Probiotic stool secretory immunoglobulin A modulation in children with gastroenteritis: a randomized clinical trial.

BACKGROUND: We previously conducted the Probiotic Regimen for Outpatient Gastroenteritis Utility of Treatment (PROGUT) study, which identified no improvements in children with acute gastroenteritis (AGE) administered a probiotic. However, the aforementioned study did not evaluate immunomodulatory benefits. OBJECTIVES: The object of this study was to determine if stool secretory immunoglobulin A (sIgA) concentrations in children with AGE increase more among participants administered a Lactobacillus rhamnosus/helveticus probiotic compared with those administered placebo. METHODS: This a priori planned multicenter, randomized, double-blinded, placebo-controlled ancillary study enrolled children presenting for emergency care who received a 5-d probiotic or placebo course. Participants submitted stool specimens on days 0, 5, and 28. The primary endpoint was the change in stool sIgA concentrations on day 5 compared with baseline. RESULTS: A total of 133 (n = 66 probiotic, 67 placebo) of 886 PROGUT participants (15.0%) provided all 3 specimens. Median stool sIgA concentrations did not differ between the probiotic and placebo groups at any of the study time points: day 0 median (IQR): 1999 (768, 4071) compared with 2198 (702, 5278) (P = 0.27, Cohen's d = 0.17); day 5: 2505 (1111, 5310) compared with 3207 (982, 7080) (P = 0.19, Cohen's d = 0.16); and day 28: 1377 (697, 2248) compared with 1779 (660, 3977) (P = 0.27, Cohen's d = 0.19), respectively. When comparing measured sIgA concentrations between days 0 and 5, we found no treatment allocation effects [ß: -0.24 (-0.65, 0.18); P = 0.26] or interaction between treatment and specimen collection day [ß: -0.003 (-0.09, 0.09); P = 0.95]. Although stool sIgA decreased between day 5 and day 28 within both groups (P < 0.001), there were no differences between the probiotic and placebo groups in the median changes in sIgA concentrations when comparing day 0 to day 5 median (IQR) [500 (-1135, 2362) compared with 362 (-1122, 4256); P = 0.77, Cohen's d = 0.075] and day 5 to day 28 [-1035 (-3130, 499) compared with -1260 (-4437, 843); P = 0.70, Cohen's d = 0.067], respectively. CONCLUSIONS: We found no effect of an L. rhamnosus/helveticus probiotic, relative to placebo, on stool IgA concentrations. This trial was registered at clinicaltrials.gov as NCT01853124.

Lactobacillus helveticus SBT2171 Induces A20 Expression via Toll-Like Receptor 2 Signaling and Inhibits the Lipopolysaccharide-Induced Activation of Nuclear Factor-kappa B and Mitogen-Activated Protein Kinases in Peritoneal Macrophages.

Lactobacillus helveticus SBT2171 (LH2171) has been reported to ameliorate the development of autoimmune diseases, such as collagen-induced arthritis and experimental autoimmune encephalitis in mice and inhibit interleukin (IL)-6 production in antigen-presenting cells in vitro. Regulation of cytokine production by antigen-presenting cells might be critical for the anti-inflammatory function of LH2171 in autoimmune diseases. However, the mechanism and contributing components of LH2171-mediated inhibition of IL-6 production are unclear. Here, we examined the anti-inflammatory effects of LH2171 in lipopolysaccharide (LPS)-stimulated peritoneal macrophages, as a model of antigen-presenting cells, necessary for the pathogenesis of autoimmune diseases. LH2171 significantly reduced LPS-induced expression and secretion of IL-6 and IL-1ß cytokines. It also inhibited activation of nuclear factor-kappa B and mitogen-activated protein kinases (NF-κB/MAPKs). Moreover, LH2171 induced gene expression of several negative regulators of NF-κB/MAPKs. Among these regulators, A20 was strongly up-regulated at the mRNA and protein levels upon LH2171 treatment. The cell wall fraction of LH2171 also demonstrated a similar increase in A20 gene expression and exerted an anti-inflammatory effect. These results suggest that the cell wall may be one of the anti-inflammatory components of LH2171. Since cell wall components of Gram-positive bacteria are recognized by toll-like receptor 2 (TLR2), we investigated whether the anti-inflammatory effect of LH2171 was mediated by TLR2 signaling. Specifically, LH2171-mediated IL-6 suppression and A20 upregulation in wild-type macrophages were reversed and significantly reduced in TLR2 knock-out macrophages. These results suggest that LH2171 induces A20 expression via TLR2 signaling, inhibiting the activation of NF-κB/MAPKs and cytokine production in antigen-presenting cells. This might contribute to the anti-inflammatory activity of LH2171 on autoimmune diseases.

Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules.

Host intestinal epithelial cells (IEC) present at the gastrointestinal interface are exposed to pathogenic and non-pathogenic bacteria and their products. Certain probiotic lactic acid bacteria (LAB) have been associated with a range of host-immune modulatory activities including down-regulation of pro-inflammatory gene expression and cytokine production by IEC, with growing evidence suggesting that these bacteria secrete bioactive molecules with immunomodulatory activity. The aim of this study was to determine whether two lactobacilli with immunomodulatory activity [Lactobacillus rhamnosus R0011 (Lr) and Lactobacillus helveticus R0389 (Lh)], produce soluble mediators able to influence IEC responses to Pattern Recognition Receptor (PRR) ligands and pro-inflammatory cytokines [Tumor Necrosis Factor α (TNFα), Interleukin-1ß (IL-1ß)], signals inducing IEC chemokine production during infection. To this end, the effects of cell-free supernatants (CFS) from Lr and Lh on IEC production of the pro-inflammatory chemokines interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant 1 (CINC-1) induced by a range of host- or pathogen-derived pro-inflammatory stimuli were determined, and the impact on human HT-29 IEC and a primary IEC line (rat IEC-6) was compared. The Lr-CFS and Lh-CFS did not significantly modulate basal IL-8 production from HT-29 IECs or CINC-1 production from IEC-6 cells. However, both Lr-CFS and Lh-CFS significantly down-regulated IL-8 production from HT-29 IECs challenged with varied PRR ligands. Lr-CFS and Lh-CFS had differential effects on PRR-induced CINC-1 production by rat IEC-6 IECs, with no significant down-regulation of CINC-1 observed from IEC-6 IECs cultured with Lh-CFS. Further analysis of the Lr-CFS revealed down-regulation of IL-8 production induced by the pro-inflammatory cytokines IL-1ß and TNFα Preliminary characterization of the bioactive constituent(s) of the Lr-CFS indicates that it is resistant to treatment with DNase, RNase, and an acidic protease, but is sensitive to alterations in pH. Taken together, these results indicate that these lactobacilli secrete bioactive molecules of low molecular weight that may modulate host innate immune activity through interactions with IEC.

Survey on the phage resistance mechanisms displayed by a dairy Lactobacillus helveticus strain.

In this study the presence and functionality of phage defence mechanisms in Lactobacillus helveticus ATCC 10386, a strain of dairy origin which is sensitive to ΦLh56, were investigated. After exposure of ATCC 10386 to ΦLh56, the whole-genome sequences of ATCC 10386 and of a phage-resistant derivative (LhM3) were compared. LhM3 showed deletions in the S-layer protein and a higher expression of the genes involved in the restriction/modification (R/M) system. Genetic data were substantiated by measurements of bacteriophage adsorption rates, efficiency of plaquing, cell wall protein size and by gene expression analysis. In LhM3 two phage resistance mechanisms, the inhibition of phage adsorption and the upregulation of Type I R/M genes, take place and explain its resistance to ΦLh56. Although present in both ATCC 10386 and LhM3 genomes, the CRISPR machinery did not seem to play a role in the phage resistance of LhM3. Overall, the natural selection of phage resistant strains resulted successful in detecting variants carrying multiple phage defence mechanisms in L. helveticus. The concurrent presence of multiple phage-resistance systems should provide starter strains with increased fitness and robustness in dairy ecosystems.

Lactobacillus helveticus Lafti L10 Supplementation Modulates Mucosal and Humoral Immunity in Elite Athletes: A Randomized, Double-Blind, Placebo-Controlled Trial.

Michalickova, DM, Kostic-Vucicevic, MM, Vukasinovic-Vesic, MD, Stojmenovic, TB, Dikic, NV, Andjelkovic, MS, Djordjevic, BI, Tanaskovic, BP, and Minic, RD. Lactobacillus helveticus Lafti L10 supplementation modulates mucosal and humoral immunity in elite athletes: a randomized, double-blind, placebo-controlled trial. J Strength Cond Res 31(1): 62-70, 2017-To test the influence of probiotic supplementation on humoral immune response, a double-blind, placebo-controlled trial was conducted. Thirty athletes (24 males and 6 females, females: V[Combining Dot Above]O2max 38.2 ± 4.9 ml·kg·min, age 23.2 ± 1.4 years; males: V[Combining Dot Above]O2max 57.5 ± 9.2 ml·kg·min, age 24.0 ± 2.4 years, mean ± SD) were randomized either to the probiotic group (Lactobacillus helveticus Lafti L10, 2 × 10 colony-forming units) or to the placebo group. Serum and saliva samples were collected at the baseline and after 14 weeks. Total and specific antibacterial antibody levels of IgM, IgG, and IgA classes were determined for different bacteria in the serum, and in saliva, total and specific antibacterial IgA levels were examined. Total IgM was elevated in both probiotic (18%, 15-20%; mean, 90% confidence interval; p = 0.02) and placebo group (35%, 22-47%; p = 0.02), without observed differences in changes between the groups. No significant changes in IgM levels specific for tested bacteria were found. Total IgG level was constant in both groups. A significant (16%, -2.8 to 35%, p = 0.04) reduction of anti-Enterococcus faecalis IgG was noted in the placebo group, in comparison with the probiotic group. There was a substantial decrease in total IgA level in the placebo group, when measured either in serum (15%, 12-18%, p = 0.04) or in saliva (35%, -1.4 to 53%, p = 0.03). Significantly reduced levels of serum anti-lactic acid bacteria IgA antibodies in the placebo group compared with the probiotic group were detected for Lactobacillus rhamnosus LA68 (24%, 5.8-42%, p = 0.02) and for L. rhamnosus LB64 (15%, 2.7-27%, p = 0.02). Probiotic administration could have beneficial effects on systemic humoral and mucosal immune responses.

Bifidobacterium bifidum R0071 decreases stress-associated diarrhoea-related symptoms and self-reported stress: a secondary analysis of a randomised trial.

Psychological stress is associated with gastrointestinal (GI) distress. This secondary analysis from a randomised, double-blind, placebo-controlled study examined whether three different probiotics could normalise self-reported stress-associated GI discomfort and reduce overall self-reported stress. Undergraduate students (n=581) received Lactobacillus helveticus R0052, Bifidobacterium longum ssp. infantis R0033, Bifidobacterium bifidum R0071, or placebo. Participants self-reported 2 outcomes for a 6-week period, which included final academic exams: daily level of stress (0=no stress to 10=extremely stressed) and weekly three diarrhoea-related symptoms (DS, 1=no discomfort to 7=severe discomfort) using the GI Symptom Rating Scale. Self-reported stress was positively related to DS (P=0.0068). Mean DS scores were lower with B. bifidum versus placebo at week 2 at the average level of stress and the average body mass index (BMI). DS scores were lower with B. bifidum at week 5 versus week 0 and 1 and with B. infantis R0033 at week 6 versus week 0. DS scores were higher when antibiotics were used in the prior week with placebo (P=0.0092). DS were not different with or without antibiotic use with the probiotics. Only B. bifidum had an effect on self-reported stress scores (P=0.0086). The self-reported stress score was also dependent on hours of sleep per day where it decreased by 0.13 for each additional hour of sleep. During a stressful period, B. bifidum R0071 decreases DS and self-reported stress scores. This trial was registered at clinicaltrials.gov as NCT01709825.

Differential effects of lactobacilli on activation and maturation of mouse dendritic cells.

Lactic acid bacteria (LAB) are of interest because of their potential to modulate immune responses. The effects of LAB range from regulation to stimulation of the immune system. A series of studies were performed in vitro to study the effects of six lactic acid bacteria (LAB), Lactobacillus helveticus LH-2, Lactobacillus acidophilus La-5, La-115, La-116 and La-14, and Lactobacillus salivarius, on maturation and activation of mouse dendritic cells. Production of tumour necrosis factor (TNF)-?, interleukin (IL)-6 and IL-10 by dendritic cells (DCs) was determined after treating cells with live LAB. The expression of DC maturation markers, CD80 and CD40, was also measured using flow cytometry after stimulation with LAB. In addition, the expression of Toll-like receptors (TLRs) 2, 4 and 9 by DCs stimulated with LAB was measured. Our results revealed that LAB act differentially on pro-inflammatory and anti-inflammatory cytokine production and induction of co-stimulatory molecules by DCs. Specifically, L. salivarius was found to be the most effective LAB to induce pro-inflammatory cytokine production and expression of co-stimulatory molecules. Moreover, La-14, La-116 and La-5 induced moderate maturation and activation of DCs. On the other hand, LH-2 and La-115 were the least effective lactobacilli to induce DC responses. The present study also revealed that L. salivarius was able to induce the expression of TLR2, 4 and 9 by DCs. In conclusion, various strains and species of LAB can differentially regulate DC activation and maturation, providing further evidence that these bacteria may have the ability to influence and steer immune responses in vivo.

Multistrain probiotic modulation of intestinal epithelial cells' immune response to a double-stranded RNA ligand, poly(i·c).

A commercially available product containing three probiotic bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033, and Bifidobacterium bifidum R0071) was previously shown in animal trials to modulate both TH1 and TH2 immune responses. Clinical studies on this combination of bacteria have also shown positive health effects against seasonal winter diseases and rotavirus infection. The goal of this study was to use a well-established in vitro intestinal epithelial (HT-29) cell model that has been shown to constitutively express double-stranded RNA (dsRNA) sensors (Toll-like receptor 3 [TLR3], retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and dsRNA-activated protein kinase). By using the HT-29 cell model, we wanted to evaluate whether or not this combination of three bacteria had the capacity to immune modulate the host cell response to a dsRNA ligand, poly(I·C). Using a custom-designed, two-color expression microarray targeting genes of the human immune system, we investigated the response of HT-29 cells challenged with poly(I·C) both in the presence and in the absence of the three probiotic bacteria. We observed that the combination of the three bacteria had a major impact on attenuating the expression of genes connected to proinflammatory TH1 and antiviral innate immune responses compared to that obtained by the poly(I·C)-only challenge. Major pathways through which the multistrain combination may be eliciting its immune-modulatory effect include the TLR3 domain-containing adapter-inducing beta interferon (TRIF), mitogen-activated protein kinase, and NF-κB signaling pathways. Such a model may be useful for selecting potential biomarkers for the design of future clinical trials.

Lactobacillus helveticus MIMLh5-specific antibodies for detection of S-layer protein in Grana Padano protected-designation-of-origin cheese.

Single-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein of Lactobacillus helveticus MIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein of L. helveticus MIMLh5 and one was also capable of binding to the S-layer protein of L. helveticus ATCC 15009. All five anti-S-layer scFvs were expressed in Escherichia coli XL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein of L. helveticus MIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

Immune signalling responses in intestinal epithelial cells exposed to pathogenic Escherichia coli and lactic acid-producing probiotics.

Enterohaemorrhagic Escherichia coli O157:H7 and adherent-invasive Escherichia coli are two groups of enteric bacterial pathogens associated with haemorrhagic colitis and Crohn's Disease, respectively. Bacterial contact with host epithelial cells stimulates an immediate innate immune response designed to combat infection. In this study, immune responses of human epithelial cells to pathogens, either alone or in combination with probiotic bacteria were studied. Industrially prepared Lactobacillus helveticus strain R0052 was first examined by microarray analysis and then compared to broth-grown strains of R0052 and Lactobacillus rhamnosus strain GG using quantitative realt-time polymerase chain reaction. Results showed host immune activation responses increased following pathogen exposure, which were differentially ameliorated using probiotics depending on both the preparation of probiotics employed and conditions of exposure. These findings provide additional support for the concept that specific probiotic strains serve as a promising option for use in preventing the risk of enteric bacterial infections.

S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity.

The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.

Lactobacillus helveticus HY7801 ameliorates vulvovaginal candidiasis in mice by inhibiting fungal growth and NF-κB activation.

The anti-inflammatory effects of hydrogen peroxide-producing lactic acid bacteria (LAB) against Candida albicans-induced vulvovaginal candidiasis in ß-estradiol-immunosuppressed mice were examined. Oral and intravaginal treatment with these LABs significantly decreased the level of viable C. albicans within the vaginal cavity as well as the quantitated myeloperoxidase activity in the vaginal tissues when compared with control untreated mice. Out of all of the LABs tested, Lactobacillus helveticus HY7801 (LH) most potently inhibited vulvovaginal candidiasis. LH also inhibited the expression of the pro-inflammatory cytokines including TNF-α, IL-1ß and IL-6, and inflammatory enzymes, COX-2 and iNOS, as well as the activation of NF-κB. However, the addition of LH led to an increase in IL-10 cytokine expression in the vaginal tissues. In addition, the decrease of Lactobacillaceae and the increase of Pasteurellaceae caused by treatment with C. albicans were reversed with oral and intravaginal administration of LH, suggesting a potential shift in the vaginal microflora present. Addition of LH was toxic to C. albicans in vitro when cultured with HeLa cells. Oral administration of LH inhibited lipopolysaccharide (LPS)-induced TNF-α and IL-1ß expressions in ß-estradiol-immunosuppressed mice but reversed the expression of anti-inflammatory cytokine IL-10 in comparison to levels observed in the normal control group. LH also inhibited the expression of the pro-inflammatory cytokines, TNF-α and IL-1ß, and the activation of NF-κB in LPS-stimulated peritoneal macrophages. Based on these findings, LH may ameliorate vulvovaginal candidiasis by suppressing the NF-κB pathway, as well as through inhibition of the growth of C. albicans.

In vitro functional and immunomodulatory properties of the Lactobacillus helveticus MIMLh5-Streptococcus salivarius ST3 association that are relevant to the development of a pharyngeal probiotic product.

The use of proper bacterial strains as probiotics for the pharyngeal mucosa is a potential prophylactic strategy for upper respiratory tract infections. In this context, we characterized in vitro the functional and immunomodulatory properties of the strains Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 that were selected during previous investigations as promising pharyngeal probiotics. In this study, we demonstrated in vitro that strains MIMLh5 and ST3, alone and in combination, can efficiently adhere to pharyngeal epithelial cells, antagonize Streptococcus pyogenes, and modulate host innate immunity by inducing potentially protective effects. In particular, we found that the strains MIMLh5 and ST3 activate U937 human macrophages by significantly inducing the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α). Nonetheless, the induction of the anti-inflammatory interleukin-10 (IL-10) by MIMLh5 or ST3 was never lower than that of TNF-α, suggesting that these bacteria can potentially exert a regulatory rather than a proinflammatory effect. We also found that the strains MIMLh5 and ST3 induce cyclooxygenase 2 (COX-2) expression and demonstrated that toll-like receptor 2 (TLR-2) participates in the recognition of the strains MIMLh5 and ST3 by U937 cells. Finally, we observed that these microorganisms grow efficiently when cocultured in milk, suggesting that the preparation of a milk-based fermented product containing both MIMLh5 and ST3 can be a practical solution for the administration of these bacteria. In conclusion, we propose the combined use of L. helveticus MIMLh5 and S. salivarius ST3 for the preparation of novel products that display probiotic properties for the pharyngeal mucosa.

Functionality of the S-layer protein from the probiotic strain Lactobacillus helveticus M92.

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.

A dairy bacterium displays in vitro probiotic properties for the pharyngeal mucosa by antagonizing group A streptococci and modulating the immune response.

The probiotic approach represents an alternative strategy in the prevention and treatment of infectious diseases, not only at the intestinal level but also at other sites of the body where the microbiota plays a role in the maintenance of physiological homeostasis. In this context, we evaluated in vitro the potential abilities of probiotic and dairy bacteria in controlling Streptococcus pyogenes infections at the pharyngeal level. Initially, we analyzed bacterial adhesion to FaDu hypopharyngeal carcinoma cells and the ability to antagonize S. pyogenes on FaDu cell layers and HaCat keratinocytes. Due to its promising adhesive and antagonistic features, we studied the dairy strain Lactobacillus helveticus MIMLh5, also through in vitro immunological experiments. First, we performed quantification of several cytokines and measurement of NF-κB activation in FaDu cells. MIMLh5 efficiently reduced the induction of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-α), in a dose-dependent manner. After stimulation of cells with IL-1ß, active NF-κB was still markedly lowered. Nevertheless, we observed an increased secretion of IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) under these conditions. These effects were associated with the ability of MIMLh5 to enhance the expression of the heat shock protein coding gene hsp70. In addition, MIMLh5 increased the GM-CSF/G-CSF ratio. This is compatible with a switch of the immune response toward a TH1 pathway, as supported by our observation that MIMLh5, once in contact with bone marrow-derived dendritic cells, triggered the secretion of TNF-α and IL-2. In conclusion, we propose MIMLh5 as a potential probiotic bacterium for the human pharynx, with promising antagonistic and immunomodulatory properties.

Milk fermented by Lactobacillus helveticus R389 and its non-bacterial fraction confer enhanced protection against Salmonella enteritidis serovar Typhimurium infection in mice.

Bacterial infections in the gastrointestinal tract represent a major global health problem, even in the presence of normally effective mucosal immune mechanisms. Milk fermented by Lactobacillus helveticus R389 (FM) or its non-bacterial fraction obtained by milk fermentation at controlled pH 6 (NBF) are able to activate the small intestine mucosal immune response according to previous studies. In this work we aimed at comparing their protection capacity against an infection by Salmonella enteritidis serovar Typhimurium and at studying the mechanisms involved. In a completely randomized design, BALB/c mice received FM or NBF for 2, 5 or 7 consecutive days, followed by a single oral challenge with S. Typhimurium (10(7) cells/mouse). The increase in the number of IgA+ cells in the lamina propria of the small intestine, after the feeding periods, was accompanied by an increase in the luminal content of total S-IgA. However, no antibodies were produced against the NBF. In mice given the FM or the NBF for 7 consecutive days, lower levels of liver colonization on day 7 post-challenge with S. Typhimurium, higher luminal contents of specific anti-Salmonella S-IgA, higher percentages of survival to infection and lower numbers of MIP-1alpha+ cells in the lamina propria were observed. In this work we observed that in both the FM or the NBF there are active principles that confer enhanced protection against S. Typhimurium infection. However, the mechanisms underlying mucosal immunomodulation and protection are different. In those mechanisms, the mucosal immune response would seem to be more involved than the competitive or exclusion mechanisms between L. helveticus R389 and S. enteritidis serovar Typhimurium.

Mucosal immunomodulation by the non-bacterial fraction of milk fermented by Lactobacillus helveticus R389.

The health promoting effects ascribed to probiotic bacteria and fermented dairy products arise not only from bacteria themselves but also from metabolites derived from milk fermentation. Exopolysaccharides produced during milk fermentation and peptides derived from major milk proteins, when released during fermentation, are potential modulators of various regulatory processes in the body. The aim of this work was to increase the knowledge of the previously observed immunomodulating capacity of milk fermented by Lactobacillus helveticus R389 by the study of the mucosal immunomodulation exerted by the non-bacterial fraction of the milk fermented at a constant pH6 (NBFpH6). The effects on IL-6 production by small intestine epithelial cells, the profile of IgA+ and cytokine+ cells (IL-2, IL-6, IL-10, TNF-alpha and IFN-gamma) induced in the gut lamina propria, and the levels of total and specific secretory IgA in the lumen of BALB/c mice that received NBFpH6 for 2, 5 or 7 days were examined. There was an increase in the number of IgA+, IL-10+, IL-2+ and IL-6+ cells after all feeding periods. Total S-IgA in the small intestine lumen increased in mice that received NBFpH6 for 2 days. However, no specific antibodies against NBFpH6 were detected. Feeding of NBFpH6 for 7 days significantly (P<0.05) enhanced IL-6 secretion by small intestine epithelial cells. NBFpH6 induced a non-specific mucosal response that was down-regulated for protective immunity, enhancing IL-6 production by epithelial cells and IgA production in the small intestine. These events improve the immunological defenses at the intestinal level, increasing host protection against pathologies. Because mucosal immune responses induced by certain dietary antigens play a large part in the prevention of gastrointestinal diseases, the oral administration of a mucosal adjuvant such as NBFpH6 may positively affect the milieu of the intestinal lumen. The opportunity exists then to manipulate the constituents of the lumen of the intestine through dietary means, thereby enhancing the health condition of the host.

Effect of milk fermented with a Lactobacillus helveticus R389(+) proteolytic strain on the immune system and on the growth of 4T1 breast cancer cells in mice.

Previous studies on a murine model have demonstrated that the administration of Lactobacillus helveticus and Lactobacillus casei inhibits the development of fibrosarcoma and colon carcinoma, respectively. The aim of this work was to study the beneficial effects of the consumption of milk fermented by L. helveticus on a murine model for mammary carcinoma. Female BALB/c mice were challenged by a single subcutaneous injection of tumoral cells (American Type Culture Collection 4T1) in the left mammary gland. Prior to tumour injection, mice were fed for two, five or seven consecutive days with fermented milk. The following factors were monitored for 2 months: rate of tumour development, histological studies, apoptosis, phagocytic index, peritoneal macrophages, determination of beta-glucuronidase enzyme in peritoneal macrophages, determination of gamma-interferon (INFgamma) and tumour necrosis factor-alpha (TNF-alpha) in blood serum, determination of CD4+, CD8+, interleukin-6 (IL-6), IL-10, TNF-alpha and INFgamma by immunoperoxidase, and measurement of beta-glucuronidase activity in intestinal fluid. The administration of L. helveticus delayed the development of the tumour in all cases, a 2- or 7-day feeding period being most effective. This work demonstrates that milk fermented with L. helveticus decreases the growth rate of mammary tumours. The effect was mediated by increased apoptosis and decreased production of pro-inflammatory cytokines, in particular IL-6, implicated in oestrogen synthesis.