Scientific evidence

To examine whether long-term consumption of fermented milk containing a specific Lactobacillus casei may improve the health status of preschool children suffering from allergic asthma and/or rhinitis a randomized, prospective, double blind, controlled trial was conducted in 187 children 2-5 y of age. The children received for 12 mo either fermented milk (100 mL) containing Lactobacillus casei (10(8) cfu/mL) or placebo. The time free from and the number of episodes of asthma/rhinitis after starting intervention were the outcome measures. The number of fever or diarrhea episodes and the change in serum immunoglobulin were further assessed. No statistical difference between intervention and control group occurred in asthmatic children. In children with rhinitis, the annual number of rhinitis episodes was lower in the intervention group, mean difference (95% CI), -1.6 (-3.15 to -0.05); the mean duration of an episode of diarrhea was lower in the intervention group, mean difference -0.81 (-1.52 to -0.10) days. While long-term consumption of fermented milk containing Lactobacillus casei may improve the health status of children with allergic rhinitis no effect was found in asthmatic children.

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. TRIAL REGISTRATION: National Research Register N0016106821.

PURPOSE: To determine whether a probiotic drink containing Lactobacillus casei DN-114 001 reduces the incidence of radiation-induced diarrhea in patients with gynecologic cancer. METHODS AND MATERIALS: Patients who were undergoing pelvic radiotherapy (45-50 Gy, conventional fractionation) for either cervical carcinoma (radiotherapy and weekly cisplatin) or endometrial adenocarcinoma (postoperative radiotherapy) were randomly assigned to a probiotic drink or placebo, in a double-blind fashion. The probiotic drink consisted of liquid yogurt containing L. casei DN-114 001 at 10(8) CFU/g. The patients recorded the daily the number of bowel movements and scored the stool consistency using the Bristol scale. Diarrhea was graded weekly according the Common Toxicity Criteria system. The primary endpoint was to reduce the incidence of diarrhea, defined by a Common Toxicity Criteria Grade of 2 or greater or the need for loperamide. RESULTS: A total of 85 patients were enrolled. Grade 2 or greater diarrhea and/or the use of loperamide was observed in 24 of 41 patients in the placebo group and 30 of 44 in the probiotic group (p = 0.568). No differences were found in the median time to the presentation of the primary endpoint. Probiotic intervention had a significant effect on stool consistency (p = 0.04). The median time for patients to present with Bristol scale stools of Type 6 or greater was 14 days for patients receiving the probiotic drink vs. 10 days for those receiving placebo. CONCLUSION: Nutritional intervention with the probiotic drink containing L. casei DN-114 001 does not reduce the incidence of radiation-induced diarrhea as defined by a Common Toxicity Criteria Grade 2 or greater. However, it had a significant effect on stool consistency as measured by the Bristol scale.

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: Multicenter, prospective, randomized, double-blind controlled study. Eighty-six 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.

Probiotics are being increasingly studied for their ability to enhance host resistance to, and recovery from, infection. The probiotic strain Lactobacillus casei DN-114001 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-114001 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 0.5 C treatment group vs. 38.5 0.6 C 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.

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 10(8) of each Lactobacillus casei DN-114001, Lactobacillus bulgaricus and Streptococcus thermophilus per gram. Group 2 was given Indian Dahi (Lf 40) containing 10(8) 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.

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 (10(8) 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.

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 (10(8) 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).

The healthy action of probiotics is not only due to their nutritional properties and their influence on the gastrointestinal environment, but also to their action on the immune system. The aim of the present study was to determine if 6 weeks of probiotic intake would be able to modulate the immune system in women who had recently delivered and were breast-feeding. The design consisted of a randomised, controlled and double-blind nutritional intervention study with parallel groups with a sample size of 104 women. The main variable is the T helper type 1/T helper type 2 (Th1/Th2) profile determined by measuring interferon-gamma (Th1) and IL-4 (Th2) values in peripheral blood by flow cytometry. The modifications of cytokines were evaluated in maternal milk by cytometric bead array in a flow cytometer and ELISA at three stages of breast-feeding: colostrum, early milk (10 d) and mature milk (45 d). Additionally, the anthropometry and infectious and allergic episodes in the newborn were followed up throughout the first 6 months of life. After the consumption of milk fermented with Lactobacillus casei during the puerperium, we observed a nonsignificant increase in T and B lymphocytes and a significant increase in natural killer cells. A decrease in the pro-inflammatory cytokine TNF-alpha in maternal milk and fewer gastrointestinal disturbances were also observed in the breast-fed child of the mothers who consumed L. casei. The intake of milk fermented with L. casei during the lactation period modestly contributes to the modulation of the mother's immunological response after delivery and decreases the incidence of gastrointestinal episodes in the breast-fed child.

BACKGROUND: Lactic acid bacteria have been shown to stimulate the secretion of cytokines by lymphocytes and monocytes in a strain-dependent manner. Therefore, in this study, the effect of a daily intake of probiotic yogurt on cytokine production in young healthy women was compared with that of a conventional product. METHODS: For 2 weeks each, subjects consumed 100 g, then 200 g of either a probiotic or a conventional, commercially available yogurt, both containing Lactobacillus bulgaricus and Streptococcus thermophilus with additional Lactobacillus casei DN 114 001 in the probiotic product. Cytokine production in blood culture following stimulation with phytohaemmaglutinin and lipopolysaccharide was measured using Cytometric Bead Array and enzyme-linked immunosorbent assay. RESULTS: Stimulated production of tumour necrosis factor-alpha increased significantly following consumption of conventional or probiotic yogurt (+63% and +24% compared with baseline, respectively, P < 0.001). There was also a significantly higher production of interleukin (IL)-1beta in the conventional (+40%, P = 0.006) and of interferon gamma in the probiotic group (+108%, P < 0.05). IL-10 decreased following consumption of the probiotic product, but increased significantly after intake cessation (+129%, P < 0.001). No significant differences in cytokine responses between the conventional and the probiotic yogurt were observed. CONCLUSION: Both conventional and probiotic yogurt enhanced the stimulated production of pro-inflammatory cytokines.

This study examined the effect of a probiotics supplementation on respiratory tract infection (RTI) and immune and hormonal changes during the French Commando training (3-week training followed by a 5-day combat course). Cadets (21 +/- 0.4 years) received either a probiotics (n = 24) or a placebo (n = 23) supplementation over the training period. We found no difference in the RTI incidence between groups but a significantly greater proportion of rhinopharyngitis in the probiotic group (p < 0.05). Among immune parameters, the major finding was an immunoglobulin A decrease after the combat course only in the placebo group (p < 0.01), but the difference between the two groups was not significant. A greater increase in dehydroepiandrostane sulfate was observed in the probiotics group after the combat course (p < 0.05). This study suggested that the benefits of a probiotics supplementation in a multistressor environment relied mainly on its capacity to prevent the infection to spread throughout the respiratory tract.

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. Copyright 2006 S. Karger AG, Basel.

BACKGROUND: A suppressed immune response has been documented in students under examination stress. AIMS: The current study aimed to evaluate the effect of milk fermented with yogurt cultures plus Lactobacillus casei DN-114001 (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 10(3)/mm(3)) and increased in the treatment group (0.37+/-0.11 cells x 10(3)/mm(3)). 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/mm(3)),while remaining similar in the treatment group (17.29+/-17.27 cells/mm(3)). During the study, mean serum cortisol increased 4.30+/-0.98 microg/dL in the control group, and 1.75+/-1.05 microg/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-114001 was able to modulate the number of lymphocytes and CD56 cells in subjects under academic examination stress.

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.

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.

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 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 induce immune system depresion of NK cells. Twenty-five athletes were selected out of 94 for their sinificant decrease in NK cells concentration compared with a normal basal concentration in plasma 2 h 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-1B, IL-6, IL-2, IFNy, IgA, IgM, IgG, NK cells, CD8, CD4, CD3 and sIL-2 receptor. A significant smaller decrease of NK cell concentration after 2 h was found in the fermented milk feeding phase vs. the standard milk period.

Immunosuppressive and antibacterial regimens in children after liver transplantation create a gut microflora imbalance that can be indirectly measured by the activity of fecal enzymes. The aim of this study was to specify the influence of diet supplementation with probiotic Lactobacillus casei DN on the activity of beta-glucuronidase, beta-glucosidase, and urease. Twenty-five children after liver transplantation (13 girls, 12 boys) ages 3 to 17 years were enrolled in the study. Two months after bacteria application the levels of all 3 enzymes decreased, reaching statistical significance for beta-glucuronidase and beta-glucosidase. Complete rebound in enzyme activity was observed months after the end of probiotic supplementation. We concluded that Lactobacillus casei DN-114001 consumption decreased fecal enzyme activity, a beneficial effect limited to the period of bacteria intake.

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 acetate, butyrate, propionate, and lactate; and bacterial enzyme activity of beta-galactosidase and alpha-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 beta-glucuronidase and beta-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.

Lactobacillus casei DN-114 001 is a probiotic strain able to interact with the immune system and to interfere with gastrointestinal pathogens. The derived strain DN-114 001Rif was studied during its transit through the upper and distal intestine of human volunteers. Seven volunteers participated in the study, which involved intestinal intubation to sample ileal contents and collection of fecal samples, with a wash-out period of 8 days between the 2 steps. The retrieval of the probiotic was analyzed in the ileum every 2 h for 8 h following the ingestion of one dose of the test product and in the feces prior to, during, and after daily consumption of the test product for 8 days. Persistence of the probiotic amplifiable DNA was assessed using temporal temperature gradient gel electrophoresis and real-time PCR. Fluorescent in situ hybridization allowed analysis of the composition of the dominant digestive microbiota. The ingestion of L. casei DN-114 001Rif led to a significant and transient increase of its amplifiable DNA in ileal and fecal samples. This is related to a high stability in the composition of dominant groups of the gut microbiota. Data from ileal samples are scarce and our study confirms the potentiality for interaction between probiotics and the human immune system.

A human trial was carried out to assess the ileal and fecal survival of Lactobacillus casei DN-114 001 ingested in fermented milk. Survival rates were up to 51.2% in the ileum and 28.4% in the feces. The probiotic bacterium has the capacity to survive during its 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.

Pediatric Academic Societies Annual Meeting; 2-6 May 2008 Honolulu Hawai, USA

2nd Congress of the European Academy of Pediatrics (EAP); 24-28 Oct 2008 Nice, France

6th meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP9-11); 9-11 Nov 2008 London Ontario, Canada

XII Congress of Pediatricians of Russia; 19-22 Feb 2008 Moscow, Russia

2nd European Conference on Probiotics and their Applications; 15-17 Oct 2008 Cracow, Poland

World Congress of Sports Medicine; 18-23 Nov 2008 Barcelona, Spain

European Union Geriatrics Society (EUGMS); 3-6 Sept 2008 Copenhagen, Denmark

3rd European Influenza Conference; 14-17 Sept 2008 Villamoura, Portugal

British Society of Immunology; 18 Nov 2008 Glasgow Scotland, UK

2nd Vaccine Congress; 7-9 Dec 2008 Boston, USA

Probiotics are living microorganisms which, when ingested in adequate amounts, exert health benefits toward the host. For instance, probiotics might act through reinforcement of the intestinal epithelial barrier function. The goal of the present study was to determine whether Lactobacillus casei DN-114 001 could abrogate the increase in paracellular permeability induced by enteropathogenic Escherichia coli. We used the human colon T84 cell line infected with a wild-type enteropathogenic E. coli (strain E2348/69). 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 the presence of pathogenic bacteria 3 h after the beginning of the infection). We also investigated the effect of L. casei on enteropathogenic E. coli adhesion. L. casei DN-114 001 inhibited, in a dose-dependent-manner, the decrease in enteropathogenic E. coli-induced transepithelial electrical resistance and zonula occludens-1 redistribution using two different infection procedures. However, L. casei did not inhibit pathogenic strain adhesion. L. casei DN-114 001 inhibited the increase in EPEC-induced paracellular permeability. This property could partially explain the previously observed health benefits of this probiotic for human natural defenses, such as those associated with prevention of diarrhea.

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.

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 yoghurt 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 Paneth 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.

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 dehydrogenase activities, cyclic AMP production, and differentiation of levels of the IEC-6 strain were evaluated between the 15th and 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 dehydrogenase 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 dehydrogenase. 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.

BACKGROUND: Microbial colonization of the intestine after birth is an important step for the development of the gut immune system. The acquisition of passive immunity through breast-feeding may influence the pattern of bacterial colonization in the newborn. The aim of this work was to evaluate the effect of the administration of a probiotic fermented milk (PFM) containing yogurt starter cultures and the probiotic bacteria strain Lactobacillus casei DN-114001 to mothers during nursing or their offspring, on the intestinal bacterial population and on parameters of the gut immune system. RESULTS: Fifteen mice of each group were sacrificed at ages 12, 21, 28 and 45 days. Large intestines were taken for determination of intestinal microbiota, and small intestines for the study of secretory-IgA (S-IgA) in fluid and the study of IgA+ cells, macrophages, dendritic cells and goblet cells on tissue samples. The consumption of the PFM either by the mother during nursing or by the offspring after weaning modified the development of bifidobacteria population in the large intestine of the mice. These modifications were accompanied with a decrease of enterobacteria population. The administration of this PFM to the mothers improved their own immune system and this also affected their offspring. Offspring from mice that received PFM increased S-IgA in intestinal fluids, which mainly originated from their mother's immune system. A decrease in the number of macrophages, dendritic cells and IgA+ cells during the suckling period in offspring fed with PFM was observed; this could be related with the improvement of the immunity of the mothers, which passively protect their babies. At day 45, the mice reach maturity of their own immune system and the effects of the PFM was the stimulation of their mucosal immunity. CONCLUSION: The present work shows the beneficial effect of the administration of a PFM not only to the mothers during the suckling period but also to their offspring after weaning and until adulthood. This effect positively improved the intestinal microbiota that are related with a modulation of the gut immune response, which was demonstrated with the stimulation of the IgA + cells, macrophages and dendritic cells.

BACKGROUND: The interaction of commensal bacteria with the intestinal immune system is an essential factor in the development of inflammatory bowel disease (IBD). The study of isolated commensal bacteria's effects on the mucosal immune response might be relevant for a better understanding of pathophysiological mechanisms in IBD. METHODS: We investigated the immune responses to signals from the commensal Escherichia coli ATCC 35345 and the probiotic Lactobacillus casei DN-114 001 in Crohn's disease (CD) mucosa. Ileal specimens were obtained during surgery from CD patients. Mucosal explants were incubated with L. casei or its genomic DNA; TNF-alpha, IFN-gamma, IL-2, IL-6, IL-8, and CXCL1 were measured in the supernatant. Second, tissue expression of key proinflammatory cytokines (IL-6, TGF-beta, IL-23p19, IL-12p35, IL-17F), and chemokines (IL-8, CXCL1, CXCL2) was evaluated after incubation with L. casei or E. coli. Finally, combination experiments were carried out by incubating both strains with mucosal explants at different timepoints. RESULTS: Live L. casei significantly decreased secretion of TNF-alpha, IFN-gamma, IL-2, IL-6, IL-8, and CXCL1 by CD mucosa, but the effect was not reproduced by L. casei DNA. Second, live L. casei downregulated expression of IL-8, IL-6, and CXCL1 and did not modify expression of IL-23p19, IL-12p35, and IL-17F. In contrast, E. coli significantly upregulated expression of all these cytokines. Interestingly, combination experiments revealed the ability of L. casei to prevent and counteract the proinflammatory effects of E. coli. CONCLUSIONS: Live L. casei can counteract the proinflammatory effects of E. coli on CD inflamed mucosa by specific downregulation of key proinflammatory mediators.

The effect of the long-term administration of commercial fermented milk containing probiotic bacteria in the mucosal immune response and peritoneal macrophages was analyzed. BALB/c mice were fed with fermented milk for 98 consecutive days. Small and large intestines were removed for histology; IgA, CD4, CD8 cells and cytokines-producing cells were counted. The influence on the immune cells associated with bronchus and mammary glands as well as on peritoneal macrophages was also analyzed. Continuous oral administration of fermented milk increased IgA+ cells in both parts of the intestine (small and large intestine). IL-10, a regulatory cytokine, increased in the intestinal cells in most samples. TNFalpha, IFNgamma and IL-2 producing cells were also enhanced. Values for CD4 and CD8(+) cell populations in lamina propria of the intestine were increased in relation to the control throughout the assay. No modifications in the histology of intestines were observed. Long-term consumption of fermented milk enhanced intestinal mucosa immunity, mediated by IgA+ cells and by cytokine production. This improvement of gut immunity was maintained and down-regulated by cytokines such as IL-10, preventing gut inflammatory immune response. The effect of this fermented milk on mucosal sites distant to the gut, such as bronchus and mammary glands, showed that in both tissues the increase in IgA+ cells was only observed at the beginning of the continuous consumption and no modifications in the number of cytokine positive cells were found. Similar observations were found when phagocytic activity of peritoneal macrophages was measured. It was demonstrated that the most evident effect of long-term consumption of fermented milk was observed in the intestine. Immunodulatory effects and the maintenance of intestinal homeostasis without secondary effects after long-term administration of fermented milk were also observed.

Dendritic cells (DCs) orchestrate the immune response establishing immunity versus tolerance. These two opposite functions may be dictated by DC maturation status with maturity linked to immunogenicity. DCs directly interact with trillions of noninvasive intestinal bacteria in vivo, a process that contributes to gut homeostasis. We here evaluated the maturation program elicited in human DCs by direct exposure to commensal-related bacteria (CB) in the absence of inflammatory signals. We showed that eight gram(+) and gram(-) CB strains up-regulated costimulatory molecule expression in DCs and provoked a chemokine receptor switch similar to that activated by gram(+) pathogens. CB strains may be classified into three groups according to DC cytokine release: high IL-12 and low IL-10; low IL-12 and high IL-10; and low IL-12 and IL-10. All CB-treated DCs produced IL-1beta and IL-6 and almost no TGF-beta. Yet, CB instructed DCs to convert naive CD4+ T cells into hyporesponsive T cells that secreted low or no IFN-gamma, IL-10, and IL-17 and instead, displayed suppressor function. These data demonstrate that phenotypic DC maturation combined to an appropriate cytokine profile is insufficient to warrant Th1, IL-10-secreting T regulatory Type 1 (Tr1), or Th17 polarization. We propose that commensal flora and as such, probiotics manipulate DCs by a yet-unidentified pathway to enforce gut tolerance.

Probiotic fermented milk products have the capacity to modulate many immunological mechanisms. Several attempts have been made to alter the progression of various atopic and inflammatory disorders in which the immune system plays a major role. We studied this issue in an animal model of the antiphospholipid syndrome (APS) by supplementing the animals' daily intake with a probiotic mixture. We studied the effects of nutritional supplementation of a commercial product that consists of 10(8)/ml Lactobacillus casei (Actimel) on Balb/c mice that were immunized with beta-2- glycoprotein (beta2GPI) in order to induce a familiar murine model of APS. As controls, we used similar animals that were fed with either yogurt or sham solution as a supplement. We analyzed the effect of Actimel on the concentrations of interleukin (IL)-10 interferon gamma (IFNgamma) as well as the extent of the primary T cell response to beta2GPI, and the levels of autoantibodies to beta2GPI determined by ELISA. Two weeks after priming (in the hind footpad) of Balb/c mice with beta2GPI, we analyzed the cytokine profile of the animals by measuring the concentration of IL-10 and IFNgamma in the supernatants of lymphocytes that were extracted from the popliteal lymph nodes. Following stimulation with 10 microg/mL of beta2GPI, we noticed significant (P < 0.05) suppression of IL-10 production by the stimulated lymphocytes in the animals fed with Actimel and yogurt in comparison to sham solution (73.42 +/- 29.4, 84.7 +/- 8, 196 +/- 41.62 pg/mL, respectively). Both dairy products enhanced the secretion of IFNgamma from 657 +/- 47.09 pg/mL to 896 +/- 78.1, and 933 +/- 76.7 (P < 0.01), respectively; similarly they also accelerated by a mild degree the level of the T cell primary response to beta2GPI measured by [3H]thymidine incorporation. The level of autoantibodies to beta2GPI was suppressed in mice fed with actimel and yogurt in a significant manner (P < 0.05). Actimel as well as yogurt confer an immunological impact on Balb/c mice immunized with beta2GPI. Actimel was able not only to enhance IFNgamma secretion but also to inhibit IL-10 production.

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.

Lactobacillus casei has been shown to attenuate the severity of experimental colitis. The objective of the present study was to determine whether the effects of L. casei on colitis are related to modulation of leukocyte recruitment into the inflamed intestine. Rats with a colonic segment excluded from fecal transit were surgically prepared. The segment was decontaminated with antibiotics and recolonized with normal flora isolated from the inflamed rat colon, associated or not to L. casei. Control and colitic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] animals were studied. Leukocyte-endothelial cell interactions were characterized in the colonic microcirculation by intravital microscopy, and ICAM-1 and VCAM-1 expression was measured by the radiolabeled antibody technique. Compared with the noninflamed colonic segment, induction of colitis by TNBS provoked a marked increase in the number of leukocytes firmly adherent to the venular wall (0.5 +/- 0.1 vs. 2.1 +/- 0.6 leukocytes/100 mum, P < 0.01). Colonization with L. casei significantly reduced the number of adherent leukocytes (1.3 +/- 0.4 leukocytes/100 mum; P < 0.05) but did not affect the increased rolling interactions associated with the induction of colitis. Compared with the noncolitic group, induction of colitis was associated with a marked increase in ICAM-1 expression (117 +/- 4 vs. 180 +/- 3 ng antibody/g tissue) that was abrogated when the colitic segment was colonized by L. casei (117 +/- 3 ng antibody/g tissue, P < 0.05). However, L. casei administration did not modify VCAM-1 upregulation in colitic animals. L. casei attenuates leukocyte recruitment observed in experimental colitis induced by TNBS. This effect is possibly related to abrogation of ICAM-1 upregulation.

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.

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.

The mechanism(s) underlying the antibacterial activity of probiotic Lactobacillus strains appears to be multifactorial and includes lowering of the pH and the production of lactic acid and of antibacterial compounds, including bacteriocins and nonbacteriocin, non-lactic acid molecules. Addition of Dulbecco's modified Eagle's minimum essential medium to the incubating medium delays the killing activity of lactic acid. We found that the probiotic strains Lactobacillus johnsonii La1, Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029, L. casei DN-114 001, and L. rhamnosus GR1 induced a dramatic decrease in the viability of Salmonella enterica serovar Typhimurium SL1344 mainly attributable to non-lactic acid molecule(s) present in the cell-free culture supernatant (CFCS). These molecules were more active against serovar Typhimurium SL1344 in the exponential growth phase than in the stationary growth phase. We also showed that the production of the non-lactic acid substance(s) responsible for the killing activity was dependent on growth temperature and that both unstable and stable substances with killing activity were present in the CFCSs. We found that the complete inhibition of serovar Typhimurium SL1344 growth results from a pH-lowering effect.

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.

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 CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation 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 coincubation and 43% to 62% in preincubation 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 to 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.

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.

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus 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 reverse transcriptase quantitative PCR (RT-QPCR) 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 3-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 h 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.

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 digestive tract of mice harboring human microbiota (known as human-microbiota-associated mice). With the same genetic system and animal model, we here investigate for the first time the ability of L. 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 h postingestion. No luciferase activity was observed in these digestive tract compartments after the first hour. At later times, the bulk of bacteria had transited to the ileum and the cecum. Luciferase synthesis was detected between 1.5 and 2.0 h postingestion at the ileal level and from 1.5 h to at least 6.0 h postingestion in the cecum, where the activity remained at a maximum level. These results demonstrate that ingested L. casei (derivative of the DN-114 001 strain) administered via a fermented milk has already reinitiated protein synthesis when it reaches the ileal and cecal compartments.

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 2,4-dinitrofluorobenzene, a model of allergic contact dermatitis mediated by CD8+ CTL and controlled by CD4+ regulatory T cells. Daily oral administration of fermented milk containing L. casei or L. casei alone decreased skin inflammation by inhibiting the priming/expansion of hapten-specific IFN-gamma-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. Copyright 2004 Wiley-VCH Verlag GmbH & Co.

OBJECTIVE: The human intestine harbors a complex microbial ecosystem, and the mucosa is the interface between the immune system and the luminal environment. The aim of this study was to elucidate whether host-bacteria interactions influence mucosal cytokine production. METHODS: Macroscopically normal colonic specimens were obtained at surgery from eight patients with neoplasm, and inflamed ileal specimens were obtained from two patients with Crohn's disease. Mucosal explants were cultured for 24 h with either nonpathogenic Escherichia coli ECOR-26, Lactobacillus casei DN-114 001, L. casei DN-114 056, L. casei ATCC-334, or Lactobacillus bulgaricus LB-10. Each study included blank wells with no bacteria. Tissue and bacteria viability were confirmed by LDH release and culture. Concentration of tumor necrosis factor (TNF)alpha, transforming growth factor beta1, interleukin (IL)-8, and IL-10 was measured in supernatants. In parallel experiments, neutralizing anti-TNFalpha antibody was added to the culture. RESULTS: Co-culture of mucosa with bacteria did not modify LDH release. Co-culture with L. casei strains significantly reduced TNFalpha release, whereas E. coli increased it. These effects were observed both in normal and inflamed mucosa. In combination studies, L. casei DN-114 001 prevented TNFalpha stimulation by E. coli. L. casei DN-114 001 also reduced IL-8 release via a TNFalpha-independent pathway. L. casei DN-114 056 or E. coli increased IL-10 release in the presence of neutralizing anti-TNFalpha. CONCLUSIONS: Nonpathogenic bacteria interact with human intestinal mucosa and can induce changes in cytokine production that are strain specific.

BACKGROUND AND AIMS: Tumour necrosis factor alpha (TNF-alpha) plays a key role in the pathogenesis of intestinal inflammation in Crohn's disease. The effect of bacteria on TNF-alpha release by intestinal mucosa was investigated. METHODS: Ileal specimens were obtained at surgery from 10 patients with Crohn's disease (ileal stricture) and five disease controls undergoing right hemicolectomy (caecal cancer). Mucosal explants from each specimen were cultured for 24 hours with either non-pathogenic Escherichia coli, Lactobacillus casei DN-114001, L bulgaricus LB10, or L crispatus (each study contained blank wells with no bacteria). Tissue and bacterial viability was confirmed by lactate dehydrogenase (LDH) release and culture. Concentrations of TNF-alpha were measured in supernatants and the phenotype of the intestinal lymphocytes was analysed by flow cytometry. RESULTS: Coculture of mucosa with bacteria did not modify LDH release. Release of TNF-alpha by inflamed Crohn's disease mucosa was significantly reduced by coculture 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. Coculture with L casei and L bulgaricus reduced the number of CD4 cells as well as TNF-alpha expression among intraepithelial lymphocytes from Crohn's disease mucosa. None of the bacteria induced changes in non-inflamed mucosa. CONCLUSIONS: Probiotics interact with immunocompetent cells using the mucosal interface and modulate locally the production of proinflammatory cytokines.

Live Lactobacillus casei is present in fermented dairy products and has 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 into 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 1 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-day-old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. 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.

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.

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% nonfermented 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 the 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, glycolytic activities, short-chain fatty acids (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 Streptoccus 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 the others. beta-Glucuronidase (EC 3.2.1.31) activity was lower in rats fed LcFM and Y than in those fed M and LcYFM, whereas beta-galactosidase (3.2.1.23), alpha-glucosidase (EC 3.2.1 20) and beta-glucosidase (EC 3.2.1.21) activities were higher in the LcYFM group compared with the others. Methane excretion was higher in rats fed Y than in other groups. 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.