Highlights • Faecalibacterium prausnitzii is an ubiquitary bacterium of intestinal microbiota of all animals. • F. prausnitzii is a dominant member of human intestinal microbiota of healthy adults. • ...F. prausnitzii is a sensor of health especially for active Crohn's disease patients. • Beneficial mechanisms and roles of F. prausnitzii in microbiota are still unclear. • F. prausnitzii is a potential novel probiotic bacterium for human diseases as IBD and IBS.
Lactic acid bacteria (LAB) are promising vectors of choice to deliver active molecules to mucosal tissues. They are recognized as safe by the World Health Organization and some strains have probiotic ...properties. The wide range of potential applications of LAB-driven mucosal delivery includes control of inflammatory bowel disease, vaccine delivery, and management of auto-immune diseases. Because of this potential, strategies for the display of proteins at the surface of LAB are gaining interest. To display a protein at the surface of LAB, a signal peptide and an anchor domain are necessary. The recombinant protein can be attached to the membrane layer, using a transmembrane anchor or a lipoprotein-anchor, or to the cell wall, by a covalent link using sortase mediated anchoring via the LPXTG motif, or by non-covalent liaisons employing binding domains such as LysM or WxL. Both the stability and functionality of the displayed proteins will be affected by the kind of anchor used. The most commonly surfaced exposed recombinant proteins produced in LAB are antigens and antibodies and the most commonly used LAB are lactococci and lactobacilli. Although it is not necessarily so that surface-display is the preferred localization in all cases, it has been shown that for certain applications, such as delivery of the human papillomavirus E7 antigen, surface-display elicits better biological responses, compared to cytosolic expression or secretion. Recent developments include the display of peptides and proteins targeting host cell receptors, for the purpose of enhancing the interactions between LAB and host. Surface-display technologies have other potential applications, such as degradation of biomass, which is of importance for some potential industrial applications of LAB.
Summary
Allergic diseases affect up to 30% of the western population, and their prevalence is increasing. Probiotics are able to modulate the mucosal immune response, and clinical trials demonstrated ...that specific strains, especially lactic acid bacteria (LAB) ones, reduce allergic symptoms. Moreover, the use of recombinant probiotics has been evaluated as possible strategies for the immunotherapy of allergic diseases. The production and delivery of allergens by recombinant LAB in concert with their ability to induce a Th1‐type immune response have been shown to be a promising mucosal vaccination strategy in mouse model. The aim of this article is to review the applications of probiotics in allergy immunotherapy with a special focus on recombinant LAB delivering proteins or DNA.
Crohn's disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, ...the chemical nature of the anti-inflammatory compounds has not yet been determined.
Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice.
The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice.
A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.
Summary
Background
The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has ...clearly demonstrated any preventive or therapeutic inhibition of an allergen‐specific IgE response.
Objective
We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine β‐lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy.
Methods
Six‐week‐old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 μg/mice. Mice were then sensitized by intra‐peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations.
Results
Pre‐treatment with natural L. lactis plus BLG allowed induction of BLG‐specific T‐helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non‐colonizing LAB. Moreover, pre‐treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN‐γ productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein.
Conclusion
Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down‐regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.
We recently demonstrated that noninvasive food-grade Lactococcus lactis (L. lactis) can deliver eukaryotic expression plasmid in mammalian cells in vitro. Here, we evaluated, in vivo, whether a ...eukaryotic expression plasmid carried by lactococci can translocate to the epithelial cells of the intestinal membrane. The strain LL(pLIG:BLG1) carrying one plasmid containing a eukaryotic expression cassette encoding beta-lactoglobulin (BLG), a major allergen of cow's milk, was orally administered by gavage to mice. BLG cDNA was detected in the epithelial membrane of the small intestine of 40% of the mice and BLG was produced in 53% of the mice. Oral administration of LL(pLIG:BLG1) induced a low and transitory Th1-type immune response counteracting a Th2 response in case of further sensitization. We demonstrated for the first time the transfer of a functional plasmid to the epithelial membrane of the small intestine in mice by noninvasive food-grade lactococci.
Recombinant lactic acid bacteria (LAB), especially Lactococcus lactis, have been genetically engineered to express heterogeneous invasion proteins, such as the fibronectin binding protein A (FnBPA) ...from Staphylococcus aureus, to increase the invasion ability of the host strains, indicating a promising approach for DNA vaccine delivery. The presence of FnBPA has been also shown to be an adjuvant for co-delivered antigens, however, the underlying mechanisms are still not clear. To explore the above underlying mechanisms, in this study, we constructed a novel Lactobacillus plantarum strain with surface displayed FnBPA, which could significantly improve the adhesion and invasion ratios of L. plantarum strain on a porcine intestinal epithelial cell line (IPEC-J2) about two-fold compared with the empty vector. At the same time, the presence of FnBPA significantly stimulated the differentiation of bone marrow-derived dendritic cells (DCs) and increased the secretion of interleukin (IL)-6 and mRNA level of IL-6 gene, which were proved by flow cytometry, enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription PCR (qRT-PCR). With regard to in vivo study, the presence of FnBPA significantly stimulated the differentiation of DCs in the Peyer’s patch (PP) and the percentages of IL-4+ and IL-17A+ T helper (Th) cells of splenocytes in flow cytometry assay. In consistent with these results, the levels of IL-4 and IL-17A in serum as measured via ELISA also increased in mice treated with FnBPA+
L. plantarum. Finally, the FnBPA strain increased the production of B220+ B cells in mesenteric lymph node (MLN) and PP and the levels of FnBPA-specific IgG and sIgA antibodies, indicating the its possible application in vaccine field. This study demonstrated that the invasive L. plantarum with surface displayed FnBPA could modulate host immune response by stimulating the differentiation of DCs and Th cells which could possibly be responsive for the adjuvant effects of FnBPA.
Inflammatory bowel diseases (IBD) describe a group of complex intestinal disorders characterized by inflammation in the gastrointestinal tract. Current treatments for IBD include the use of ...antiinflammatory drugs; furthermore, recombinant lactic acid bacteria have been used as a therapeutic vehicle for anti-inflammatory agents in IBD models. Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokines; however, its oral administration is limited because it is quickly degraded in the gastrointestinal tract and systemic treatments have led to undesirable side effects. In this study, an engineered invasive strain of Lactococcus (L.) lactis producing Fibronectin Binding Protein A (FnBPA+), from Staphylococcus aureus capable of delivering, directly inside eukaryotic cells, an eukaryotic DNA expression vector containing the ORF coding for IL-10 of Mus musculus (pValac:il-10) was developed and its functionality was evaluated using in vitro and in vivo assays. Functionality of the plasmid and the invasive strain was demonstrated by transfection and invasiveness assays using cell cultures and in vivo in mice by fluorescence microscopy. TNBS inoculated mice that received this novel strain showed lower damage scores in their large intestines (at both macroscopic and microscopic levels), lower microbial translocation to liver, and increased anti-inflammatory/pro-inflammatory cytokine ratios compared to mice that received L. lactis FnBPA+ without the pValac:il-10 plasmid. The effectiveness was demonstrated of this novel DNA delivery therapeutic strategy in the prevention of inflammation using a murine model of colitis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background
Bovine β‐lactoglobulin (Blg) is a major cow's milk allergen. It is the main whey protein, without any counterpart in human milk. Blg chemical hydrolysates appeared to retain most of the ...immunoreactivity of the native protein. Allergenicity of Blg has already been shown to be associated with the four peptides derived from cyanogen bromide cleavage of Blg.
Objectives
To map the major allergenic epitopes (e.g. regions of the molecule able to bind IgE) on Blg using specific IgE from sera of 46 milk‐allergic patients as a probe.
Methods
Direct and competitive inhibition enzyme immunoassays involving immobilized native protein or purified peptides derived from Blg tryptic cleavage.
Results
Several peptides capable of specifically binding human IgEs were identified and were classified according to the intensity and frequency of the responses. The major epitopes appeared to be fragments (41–60), (102–124) and (149–162) recognized by 92, 97 and 89% of sera, respectively, whilst a second group which contained the fragments (1–8) and (25–40) was recognized by 58 and 72% of the population. A third group, comprising peptides (9–14), (84–91) and (92–100), was still detected by more than 40% of sera.
Conclusion
Three peptides were identified as major epitopes, recognized by a large majority of human IgE antibodies. Numerous other epitopes are scattered all along the Blg sequence.
Identifying environmental damage due to anthropogenic activities is a focal point for scientists and policy makers like those involved in the European Water Framework Directive (WFD). Many of these ...approaches focus on ecological endpoints for assessing environmental perturbations, which lead to policies emphasizing mitigation rather than prevention. Biomarkers provide early-warning indicators of stress but it is necessary to distinguish their natural variations from those induced by chemical stress. The global aim of this study was to establish a baseline assessment criterion (BAC) using historical data in a reference site to define toxicity thresholds. We have developed a multiple polynomial regression model (MPR) accounting the influence of salinity, temperature and size of individual on energetic reserves (glycogen and lipids) in the marine polychaete Hediste diversicolor. The model identified a complex, orthogonal relationship between confounding factors and glycogen and a linear relationship between lipids and size of individuals.
Display omitted
•Establishment of thresholds for energetic reserve levels•Multiple polynomial regression model to characterize the influence of confounding factors•Facilitation of sub-individual, multimarker approaches into environmental monitoring surveys
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