New probiotic strains for inflammatory bowel disease management identified by combining in vitro and in vivo approaches.

Alterations in the gut microbiota composition play a key role in the development of chronic diseases such as inflammatory bowel disease (IBD). The potential use of probiotics therefore gained attention, although outcomes were sometimes conflicting and results largely strain-dependent. The present study aimed to identify new probiotic strains that have a high potential for the management of this type of pathologies. Strains were selected from a large collection by combining different in vitro and in vivo approaches, addressing both anti-inflammatory potential and ability to improve the gut barrier function. We identified six strains with an interesting anti-inflammatory profile on peripheral blood mononuclear cells and with the ability to restore the gut barrier using a gut permeability model based on Caco-2 cells sensitized with hydrogen peroxide. The in vivo evaluation in two 2,4,6-trinitrobenzene sulfonic acid-induced murine models of colitis highlighted that some of the strains exhibited beneficial activities against acute colitis while others improved chronic colitis. Bifidobacterium bifidum PI22, the strain that exhibited the most protective capacities against acute colitis was only slightly efficacious against chronic colitis, while Bifidobacterium lactis LA804 which was less efficacious in the acute model was the most protective against chronic colitis. Lactobacillus helveticus PI5 was not anti-inflammatory in vitro but the best in strengthening the epithelial barrier and as such able to significantly dampen murine acute colitis. Interestingly, Lactobacillus salivarius LA307 protected mice significantly against both types of colitis. This work provides crucial clues for selecting the best strains for more efficacious therapeutic approaches in the management of chronic inflammatory diseases. The strategy employed allowed us to identify four strains with different characteristics and a high potential for the management of inflammatory diseases, such as IBD.

Mortality and translocation assay to study the protective capacity of Bifidobacterium lactis INL1 against Salmonella Typhimurium infection in mice.

The mouse has been largely used for the study of the protective capacity of probiotics against intestinal infections caused by Salmonella. In this work we aimed at comparing the mortality and translocation assay for the study of the protective capacity of the human breast milk-derived strain Bifidobacterium animalis subsp. lactis INL1 on a model of gut infection by Salmonella enterica subsp. enterica serovar Typhimurium. Different doses of S. Typhimurium FUNED and B. animalis subsp. lactis INL 1 were administered to Balb/c mice in a mortality or a translocation assay. The survival of the control group in the mortality assay resulted to be variable along experiments, and then we preferred to use a translocation assay where the preventive administration of 109 cfu of bifidobacteria/mouse for 10 consecutive days significantly reduced the number of infected animals and the levels of translocation to liver and spleen, with enhanced secretory immunoglobulin A and interleukin 10 production in the small and large intestine, respectively. Ten days of B. animalis subsp. lactis strain INL1 administration to mice significantly reduced both the incidence and the severity of Salmonella infection in a mouse model of translocation. This work provided the first evidence that a translocation assay, compared to a mortality assay, could be more useful to study the protective capacity of probiotics against Salmonella infection, as more information can be obtained from mice and less suffering is conferred to animals due to the fact that the mortality assay is shorter than the latter. These facts are in line with the guidelines of animal research recently established by the National Centre for the Replacement, Refinement & Reduction of Animals in Research.

Genes involved in obesity: Adipocytes, brain and microflora.

The incidence of obesity and related metabolic disorders such as cardiovascular diseases and type 2 diabetes, are reaching worldwide epidemic proportions. It results from an imbalance between caloric intake and energy expenditure leading to excess energy storage, mostly due to genetic and environmental factors such as diet, food components and/or way of life. It is known since long that this balance is maintained to equilibrium by multiple mechanisms allowing the brain to sense the nutritional status of the body and adapt behavioral and metabolic responses to changes in fuel availability. In this review, we summarize selected aspects of the regulation of energy homeostasis, prevalently highlighting the complex relationships existing between the white adipose tissue, the central nervous system, the endogenous microbiota, and nutrition. We first describe how both the formation and functionality of adipose cells are strongly modulated by the diet before summarizing where and how the central nervous system integrates peripheral signals from the adipose tissue and/or the gastro-intestinal tract. Finally, after a short description of the intestinal commensal flora, rangingfrom its composition to its importance in immune surveillance, we enlarge the discussion on how nutrition modified this perfectly well-balanced ecosystem.

Mucosal immune responses and protection against tetanus toxin after intranasal immunization with recombinant Lactobacillus plantarum.

The use of live microorganisms as an antigen delivery system is an effective means to elicit local immune responses and thus represents a promising strategy for mucosal vaccination. In this respect, lactic acid bacteria represent an original and attractive approach, as they are safe organisms that are used as food starters and probiotics. To determine whether an immune response could be elicited by intranasal delivery of recombinant lactobacilli, a Lactobacillus plantarum strain of human origin (NCIMB8826) was selected as the expression host. Cytoplasmic production of the 47-kDa fragment C of tetanus toxin (TTFC) was achieved at different levels depending on the plasmid construct. All recombinant strains proved to be immunogenic by the intranasal route in mice and able to elicit very high systemic immunoglobulin G (IgG1, IgG2b, and IgG2a) responses which correlated to the antigen dose. No significant differences in enzyme-linked immunosorbent assay IgG titers were observed when mice were immunized with live or mitomycin C-treated recombinant lactobacilli. Nevertheless, protection against the lethal effect of tetanus toxin was obtained only with the strains producing the highest dose of antigen and was greater following immunization with live bacteria. Significant TTFC-specific mucosal IgA responses were measured in bronchoalveolar lavage fluids, and antigen-specific T-cell responses were detected in cervical lymph nodes, both responses being higher in mice receiving a double dose of bacteria (at a 24-h interval) at each administration. These results demonstrate that recombinant lactobacilli can induce specific humoral (protective) and mucosal antibodies and cellular immune response against protective antigens upon nasal administration.

The superantigenic toxin of Yersinia pseudotuberculosis: a novel virulence factor?

Recently, a superantigenic toxin designated YPM (Yersinia pseudotuberculosis-derived mitogen) was characterized in the supernatant of Y. pseudotuberculosis, a Gram-negative bacterium involved in human enteric infection. To assess the role of YPM in pathophysiology of Y. pseudotuberculosis, a superantigen-deficient mutant was constructed and its virulence was tested in a murine model of infection and compared with the virulence of the wild-type strain (wt). Determination of the survival rate after intravenous inoculation of mice clearly demonstrated a higher survival rate when animals were infected with the superantigen-deficient strain. This decreased virulence of the mutant strain could not be explained by a lower bacterial growth rate in spleen, liver or lung of infected animals. Therefore, production of IFNgamma, TNFalpha, IL-2, IL-6 and IL-10 was followed during the course of infection by cytokine assay in the blood and mRNA detection in the spleen. IL-6 and IFNgamma were the two major cytokines detected whereas TNFalpha production was never observed.

Adaptation of the nisin-controlled expression system in Lactobacillus plantarum: a tool to study in vivo biological effects.

The potential of lactic acid bacteria as live vehicles for the production and delivery of therapeutic molecules is being actively investigated today. For future applications it is essential to be able to establish dose-response curves for the targeted biological effect and thus to control the production of a heterologous biopeptide by a live lactobacillus. We therefore implemented in Lactobacillus plantarum NCIMB8826 the powerful nisin-controlled expression (NICE) system based on the autoregulatory properties of the bacteriocin nisin, which is produced by Lactococcus lactis. The original two-plasmid NICE system turned out to be poorly suited to L. plantarum. In order to obtain a stable and reproducible nisin dose-dependent synthesis of a reporter protein (beta-glucuronidase) or a model antigen (the C subunit of the tetanus toxin, TTFC), the lactococcal nisRK regulatory genes were integrated into the chromosome of L. plantarum NCIMB8826. Moreover, recombinant L. plantarum producing increasing amounts of TTFC was used to establish a dose-response curve after subcutaneous administration to mice. The induced serum immunoglobulin G response was correlated with the dose of antigen delivered by the live lactobacilli.

Lactic acid bacteria as live vaccines.

Mucosal routes for vaccine delivery offer several advantages over systemic inoculation from both immunological and practical points of view. The development of efficient mucosal vaccines therefore represents a top prority in modern vaccinology. One way to deliver protective antigens at the mucosal surfaces is to use live bacterial vectors. Until recently most of these were derived from attenuated pathogenic microorganisms. As an alternative to this strategy, non-pathogenic food grade bacteria such as lactic acid bacteria (LAB) are being tested for their efficacy as live antigen carriers. The LABVAC european research network is presently comparing the vaccine potential of Lactococcus lactis, Streptococcus gordonii and Lactobacillus spp. To date, it has been shown that systemic and mucosal antigen-specific immune responses can be elicited in mice through the nasal route using the three LAB systems under study. Data on successful oral and vaginal immunisations are also accumulating for L. lactis and S. gordonii, respectively. Moreover, the immune responses can be potentiated by co-expression of interleukins. Future areas of research include improvement of local immunisation efficiency, analysis of in vivo antigen production, unravelling of the Lactobacillus colonisation mechanisms and construction of biologically contained strains.

Influence of cold stress on the preliminary enrichment time needed for detection of enterohemorrhagic Escherichia coli in ground beef by PCR.

The influence of cold stress at 4 and 0 degree C on the detection time as assessed by impedance technology (Bactometer; Biomérieux, Marcy l'Etoile, France) of different enterohemorrhagic Escherichia coli (EHEC) strains was determined. Although there is some variation in susceptibility among EHEC strains, prolonged exposure of EHEC to cold stress, i.e., 4 and 5 days at 4 and 0 degree C, respectively, in general significantly increased their detection time. This reflects an increase of the lag-phase time caused by cold stress. Two EHEC strains were selected to determine the minimum preliminary enrichment time that would ensure a positive PCR detection of low numbers of verotoxin-producing E. coli (VTEC; 2 to 2 x 10(5) CFU/25 g) inoculated into ground beef (25 g) and stored at 4 or -20 degrees C for 8 and 14 days, respectively. Incubation times of 6 and 9 h of 1 to 10 CFU/g and 1 to 10 CFU/25 g, respectively, were sufficient for PCR detection of VTEC in ground beef when analysis was performed immediately after inoculation (no cold stress). When cells are exposed to cold stress (4 or -20 degrees C) a 24-h enrichment period is recommended. Restriction of enrichment time to 9 h under these circumstances decreases the sensitivity of PCR detection to 80 CFU/g. Hence, to obtain maximum sensitivity, PCR detection of VTEC in naturally contaminated ground beef should be performed after 24 h of enrichment.

NF kappa B upstream regulatory sequences of the HIV-1 LTR are involved in the inhibition of HIV-1 promoter activity by the NS proteins of autonomous parvoviruses H-1 and MVMp.

To investigate parvoviral interference with human immunodeficiency virus type 1 (HIV-1) in human cells that are normally susceptible to HIV-1 infection, nonstructural (NS) proteins of the parvoviruses H-1 virus and minute virus of mice were studied for their effect on the activity of the HIV-1 promoter in a variety of CD4+ cells. Transient cotransfection assays revealed a reduced HIV-1 promoter activity in the presence of parvoviral NS proteins. Stimulation of the HIV-1 promoter by phorbol 12-myristate 13-acetate (PMA) led to an increase in its sensitivity to NS-induced suppression. The inhibitory effect of NS polypeptides depended, at least in part, on the presence of the NF kappa B motifs of the HIV-1 long terminal repeat, suggesting an interaction of the parvoviral products with PMA-inducible cellular factors binding to these elements of the HIV-1 promoter.

Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by tumor necrosis factor-alpha.

Recent findings have indicated that megakaryocytes may be susceptible to human immunodeficiency virus (HIV) infection, suggesting a potential role for megakaryocytes as viral reservoirs in HIV-infected patients. We report that the megakaryocytic cell line Dami could be productively infected with the HTLV III-B strain of HIV-1, in 26 different experiments (results of 16 experiments are reported); productive infection lasted up to 30 weeks. Despite a lack of detectable surface expression of the CD4 molecule and very low levels of CD4 mRNA, between 40% and 60% of megakaryocytic cells produced viral proteins after contact with HIV-1. Neither cytopathogenic effects nor syncytial formation was observed. Production of high levels of functional viral particles was indicated by analysis of p24 protein levels, reverse transcriptase activity, ultrastructural studies, and the capacity of supernatants from infected Dami cells to infect the Molt-4 T-lymphocytic cell line. HIV-1 RNA and protein levels in infected Dami cells were enhanced by treatment with tumor necrosis factor-alpha (TNF-alpha), and decreased by treatment with interferon-alpha (IFN-alpha) and IFN-gamma. Transient transfection of the megakaryocytic cells with various constructs of the HIV-1 promoter (LTR) linked to the luciferase reporter gene suggested that the effect of TNF-alpha was related, as in monocytic and T-cell lines, to transactivation of the enhancer region of the HIV-1 LTR. These findings indicate that signals provided by the immune system may modulate HIV-1 expression in cells of the megakaryocytic lineage.

IgE receptor on human eosinophils (FcERII). Comparison with B cell CD23 and association with an adhesion molecule.

IgE FcR (FcERII) on human eosinophils was characterized and compared with FcERII present on B cells (CD23). Two mAb, BB10 (anti-eosinophil FcERII) and 135 (anti-CD23), bound to the major component of FcERII at 45,000 to 50,000 Mr, both on purified hypodense eosinophils and on a B cell line (WIL-2WT). The specific ligand, human myeloma IgE, was able to bind to the molecules immunoprecipitated by BB10. A cross-reactivity between BB10 and a mAb anti-Leishmania gp63, which is a "fibronectin (Fn)-like" molecule, containing the L-arginine-L-glycyl-L-aspartyl (RGD) cell attachment domain indicated the presence of such a sequence in the common structure present on eosinophil and B cell FcERII. The synthetic tetrapeptide RGDS as well as its inverted sequence (SDGR) reduced the binding of BB10 and anti-Fn mAb to eosinophils and B cells. Flow microfluorometry analysis revealed a variable binding of BB10 and anti-Fn mAb to eosinophils purified from different patients, results compatible with recent findings on the inducibility of FcERIIb. The significant inhibition of IgE-dependent cytotoxicity against parasite targets by preincubation of eosinophils with BB10, anti-Fn and anti-CD23 mAb, with anti-RGDS polyclonal antibodies or with the SDGR peptide suggested the requirement of this cell adhesion sequence for the function of low affinity FcERII. The presence of such a sequence in the C-terminal domain of B cell FcERII raised the possibility of its role in B cell adhesion or B cell growth.

IgE and inflammatory cells.

Extensive studies initiated in parasitic disease models have unequivocally established that IgE antibodies can directly interact with mononuclear phagocytes, eosinophils and platelets through specific surface receptors now identified as Fc epsilon RII. Genes coding for B cell and more recently eosinophil IgE receptors have been cloned. Studies on molecular structure indicate a close homology between Fc epsilon RII on inflammatory cells and on B cells but indications are emerging of some degree of heterogeneity among the second class of receptors for IgE. Recent studies performed in parallel on eosinophils indicate that their IgE receptors contain a sequence commonly involved in the primary structure of adhesion proteins. Interaction between antigen and cytophilically bound IgE antibodies results in the triggering of cell effector function and the release of a variety of pro-inflammatory or cytocidal mediators. Among others, one eosinophil granule protein (eosinophil peroxidase) is preferentially released by anaphylactic isotype-dependent stimuli. The main expression of IgE-dependent platelet activation appears related to the production of oxygen-derived free radicals (detected by chemoluminescence and electron magnetic resonance) together with their cytocidal properties. Taken together these findings confirm our current view that IgE receptors on inflammatory cells play a major role in the expression of cell effector function, both in defence mechanisms against several parasites and in allergic reactions.