-114 001 on the incidence and severity of winter infections (gastrointestinal and respiratory) in elderly people. We found no difference in the incidence of winter infections between groups. However, duration of all pathologies was significantly lower in the treatment group (7.0±3.2 days, n=180) than in the control group (8.7±3.7 days; n=180) (p=0.024), as was maximal temperature (38.3±0.5ºC treatment group vs. 38.5±0.6ºC control; p=0.01). The potential for a 20% reduction in the duration of winter infections that we have found warrants further investigation on a larger scale.

Abstract. The aim of this study was first to compare the effects of milks fermented with various Lactobacillus casei strains on the survival rate of mice infected with Salmonella typhimurium. Secondly, the relationship between this protective effect and the non-specific immune response was studied. For each experiment, mice were randomized into groups of eight animals. Animals were supplemented with one of the following preparations for 7 days: milk fermented with one of the three L. casei strains (Danone strain 001 (LAB-1), LAB-2 and Yakult), yogurt (YF), a mixture of LAB-1 and YF (LAB-1+YF) or milk. The survival of the animals after single oral S. typhimurium infection (LD50 dosage) was monitored for 2 weeks. All fermented milks exhibited a protective effect against S. typhimurium infection. The highest protection was obtained with LAB-1 and that of LAB-1 + YF was significantly higher than that obtained with other treatments. Circulating IgA levels, ß-glucuronidase activity of peritoneal macrophages and phagocytosis index were significantly enhanced in animals supplemented with LAB-1 and Yakult. This study shows that for one bacteria species, various strains exhibit different effects on protection and on immune parameters. The hierarchy established for survival rates does not entirely correlate with the effects noted on immune parameters. This indicates that stimulation of non-specific immunity, especially on macrophage activity, is not the only mechanism involved in protection against intestinal infections.

Abstract. Group A rotavirus is the leading cause of diarrhea among children aged 3-36 mo worldwide. Introducing fermented milk products into the infant diet has been proposed for the prevention or treatment of rotavirus diarrhea. The preventive effect of milk fermented by the Lactobacillus casei strain DN-114 001 was studied in a model of germfree suckling rats supplemented daily from d 2 of life and infected with SA11 rotavirus at d 5 (RF group). One group was supplemented with nonfermented milk (RM) and two uninfected groups (CM and CF) received either nonfermented or fermented milk. Frequency and severity of diarrhea were observed. Rats were killed at various times from 0 to 120 h postinfection (p.i.). Bacteria were measured in the intestine, and rotavirus antigens were detected by ELISA in fecal samples and in different parts of the intestine. Histologic observations were made, including vacuolation, morphology of intestinal villi and number of mucin cells. RM rats had diarrhea for 6 d; compared with the CM group, they had alterations of the intestinal mucosa characterized by cellular vacuolation 48 and 72 h p.i. and a lower number of sulfated mucin cells 72 and 96 h p.i. (P<0.05). Early supplementation with fermented milk significantly decreased the clinical signs of diarrhea from 24 to 144 h p.i. (P<0.05) and prevented rotavirus infection in all sections of the intestine. Histologic lesions of the small intestine were greatly reduced (P<0.05) and the number of mucin cells remained unchanged. The data are discussed with respect to the possibility of reducing rotavirus diarrhea in young children by consumption of fermented milk.

This study investigated the protective capacity of the oral administration of fermented milk containing the probiotic strains; Lactobacillus casei, Lb. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, against enteroinvasive Escherichia coli infection in a murine (BALB/ c mice) model. Mice were fed for 2, 5 or 7 consecutive days with fermented milk diluted to a concentration of viable Lb. casei, Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus of 10(7) cfu/ml. Phagocytic activity of peritoneal macrophages and the number of IgA+ cells in small and large intestine were determined at the end of the feeding periods. For the preventive effect against Esch. coli, animals were fed for 5 days (selected dose). Mice were challenged with an infective dose of enteroinvasive Esch. coli of 10(8) cfu/mouse. The colonization of liver and spleen and the secretory IgA specific for the pathogen in the intestinal fluid were determined (ELISA test). Results showed that the unspecific immune response enhanced itself after 5 consecutive days of the administration of this fermented milk (increase in the percentage of phagocytosis and number of IgA+ cells in the small intestine). Treated animals showed less Esch. coli colonization of liver than control mice and a higher secretory anti-Esch. coli IgA in the intestinal fluids. These results suggest that the protection against enteroinvasive Esch. coli infection observed for the fermented milk containing probiotic bacteria may be associated with an enhance of the intestinal mucosa immunity.

Goals: To determine the efficacy of triple therapy supplemented with a specially designed fermented milk product containing specific probiotic Lactobacillus casei (L. casei) DN-114 001 strain on Helicobacter pylori eradication in children. Background: Lactobacillus species possess in vitro activity against H. pylori. There are no consistent data on the impact of eradication therapy supplemented with probiotics on H. pylori cure rates in childhood in vivo. Study: Multicentre, prospective, randomized, double-blind controlled study. 86 symptomatic H. pylori-positive children were randomized either to receive the control treatment of omeprazole, amoxicillin, and clarithromycin (OAC) for 7 days or the test treatment of omeprazole, amoxicillin, and clarithromycin for 7 days supplemented with fermented milk (Actimel) containing L. casei DN-114 001 (OAC-LC), for 14 days. H. pylori status was assessed at 4 weeks following therapy using two noninvasive tests. Results: Intention-to-treat (ITT) based eradication rates for the OAC-LC group were 84.6% (95% CI, 71.2%-95.5%), and 91.6% (95% CI, 76.9%-98.2%) by per-protocol (PP) analysis. Eradication in the OAC group was 57.5% (95% CI, 42.2%-72.3%) in the ITT set and 61.3% (95% CI, 44.4%-75.0%) in the PP group. Eradication success was higher in the OAC-LC group compared with the OAC group in both ITT (P=0.0045) and PP analysis (P=0.0019). Primary resistance for clarithromycin could be determined in 21.2%. Side effects were infrequent. Drug compliance was good throughout the study. Conclusion: Supplementation with fermented milk, containing live special probiotic L. casei DN-114 001, confers an enhanced therapeutic benefit on H. pylori eradication in children with gastritis on triple therapy.

BACKGROUND: Probiotic dairy products are increasingly gaining popularity. Although the role of probiotic bacteria in the prevention and treatment of pediatric and antibiotic associated diarrhea is fairly well established, their role in the prevention of adult infectious diarrhea has not been well investigated. METHODS: Five hundred forty-one, young male military recruits were enrolled and randomly assigned to receive a yogurt containing Lactobacillus casei (n = 275) or a nonprobiotic yogurt (n = 266). The incidence and duration of diarrhea were documented and stool samples examined for bacteria and parasites. RESULTS: Five hundred and two participants were eligible for final analysis, 254 receiving probiotic yogurt and 248 in the control group. Seventy-one participants (14.14%) experienced diarrhea during the study period. The incidence of diarrhea in the probiotic group and the control group was 12.2% and 16.1%, respectively (P = .207). The mean duration of diarrhea was 3 + /- 1.95 days in the probiotic group and 2.6 +/- 1.08 days in the control group (P = .276). CONCLUSION: Our study demonstrated a nonsignificant trend for reduction of the incidence of diarrhea among healthy young adults consuming yogurt containing Lactobacillus casei . Further study is needed to evaluate the role of probiotics in adults.

OBJECTIVE: To determine the efficacy of a probiotic drink containing Lactobacillus for the prevention of any diarrhoea associated with antibiotic use and that caused by Clostridium difficile. DESIGN: Randomised double blind placebo controlled study. PARTICIPANTS: 135 hospital patients (mean age 74) taking antibiotics. Exclusions included diarrhoea on admission, bowel pathology that could result in diarrhoea, antibiotic use in the previous four weeks, severe illness, immunosuppression, bowel surgery, artificial heart valves, and history of rheumatic heart disease or infective endocarditis. INTERVENTION: Consumption of a 100 g (97 ml) drink containing Lactobacillus casei, L bulgaricus, and Streptococcus thermophilus twice a day during a course of antibiotics and for one week after the course finished. The placebo group received a longlife sterile milkshake. MAIN OUTCOME MEASURES: Primary outcome: occurrence of antibiotic associated diarrhoea. Secondary outcome: presence of C difficile toxin and diarrhoea. RESULTS: 7/57 (12%) of the probiotic group developed diarrhoea associated with antibiotic use compared with 19/56 (34%) in the placebo group (P=0.007). Logistic regression to control for other factors gave an odds ratio 0.25 (95% confidence interval 0.07 to 0.85) for use of the probiotic, with low albumin and sodium also increasing the risk of diarrhoea. The absolute risk reduction was 21.6% (6.6% to 36.6%), and the number needed to treat was 5 (3 to 15). No one in the probiotic group and 9/53 (17%) in the placebo group had diarrhoea caused by C difficile (P=0.001). The absolute risk reduction was 17% (7% to 27%), and the number needed to treat was 6 (4 to 14). CONCLUSION: Consumption of a probiotic drink containing L casei, L bulgaricus, and S thermophilus can reduce the incidence of antibiotic associated diarrhoea and C difficile associated diarrhoea. This has the potential to decrease morbidity, healthcare costs, and mortality if used routinely in patients aged over 50.

Abstract. The aim of this study was to compare the effects of milk and of various fermented milks on the composition and metabolic activities of the intestinal microflora. Groups of eight rats were fed for 6 wk a diet containing 30% non fermented milk (M), yogurt (Y), milk fermented with Lactobacillus casei (LcFM) or milk fermented with the association of L. casei DN-114 001 and yogurt starters (LcYFM). In the first study, the survival of the lactic acid bacteria from the fermented milks was assessed by bacterial enumeration in feces of germ-free rats (GF rats) fed milk or fermented milks.The metabolic activities of the lactic acid bacteria were studied in these rats by measurement of glycolytic activities and products of bacterial fermentation, i.e., acetate and lactate (isoforms L and D). In a second study, the effects of fermented milks on the composition and metabolism [gas, glycolitic activities, short-chain fatty acid (SCFA), alcohol and ammonia] of human flora were studied using human flora-associated rats (HF rats). In GF rats, the survival of L. casei in the feces did not differ between those fed the LcFM and LcYFM diets. L. bulgaricus was detected in the feces of the rats fed Y, whereas Streptococcus thermophilus was found in the feces of the LcYFM group. In HF rats, fecal concentration of Bifidobacteria was greater in the LcFM group than in others. β-glucuronidase (EC 3.2.1.31) activity was lower in rats fed LcFM and Y than in those fed M and LcYFM, whereas α-galactosidase (3.2.1.23), β-glucosidase (EC 3.2.1.20) and β-glucosidase (EC 3.2.1.21) activities were higher in the LcYFM group compared with the others. Methane excetion was higher in rats fed Y than in other group. Cecal SCFA concentrations did not differ in LcFM, Y and M groups, but total SCFA, acetate, propionate and butyrate were significantly greater in the LcYFM group. These results suggest that milk fermented with the combination of L. casei and yogurt starters leads to specific effects that are different from the simple addition of the effects found with yogurt and milk fermented with L. casei. These specific effects are potentially beneficial to human health.

Abstract. Ingestion of fermented dairy products induces changes in the equilibrium and metabolism of the intestinal microflora and may thus exert a healthful influence on the host. We compared the effects of consumption of a traditional yogurt, a milk fermented with yogurt cultures and Lactobacillus casei (YC), and a nonfermented gelled milk on the fecal microflora of healthy infants. Thirty-nine infants aged 10-18 mo were randomly assigned to one of three groups in which they received 125 g/d of one of the three products for 1 mo. The following indexes were not modified during the supplementation period or for 1 wk after the end of supplementation: total number of anaerobes, bifidobacteria, bacteroides, and enterobacteria; pH; water content; concentrations of actetate, butyrate, propionate, and lactate; and bacterial enzyme activity of ß-galactosidase and ß–glucosidase. In contrast, in the yogurt group the number of enterococci in fecal samples increased (P<0.05), whereas the percentage of branched-chain and long-chain fatty acids, which are markers of proteolytic fermentation, decreased (P<0.05). In the YC group, the percentage of children with >6 log₁₀ colony-forming units lactobacilli/g feces increased (P<0.05) whereas the potentially harmful enzyme activity of ß–glucuronidase and ß–glucosidase decreased (P<0.05). These decreases were particularly marked in those infants in the YC group in whom activity of the enzymes was initially unusually high.

Abstract. Trophic effects of milk fermented with Lactobacillus helveticus, Lactobacillus paracasei ssp paracasei, Bifidobacterium sp., or the combination of Lactobacillus bulgaricus and Streptococcus thermophilus (yogurt) were studied on the IEC-6 intestinal epithelial cell line. Incorporation of [methyl-3H]thymidine, mitochondrial deshydrogenase activities, cyclic AMP production, and differenciation of levels of the IEC-6 strain were evaluated between the 15th and the 30th passage in culture. All fermented and unfermented milks enhanced trophic responses of IEC-6 cells in a dose-dependent manner. Compared with the corresponding milks, supernatant fractions were more effective in stimulating mitochondrial deshydrogenase response. Fermented milk supernatants were also more effective than the corresponding unfermented fractions. Increases in DNA synthesis and cyclic AMP confirmed the activation observed with mitochondrial deshydrogenase. Yogurt induced the more trophic response with an increased number of the more differentiated cell morphotype. Fermentation with L. casei also demonstrated an important trophic adaptation of IEC-6 cells. Milk processing by lactic acid bacteria enhanced trophic and proliferation responses of intestinal epithelial cell line IEC-6. These results suggested that IEC-6 cells could represent an accurate and easy in vitro model for testing the trophic quality of various nutrients and for an optimization of physiological digestive functions.

Abstract. The nutritional benefits of lactic acid bacteria in fermented dairy products have been well documented, especially in terms of weight gain and feed efficiency, but not in terms of small intestine adaptation. The effects of a diet supplemented (30% wt/wt) with milk fermented either by Lactobacillus casei DN-114 001 or yogurt for 3 or 15 days were investigated in the small intestine of mice by morphometry, kinetic analysis and determination of brush-border enzyme activities. Results were compared with those obtained with standard or milk isocaloric diets. Cell proliferation and villous area were significantly increased in the proximal intestine of mice fed the fermented-milk-supplemented diets for 3 days and were associated with hypertrophy and hyperplasia of Planeth and goblet cells. Lactase-specific activity was increased by fermented-milk diets at days 3 and 15, whereas there was no variation in maltase-specific activity. Alkaline phosphatase-specific activity was increased after 3 days of the three tested diets in the whole intestine, and after 15 days in the proximal intestine. Aminopeptidase activity was increased in the distal part of the intestine after 3 days of the 3 diets. Our findings suggest that diets supplemented with fermented milks have a positive effect on the trophicity of the mucosa in the small intestine of mice.

Microflora-born bacteria or probiotic strains are able to modulate host-pathogens interactions in the gut. In vivo and in vitro studies indicate that species-specific modulations of intestinal cell glycosylation may represent a simple, general and efficient mechanism to adapt the host defense toward pathogens.

Abstract. Probiotics are living microorganisms, which, when ingested in adequate amount exert health benefits to the host. Probiotics, for instance, could act through the reinforcement of the intestinal epithelial barrier function. The goal of the present study was to determine if Lactobacillus casei DN-114 001 could abrogate the increase of paracellular permeability induced by enteropathogenic Escherichia coli. We have used the human colon T84 cell line infected with a wild type enteropathogenic Escherichia coli (strain E2348/69). The paracellular permeability was followed by monitoring transepithelial electrical resistance variations and by observing Zonula Occludens-1 distribution. Two infection procedures were used; co-incubation (the pathogenic and probiotic strains were simultaneously incubated with T84 cells) and post-infection (the probiotic was added in presence of pathogenic bacteria, three hours after the beginning of the infection). We also investigated the effect of Lactobacillus casei on enteropathogenic Escherichia coli adhesion. Lactobacillus casei DN-114 001 inhibited in a dose-dependent-manner, enteropathogenic Escherichia coli-induced transepithelial electrical resistance decrease, and Zonula Occludens-1 redistribution, using two different infection procedures. However, Lactobacillus casei did not inhibit the pathogenic strain adhesion. Lactobacillus casei DN-114 001 inhibited EPEC-induced paracellular permeability increase. This property could partially explain the previously observed health benefits of this probiotic on human natural defenses, for example, those associated to the prevention of diarrhea.

Abstract. Ileal lesions in 36.4% of patients with Crohn’s disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli, isolated from Crohn’s disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting colony-forming units. The inhibitory effects of L. casei were determined after co-incubation with adherent-invasive E. coli or after pre-incubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in co-incubation and 43% to 62% in pre-incubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli, or a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn’s disease.

Abstract. There is evidence that exhaustive exercise produces depression of the immune system, especially on the number and activity of Natural Killer (NK) cells. On the other hand, fermented milk has been shown to moderate the immune response by inducing NK activity. The present work was carried out to determine if a Lactobacillus casei (LC) fermented milk supplemented diet would provide protection of the immune system against an exercise induced immune system depression of NK cells.
Twenty-five athletes were selected out of 94 for their significant decrease in NK cell concentration compared with a normal basal concentration in plasma 2h after an exercise stress test. Subjects ingested a daily fermented milk diet with LC for one month and a standard milk diet also for one month. After each phase of dieting, a subject was investigated before, 5 min and 2 h after an exercise stress test, testing for NK cells and IL-1ß, IL-6, IL-2, IFNγ, IgA, IgM, IgG, NK cells, CD8, CD4, CD3 and sIL-2 receptor.
A significant smaller decrease of NK cell concentration after 2h was found in the fermented milk feeding phase vs. the standard milk period.

Abstract :
Background: A suppressed immune response has been documented in students under examination stress. Aim of the study: The current study aimed to evaluate the effect of milk fermented with yogurt cultures plus Lactobacillus casei DN-114 001 (Actimel®) on the immune system of subjects under academic examination stress. Methods: University students were allocated to one of two groups, receiving during 6 weeks (3 weeks prior to, as well as the 3-week duration of the examination period) either: a) a glass of semi-skimmed milk each day (control group, n = 63) or b) two 100mL portions per day of fermented milk (treatment group, n = 73). Anxiety and immunological measurements were monitored at baseline (Phase 0) and study end (Phase 1). Results : The results were expressed as the differences between the data obtained from Phase 0 and Phase 1. This was calculated by subtracting Phase 1 results from the Phase 0 and it is denominated "Treatment effect". Mean (±SE) anxiety increased significantly (P<0.05) over the 6-week study in all students, from 40.74 ± 2.50 to 61.19 ± 2.64 (in percentiles). There was no significant treatment effect since this increase was similar in the control and the treatment groups (21.65 ± 5.09 vs 19.14 ± 3.67, respectively). However, there was a significant treatment effect (P<0.05) on the mean change in absolute number of lymphocytes during the 6-week study, which decreased in the control group (-0.04 ± 0.12 cells x 103/mm³) and increased in the treatment group (0.37 ± 0.11 cells x 103/mm³). There was also a significant treatment effect (P<0.05) on the change in absolute numbers of CD56 cells during the 6-week study. Mean absolute CD56 cells significantly decreased (P<0.05) in the control group (-51.97 ± 21.33 cells/mm³), whilst remaining similar in the treatment group (17.29 ± 17.27 cells/mm³). During the study, mean serum cortisol increased 4.30 ± 0.98 µg/dL in the control group, and 1.75 ± 1.05 µg/dL in the treatment group and no significant differences were found between both values (P=0.062). Conclusions: Milk fermented with yogurt cultures plus Lactobacillus casei DN-114 001 was able to modulate the number of lymphocytes and CD56 cells in subjects under academic examination stress.

Abstract: Background: Lactic acid bacteria have been suggested as a dietary strategy to enhance immune system activity. Objective: The aim of the current work was to test the effects of a Lactobacillus casei fermented milk consumption on monocyte activity of middle-aged volunteers. Design: Forty-five healthy volunteers, 24 women and 21 men (aged: 51 - 58 years), were randomized in two groups to receive three cups per day of a fermented milk containing L. casei DN114001 (108 – 1010/g) (n = 23), or placebo (n = 22), during 8 weeks. White blood cell count and the oxidative burst capacity of monocytes and granulocytes were examined with a FACScalibur. Measurements were performed at baseline and after the nutritional intervention, at day fifty-six. Results: After the trial, no changes in immune cell proportions were detected in both groups, as well as in monocyte activity after the placebo consumption (p = 0.625). However, volunteers included in the probiotic-treated group increased (p = 0.029) their oxidative burst capacity of monocytes, and this increment inversely and significantly correlated with the intensity registered at baseline (r = -0.653, p = 0.004). Conclusions: Results showed that daily intake of fermented milk containing Lactobacillus casei was able to module the oxidative burst capacity of monocyte subset in healthy middle-aged people, particularly in subjects with lower initial levels. Thus, this nutritional strategy could be considered to maintain immune competence in ageing.

Abstract: Different lactic acid bacteria have often been administered as a dietary means to enhance immune system activity. Based on this statement, the aim of the current work was to test the effects of a Lactobacillus casei DN114001 fermented milk consumption on the immune response capacity in middle-age volunteers. Forty-five healthy volunteers, 24 women and 21 men (aged: 51-58 years), were randomized into two groups to receive three cups per day of a L. casei DN114001 (10(8)-10(10) ufc/g) fermented milk (n = 23), or placebo (n = 22), during an 8-week period. Measurements were performed before (day 0), and after the nutritional intervention (day 56). After the trial, no changes in immune cell proportions were detected, but the probiotic-treated group increased oxidative burst capacity of monocytes (probiotic group: p = 0.029; placebo group: p = 0.625), as well as NK cells tumoricidal activity (probiotic group: p = 0.023; placebo group: p = 0.125). Results showed that daily intake of fermented milk containing Lactobacillus casei DN114001 could have a positive effect in modulating the innate immune defense in healthy-middle-age people.

Daily intake of probiotic as well as conventional yogurt has a stimulating effect on cellular immunity in young healthy women.
Meyer AL, Micksche M, Herbacek I, Elmadfa I. Ann Nutr Metab. 2006;50(3):282-9. Epub 2006 Feb 23
Department of Nutritional Sciences of the University of Vienna, Vienna, Austria.

BACKGROUND/AIMS: The aim of this work was to study the effects of daily yogurt consumption on the cellular immunity of young healthy women and to compare a conventional with a probiotic product. METHODS: 33 young healthy women (22-29 years) consumed 100 g/day of either probiotic or conventional commercially available yogurt for 2 weeks and 200 g/day for another 2 weeks followed by a 2-week washout period with no fermented food at all. Before the intervention and after each phase, a complete white blood count was done, the percentage of activated CD69+ T lymphocytes after stimulation of whole blood with pokeweed mitogen was determined as well as the natural cytotoxicity of peripheral blood mononuclear cells against a human erythroleukemic target cell line (K562). All analyses were done by flow cytometry. RESULTS: In the probiotic group only, the numbers of cytotoxic T lymphocytes (CD3+CD16+CD56+) increased significantly (+30.8% with p = 0.001, +22.1 and +32.7% with p = 0.002, for T2, T3 and T4 compared to T1). There were no major changes for other cell populations, and all remained within the physiological range. In both groups, the expression of CD69 on T lymphocytes increased after yogurt consumption, especially on CD8+ (conventional: T2 +23%, T3 +27.2%, probiotic: T2 +15.7%; T3 +10.8% compared to T1) and to a lesser extent on CD4+ (conventional: T2 +7.7%, T3 +14.9%, probiotic: T2 +4% compared to T1. The cytotoxic activity also augmented following the intake, this effect persisting after cessation of consumption. However, there were no significant differences between the probiotic and the conventional yogurt group. CONCLUSION: Daily yogurt intake has a stimulating effect on cellular immune functions, but in this study the probiotic product did not perform better than the traditional one.

Abstract.
Background & Aims: In Crohn´s disease, TNFα plays a key role in the pathogenesis of altered mucosal immune function. The effect of selected bacteria on TNFα release by explants of intestinal mucosa was investigated.
Methods: Ileal specimens were obtained at surgery from 10 patients with Crohn’s disease (ileal stricture) and 4 controls undergoing right hemi-colectomy (caecal cancer). Mucosal explants from each specimen were cultured for 24 h with either non-pathogenic E. coli, Lactobacillus casei DN-114 001, L.bulgaricus LB10, or L. crispatus (each study contained blank wells with no bacteria). Tissue and bacteria viability was confirmed by LDH release and culture. TNFα was measured in supernatants, and phenotype of intestinal lymphocytes analysed by flow cytometry.
Results: Co-culture of mucosa with bacteria did not modify LDH release. Release of TNFα by inflamed Crohn’s disease mucosa was significantly reduced by co-culture with L. casei or L. bulgaricus; changes induced by L. crispatus or E. coli were not significant. The effect of L. casei and L. bulgaricus was not prevented by protease inhibitors. Co-culture with L.casei and L. bulgaricus reduced the number of CD4 cells, as well as TNFα expression in lymphocytes from Crohn’s disease mucosa. None of the bacteria induced changes in non-inflamed mucosa.
Conclusions: Probiotics may interact with immunocompetent cells using the mucosal interface and thus modulate locally the production of proinflammatory cytokines.

Abstract. The human intestine harbors a complex microbial ecosystem and the mucosa is the interface. Objective: To elucidate whether host-bacteria interactions influence mucosal cytokine production. Methods: Macroscopically normal colonic specimens were obtained at surgery from 8 patients with neoplasm, and inflamed ileal specimens from 2 patients with Crohn’s disease. Mucosal explants were cultured for 24 h with either nonpathogenic Escherichia coli ECOR-26, Lactobacillus casei DN-114001, Lactobacillus casei DN-114056, Lactobacillus casei ATCC-334 or Lactobacillus bulgaricus LB-10. Each study included blank wells with no bacteria. Tissue and bacteria viability was confirmed by LDH release and culture. Concentration of TNFα, TGFβ1, IL-8 and IL-10 was measured in supernatants. In parallel experiments, neutralising anti-TNFα antibody was added to the culture. Results: Co-culture of mucosa with bacteria did not modify LDH release. Co-culture with Lactobacillus casei strains significantly reduced TNFα release, whereas E. coli increased it. These effects were observed both in normal and inflamed mucosa. In combination studies, DN-114001 prevented TNFα stimulation by E. coli. DN-114001 also reduced IL-8 release via a TNFα-independent pathway. DN-114056 or E. coli increased IL-10 release in the presence of neutralising anti-TNFα. Conclusions: Nonpathogenic bacteria interact with human intestinal mucosa and can induce changes in cytokine production that are strain-specific.

Abstract. Probiotics, including Lactobacilli, have been postulated to alleviate allergic and inflammatory diseases, but evidence that they exert an anti-inflammatory effect by immune modulation of pathogenic T cell effectors is still lacking. The aim of this study was to examine whether L. casei could affect antigen-specific T cell mediated skin inflammation. To this end we used contact hypersensitivity to the hapten DNFB, a model of allergic contact dermatitis mediated by CD8⁺ CTL and controlled by CD4⁺ regulatory T cells. Daily oral administration of a fermented milk containing L. casei or L. casei alone decreased skin inflammation by inhibiting the priming/expansion of hapten-specific IFNα-producing CD8⁺ effector T cells. The down regulatory effect of the probiotics required the presence of CD4⁺ T cells, which control the size of the hapten-specific CD8⁺ T cell pool primed by skin sensitization. L. casei cell wall was as efficient as live L. casei to regulate both the CHS response and the hapten-specific CD8⁺ T cell response, suggesting that cell wall components contribute to the immunomodulatory effect of L. casei. This study provides the first evidence that oral administration of L. casei can reduce antigen-specific skin inflammation by controlling the size of the CD8⁺ effector pool.

BACKGROUND: Trinitrobenzene sulphonic acid (TNBS) induces chronic transmural inflammatory lesions in the rat colon. Injury is facilitated by barrier disruption and invasion of commensal bacteria. However, certain bacteria have shown anti-inflammatory properties in in vitro models. AIM: To investigate in vivo the anti-inflammatory effect of Lactobacillus casei DN-114 001. METHODS: Rats with a colonic segment excluded from faecal transit were surgically prepared. After washing the lumen with antibiotics, the excluded segment was recolonized (control group: standard flora of rat origin; test group: standard flora and L casei). Microbial colonisation was confirmed by culture of segment washing, and colitis was then induced by instillation of TNBS. One day after, intestinal lesions were blindly graded by macro- and microscopic scores, and myeloperoxidase activity measured in tissue homogenates. Translocation of bacteria to mesenteric lymph nodes, spleen and liver was investigated. RESULTS: Test rats showed a smaller area of mucosal injury than control rats (p<0.05). Maximum depth lesion scores were similar in both groups but myeloperoxidase activity was lower in test than in control rats (p<0.05). Remarkably, bacterial translocation was quantitatively lower (p<0.01) and less frequent (p<0.05) in test than in control rats. CONCLUSION: In rats colonised with L casei, mucosal injury, inflammatory response, and barrier disruption after TNBS challenge were attenuated. Bacterial communities colonising the mucosa can modify inflammatory responses to luminal challenges.

Our study examined whether repeated preventive oral administration of live probiotic bacterial strains Escherichia coli O83:K24:H31 (Ec O83), Escherichia coli Nissle 1917 O6:K5:H1 (Ec Nis) and Lactobacillus casei DN 114001 (Lc) can protect mice against dextran sodium sulfate (DSS)-induced colitis. A significant decrease in average symptom score was observed in Ec O83-, Ec Nis- and Lc-pretreated group (p < 0.05). Significant differences in body mass loss between Lc pretreated mice with DSS-induced colitis were found when compared with nontreated mice (p < 0.05). PBS pretreated mice had a significantly shorter colon than Ec O83-, Ec Nis- and Lc-pretreated mice (p < 0.05). Administration of Lc significantly decreased the severity of DSS induced histological marks of inflammation (p < 0.05). A significant difference (p < 0.05) was also found in specific IgA level against given probiotic in enteral fluid between colitic mice and healthy mice pretreated with Ec 083 and Ec Nis.

Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-kappaB pathway, particularly through stabilization of I-kappaBalpha. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-kappaBalpha.

Apoptosis of active T lymphocytes constitutes a major control mechanism of immune homeostasis and tolerance. In Crohn's disease, abnormal activation of mucosal T lymphocytes against enteric bacteria is the key event triggering intestinal inflammation. Resistance of lymphocytes to apoptosis has been proposed as the pathogenetic defect. We examined the influence of bacteria-mucosa interactions on apoptosis of mucosal T lymphocytes. Ileal specimens were obtained at surgery from 12 patients with Crohn's disease. Mucosal explants from each specimen were cultured with nonpathogenic Escherichia coli ATCC 35345, Lactobacillus casei DN-114 001, or no bacteria. Cytokine release was measured in supernatant, and mononuclear cells were isolated for phenotypic characterization and Bcl-2 family protein expression. Coculture of inflamed tissue with L. casei significantly reduced the release of interleukin (IL)-6 and tumor necrosis factor alpha (P < 0.05). In addition, coculture with L. casei significantly reduced the number of T cells displaying the IL-2 receptor in the lamina propria. Expression of the antiapoptotic protein Bcl-2 in lamina propria lymphocytes was also reduced after coculture with L. casei, and the percentage of deoxyuridine triphosphate nick-end labeling positive lymphocytes increased. The nonpathogenic E. coli strain had no significant effect. In conclusion, L. casei reduces the number of activated T lymphocytes in the lamina propria of Crohn's disease mucosa. A balanced, local microecology may restore immune homeostasis.

Abstract. Live Lactobacillus casei is present in fermented dairy products with beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine if L. casei DN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora associated (HFA) mouse model. L. casei DN-114 001 was genetically modified by the introduction of erm and lux genes encoding erythromycin resistance and luciferase respectively. For this modified strain (DN-240 041) light emission, related to luciferase expression, could easily be detected in the contents of the digestive tract. When inoculated in the digestive tract of HFA mice, L. casei (DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture of L. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable one day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5 days old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 hours of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampicin. These results suggest that in HFA mice L. casei DN-240 041 and thus probably L. casei DN-114 001 is able to initiate new protein synthesis during its transit with the diet. The beneficial properties of L. casei fermented milk for human health might be related to this protein synthesis in the digestive tract.

Abstract. Although studies on the survival of bacteria in the digestive tract have been reported in the literature, little data are available on the physiological adaptation of probiotics to the digestive environment. In previous work, a transcriptional fusion system (i.e. luciferase genes under the control of a deregulated promoter) was used to demonstrate that a derivative of the Lactobacillus casei DN-114 001 strain, ingested in a fermented milk and thus exhibiting initially a very weak metabolic activity, synthesized proteins de novo after its transit in the human microbiota-associated mice digestive tract. Using the same genetic system and animal model, we here investigate, for the first time the ability of Lactobacillus casei to reinitiate synthesis in the different digestive tract compartments. In this study, most ingested L. casei cells transited from the stomach to the duodenum-jejunum within 1 hour post-ingestion. No 11 luciferase activity was observed in these digestive tract compartments over the first hour. At 12 later times, the bulk of bacteria had transited to the ileum and the cecum. Luciferase synthesis was detected at the ileal level between 1.5 - 2.0 hours and from 1.5 hours to at least 6.0 hours in the cecum where the activity remained at a maximum level. These results demonstrate that ingested Lactobacillus casei (derivative of DN-114 001 strain) administered via a fermented milk has already reinitiated protein synthesis when it reaches the ileal and cecal compartments.

Abstract. In a previous study using fusion of the deregulated lactose promoter, lacTp*, and reporter genes, we suggested that L. casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a quantitative reverse transcriptase PCR approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth and variable luciferase activities were detected. In three-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to Human-Microbiota-Associated mice and the feces were collected 6 hours later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.

A human trial was carried out to assess ileal and faecal survival of the probiotic bacterium Lactobacillus casei DN-114 001 ingested in fermented milk. Results showed that survival rates were up to 51.2% in the ileum and 28.4% in faeces. It is concluded that L. casei can survive transit through the human gut.

The composition and activities of the faecal microbiota in twelve healthy subjects analysed in a single open study were monitored before (1-week baseline step), during (10 d supplementation step) and after (10 d follow-up step) the ingestion of a fermented milk containing Lactobacillus casei DN-114 001. Fluorescent in situ hybridisation with group-specific DNA probes, real-time PCR using L. paracasei group-specific primers and temporal temperature gradient gel electrophoresis (TTGE) using group-specific primers were carried out, together with bacterial enzyme activity and metabolite analyses to monitor the structure and activities of the faecal microbiota. L. casei DNA was detected in the faeces of all of the subjects by TTGE after 10 d supplementation. Its quantification by real-time PCR showed a 1000-fold increase during the test step compared with initial levels. No major modification in either the dominant members of the faecal microbiota or their activities was observed during the trial. In conclusion, the short-term consumption of a milk product containing L. casei DN-114 001 was accompanied by a high, transient increase in the quantity of this strain in the faeces of all of the subjects without markedly affecting biochemical or bacteriological factors.