Exopolysaccharide from Lacticaseibacillus rhamnosus induces IgA production in airways and alleviates allergic airway inflammation in mouse model.

The currently observed high prevalence of allergic diseases has been associated with changes in microbial exposure in industrialized countries. Defined bacterial components represent a new strategy for modulating the allergic immune response. We show that intranasal administration of exopolysaccharide (EPS) isolated from Lacticaseibacillus (L.) rhamnosus LOCK900 induces TGF-ß1, IgA, and regulatory FoxP3+ T-cells in the lungs of naïve mice. Using the ovalbumin mouse model, we demonstrate that intranasal administration of EPS downregulates the development of allergic airway inflammation and the Th2 cytokine response in sensitized individuals. At the same time, EPS treatment of sensitized mice, similar to EPS-induced responses in naïve mice, significantly increased the level of total, OVA-specific, and also bacteria-specific IgA in bronchoalveolar lavage and the number of IgA-producing B-cells in the lung tissue of these mice. Thus, EPS derived from L. rhamnosus LOCK900 can be considered a safe candidate for preventing the development of allergic symptoms in the lungs of sensitized individuals upon exposure to an allergen.

What happens to Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with eukaryotic cells?

BACKGROUND: The impact of probiotic strains on host health is widely known. The available studies on the interaction between bacteria and the host are focused on the changes induced by bacteria in the host mainly. The studies determining the changes that occurred in the bacteria cells are in the minority. Within this paper, we determined what happens to the selected Bifidobacterium adolescentis and Bifidobacterium longum ssp. longum in an experimental environment with the intestinal epithelial layer. For this purpose, we tested the bacteria cells' viability, redox activity, membrane potential and enzymatic activity in different environments, including CaCo-2/HT-29 co-culture, cell culture medium, presence of inflammatory inductor (TNF-α) and oxygen. RESULTS: We indicated that the external milieu impacts the viability and vitality of bacteria. Bifidobacterium adolescentis decrease the size of the live population in the cell culture medium with and without TNF-α (p < 0.001 and p < 0.01 respectively). In contrast, Bifidobacterium longum ssp. longum significantly increased survivability in contact with the eukaryotic cells and cell culture medium (p < 0.001). Bifidobacterium adolescentis showed significant changes in membrane potential, which was decreased in the presence of eukaryotic cells (p < 0.01), eukaryotic cells in an inflammatory state (p < 0.01), cell culture medium (p < 0.01) and cell culture medium with TNF-α (p < 0.05). In contrast, Bifidobacterium longum ssp. longum did not modulate membrane potential. Instead, bacteria significantly decreased the redox activity in response to milieus such as eukaryotic cells presence, inflamed eukaryotic cells as well as the culture medium (p < 0.001). The redox activity was significantly different in the cells culture medium vs the presence of eukaryotic cells (p < 0.001). The ability to ß-galactosidase production was different for selected strains: Bifidobacterium longum ssp. longum indicated 91.5% of positive cells, whereas Bifidobacterium adolescentis 4.34% only. Both strains significantly reduced the enzyme production in contact with the eukaryotic milieu but not in the cell culture media. CONCLUSION: The environmental-induced changes may shape the probiotic properties of bacterial strains. It seems that the knowledge of the sensitivity of bacteria to the external environment may help to select the most promising probiotic strains, reduce research costs, and contribute to greater reproducibility of the obtained probiotic effects.

Saccharomyces cerevisiae ß-glucan improves the response of trained macrophages to severe P. aeruginosa infections.

OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with ß-glucan might improve their response to P. aeruginosa infections. METHODS: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae ß-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo ß-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared. RESULTS: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced. CONCLUSIONS: Training of murine macrophages with S. cerevisiae ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.

Biofilm-forming strains of P. aeruginosa and S. aureus isolated from cystic fibrosis patients differently affect inflammatory phenotype of macrophages.

OBJECTIVE: Lung cystic fibrosis (CF) is characterized by chronic infections and hyperinflammatory response of neutrophils and macrophages. P. aeruginosa (PA) and S. aureus (MSSA, MRSA) are major pathogens of advanced CF. The main goal of this study was to compare the inflammatory phenotype of murine C57BL/6 macrophages exposed to PA57 with that exposed to MSSA60, both strains isolated from the same patient with severe CF. In the present study, we used C57BL/6 mice sensitive to lung infection with P. aeruginosa. METHODS: We measured the release of cytokines and the expression of phenotypic markers of murine neutrophils and macrophages exposed to bacterial cells and biofilm components (i.e., EPS) of the selected bacteria. In addition, a quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. RESULTS: Neutrophils stimulated with PA57 and MSSA60 strains produced hyperinflammatory pattern of cytokines. The pro-inflammatory impact of PA57 was significantly higher than that of MSSA60 (IL-6/IL-10 ratio: PA57 = 9.3 vs. MSSA60 = 1.7). Macrophages produced significantly lower amount of cytokines, but showed classical pattern of M1 markers (iNOS-High; arginase-1 and mannose receptor MRC1-Low). Importantly, as evidenced by proteomic analysis, PA57 and PA57-EPS were stronger inducers of M1 macrophage polarization than the MSSA60 counterparts. CONCLUSIONS: Our study demonstrated that strong biofilm P. aeruginosa strains, CF isolates, are dominant inducers of M1 macrophages, termed biofilm-associated macrophages (BAMs). We suggest that repolarization of detrimental BAMs might be a new therapeutic strategy to ameliorate the airway damage in CF.

A Novel Mechanism of Macrophage Activation by the Natural Yolkin Polypeptide Complex from Egg Yolk.

Ageing is accompanied by the inevitable changes in the function of the immune system. It provides increased susceptibility to chronic infections that have a negative impact on the quality of life of older people. Therefore, rejuvenating the aged immunity has become an important research and therapeutic goal. Yolkin, a polypeptide complex isolated from hen egg yolks, possesses immunoregulatory and neuroprotective activity. Considering that macrophages play a key role in pathogen recognition and antigen presentation, we evaluated the impact of yolkin on the phenotype and function of mouse bone marrow-derived macrophages of the BMDM cell line. We determined yolkin bioavailability and the surface co-expression of CD80/CD86 using flow cytometry and IL-6, IL-10, TGF-ß and iNOS mRNA expression via real-time PCR. Additionally, the impact of yolkin on the regulation of cytokine expression by MAPK and PI3K/Akt kinases was determined. The stimulation of cells with yolkin induced significant changes in cell morphology and an increase in CD80/CD86 expression. Using pharmaceutical inhibitors of ERK, JNK and PI3K/Akt, we have shown that yolkin is able to activate these kinases to control cytokine mRNA expression. Our results suggest that yolkin is a good regulator of macrophage activity, priming mainly the M1 phenotype. Therefore, it is believed that yolkin possesses significant therapeutic potential and represents a promising possibility for the development of novel immunomodulatory medicine.

Effective mucosal vaccines - opportunities and challenges / Efektywne szczepionki sluzówkowe ­ mozliwosci i wyzwania.

Most pathogens enter the body through the surfaces of the mucous membranes, e.g. the nose or the intestines. The mucosal immune response is essential for the effective elimination of invading pathogens. Unfortunately, most vaccines which are administered intramuscularly by injection do not induce an adequate protective immune response on mucous membranes. For example, after intramuscular injection, the level of secretory IgA antibodies is low and often insufficient to successfully combat the pathogen. On the other hand, mucosal-induced immunity produces a long-lasting effect in the form of a local and systemic response to the pathogen. Moreover, the administration of such vaccines does not generate hazardous medical waste and their application does not require the presence of qualified medical personnel. Therefore, intensive research into vaccines administered via the mucosal route is ongoing. An obstacle in the development of mucosal vaccines is the natural defense mechanisms of the mucosa, the overcoming of which requires the use of specialized adjuvants. Currently, there are no such formulations on the market.

Selenium-Containing Exopolysaccharides Isolated from the Culture Medium of Lentinula edodes: Structure and Biological Activity.

In continuation of our research on the influence of selenium incorporation on the biosynthesis, structure, and immunomodulatory and antioxidant activities of polysaccharides of fungal origin, we have isolated from a post-culture medium of Lentinula edodes a selenium (Se)-containing exopolysaccharide fraction composed mainly of a highly branched 1-6-α-mannoprotein of molecular weight 4.5 × 106 Da, with 15% protein component. The structure of this fraction resembled mannoproteins isolated from yeast and other mushroom cultures, but it was characterized by a significantly higher molecular weight. X-ray absorption fine structure spectral analysis in the near edge region (XANES) suggested that selenium in the Se-exopolysaccharide structure was present mainly at the IV oxidation state. The simulation analysis in the EXAFS region suggested the presence of two oxygen atoms in the region surrounding the selenium. On the grounds of our previous studies, we hypothesized that selenium-enriched exopolysaccharides would possess higher biological activity than the non-Se-enriched reference fraction. To perform structure-activity studies, we conducted the same tests of biological activity as for previously obtained mycelial Se-polyglucans. The Se-enriched exopolysaccharide fraction significantly enhanced cell viability when incubated with normal (human umbilical vein endothelial cells (HUVEC)) cells (but this effect was absent for malignant human cervical HeLa cells) and this fraction also protected the cells from oxidative stress conditions. The results of tests on the proliferation of human peripheral blood mononuclear cells suggested a selective immunosuppressive activity, like previously tested Se-polyglucans isolated from L. edodes mycelium. The Se-exopolysaccharide fraction, in concentrations of 10-100 µg/mL, inhibited human T lymphocyte proliferation induced by mitogens, without significant effects on B lymphocytes. As with previously obtained Se-polyglucans, in the currently tested Se-polymannans, the selenium content increased the biological activity. However, the activity of selenium exopolysaccharides in all tests was significantly lower than that of previously tested mycelial isolates, most likely due to a different mode of selenium binding and its higher degree of oxidation.

Identification of the Primary Structure of Selenium-Containing Polysaccharides Selectively Inhibiting T-Cell Proliferation.

We previously described the biosynthesis, isolation, and immunosuppressive activity of the selenium-containing polysaccharide fraction isolated from the mycelial culture of Lentinula edodes. Structural studies have shown that the fraction was a protein-containing mixture of high molar mass polysaccharides α- and ß-glucans. However, which of the components of the complex fraction is responsible for the immunosuppressive activity non-typical for polysaccharides of fungal origin has not been explained. In the current study, we defined four-polysaccharide components of the Se-containing polysaccharide fraction determined their primary structure and examined the effect on T- and B-cell proliferation. The isolated Se-polysaccharides, α-1,4-glucan (Mw 2.25 × 106 g/mol), unbranched ß-1,6-d-glucan, unbranched ß-1,3-d-glucan and ß-1,3-branched ß-1,6-d-glucan (Mw 1.10 × 105 g/mol), are not typical as components of the cell wall of L. edodes. All are biologically active, but the inhibitory effect of the isolated polysaccharides on lymphocyte proliferation was weaker, though more selective than that of the crude fraction.

Absence of Mal/TIRAP Results in Abrogated Imidazoquinolinones-Dependent Activation of IRF7 and Suppressed IFNß and IFN-I Activated Gene Production.

Activation of TLR7 by small imidazoquinoline molecules such as R848 or R837 initiates signaling cascades leading to the activation of transcription factors, such as AP-1, NF-κB, and interferon regulatory factors (IRFs) and afterward to the induction of cytokines and anti-viral Type I IFNs. In general, TLRs mediate these effects by utilizing different intracellular signaling molecules, one of them is Mal. Mal is a protein closely related to the antibacterial response, and its role in the TLR7 pathways remains poorly understood. In this study, we show that Mal determines the expression and secretion of IFNß following activation of TLR7, a receptor that recognizes ssRNA and imidazoquinolines. Moreover, we observed that R848 induces Mal-dependent IFNß production via ERK1/2 activation as well as the transcription factor IRF7 activation. Although activation of TLR7 leads to NF-κB-dependent expression of IRF7, this process is independent of Mal. We also demonstrate that secretion of IFNß regulated by TLR7 and Mal in macrophages and dendritic cells leads to the IP-10 chemokine expression. In conclusion, our data demonstrate that Mal is a critical regulator of the imidazoquinolinones-dependent IFNß production via ERK1/2/IRF7 signaling cascade which brings us closer to understanding the molecular mechanism's regulation of innate immune response.

Pseudomonas aeruginosa biofilm is a potent inducer of phagocyte hyperinflammation.

OBJECTIVE: Pseudomonas aeruginosa effectively facilitate resistance to phagocyte killing by biofilm formation. However, the cross talk between biofilm components and phagocytes is still unclear. We hypothesize that a biofilm provides a concentrated extracellular source of LPS, DNA and exopolysaccharides (EPS), which polarize neighbouring phagocytes into an adverse hyperinflammatory state of activation. METHODS: We measured the release of a panel of mediators produced in vitro by murine neutrophils and macrophages exposed to various biofilm components of P. aeruginosa cultures. RESULTS: We found that conditioned media from a high biofilm-producing strain of P. aeruginosa, PAR5, accumulated high concentrations of extracellular bacterial LPS, DNA and EPS by 72 h. These conditioned media induced phagocytes to release a hyperinflammatory pattern of mediators, with enhanced levels of TNF-α, IL-6, IL12p40, PGE2 and NO. Moreover, the phagocytes also upregulated COX-2 and iNOS with no influence on the expression of arginase-1. CONCLUSIONS: Phagocytes exposed to biofilm microenvironment, called by us biofilm-associated neutrophils/macrophages (BANs/BAMs), display secretory properties similar to that of N1/M1-type phagocytes. These results suggest that in vivo high concentrations of LPS and DNA, trapped in biofilm by EPS, might convert infiltrating phagocytes into cells responsible for tissue injury without direct contact with bacteria and phagocytosis.

Subsite heterogeneity in the profiles of circulating cytokines in colorectal cancer.

Colorectal cancers (CRCs) are treated as one entity but are in fact a heterogeneous group of diseases. If not addressed, subsite-associated variability may interfere with mechanism-targeted therapies and accuracy of potential CRC biomarkers. Little is known about the contribution of systemic inflammatory and immune mediators to subsite heterogeneity in CRC. Our purpose was to compare the profiles of key cytokines between right and left colonic and rectal CRCs. Using Luminex xMAP® technology, serum concentrations of eotaxin, IL-1ß, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12(p70), IL-13, IL-15, IL-17, IFNγ, IP-10, FGF-2, G-CSF, GM-CSF, MCP-1, MIP-1α and ß, PDGF-BB, RANTES, TNFα, and VEGF-A were determined in 104 CRC patients. We found the concentrations of IL-12(p70), IL-10, IL-1ra, IL-4, IL-6, IL-7, IL-8, G-CSF and TNFα to be significantly higher in right-sided and GM-CSF in left-sided than rectal CRCs. The concentrations of IFNγ and MIP-1α were significantly higher in right-sided CRCs as compared to cancers of other locations combined. In turn, MIP-1ß was higher in rectal CRCs as compared to colon cancers. Taken together, our results show subsite heterogeneity of CRC cancers in terms of systemic inflammatory and immune responses that ought to be taken into account when attempting immunotherapy or developing biomarkers. Additionally, more pronounced TH2 response accompanied by TH1 immunity and more prominent tumor-promoting inflammation in CRC patients with primary tumors originating from right-sided colon may constitute a molecular background of unfavorable prognosis associated with this location.

Effectiveness of Sensors Contact Metallization (Ti, Au, and Ru) and Biofunctionalization for Escherichia coli Detection.

In designing a bacteria biosensor, various issues must be addressed: the specificity of bacteria recognition, the immobilization of biomolecules that act as the bacteria receptor, and the selectivity of sensor surface. The aim of this paper was to examine how the biofunctionalized surface of Ti, Au, and Ru metals reacts in contact with strains of Escherichia coli (E. coli). The focus on metal surfaces results from their future use as electrodes in high frequency biosensors, e.g., resonant circuits or transmission-line sections. First, the surfaces of different metals were chemically functionalized with 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde or with 3-glycidylooxypropyltrimethoxysilane (GPTMS) followed by N-(5-amino-1-carboxypentyl) iminodiacetic acid (AB-NTA) and NiCl2. Secondly, the lipopolysaccharide binding protein (LBP), polyclonal anti-Escherichia coli antibody and bacteriophage protein gp37 were tested as bacteria receptors. The selectivity and specificity have been confirmed by the Enzyme-Linked Immunosorbent Assay (ELISA) and visualized by scanning electron microscopy at low landing energies. We noticed that LBP, polyclonal antibody, and gp37 were successfully immobilized on all studied metals and recognized the E. coli bacteria selectively. However, for the antibody, the highest reactivity was observed when Ti surface was modified, whereas the bacteria binding was comparable between LBP and gp37 on the functionalized Ru surfaces, independent from modification. Thus, all surfaces were biocompatible within the scope of biosensor functionality, with titanium functionalization showing the best performance.

Elevated systemic interleukin-7 in patients with colorectal cancer and individuals at high risk of cancer: association with lymph node involvement and tumor location in the right colon.

Interleukin (IL)-7 is a cytokine essential for protective immunity, and it is considered as a promising agent for cancer immunotherapy. Recent studies, however, appear to associate IL-7 with aggressiveness of solid tumors. The IL-7 has been less studied in colorectal cancer (CRC) and conditions associated with increased risk of CRC development. To explore IL-7 status in bowel diseases, it was measured immunofluorometrically in 431 individuals (110 with CRC) by using Luminex platform. A level of IL-7 in CRC patients was significantly higher than in controls, did not differ from those with adenomas, but was lower than in both active and inactive inflammatory bowel disease (IBD) cases. In CRC, IL-7 was higher in patients with lymph node and distant metastases and with tumors located in right colon. In adenomas, IL-7 elevation was associated exclusively with villous growth pattern, while in IBD, circulating IL-7 reflected clinical activity of Crohn's disease and ulcerative colitis. Systemic TNFα, IL-10, and PDGF-BB were independent predictors of circulating IL-7. In summary, our study is the first to demonstrate IL-7 elevation in CRC in association with metastatic disease and tumor location. Both associations should be considered when designing IL-7-based immunotherapies for CRC. Further studies on IL-7 functionality in CRC are necessary.

Re-classification within the serogroups O3 and O8 of Citrobacter strains.

BACKGROUND: Citrobacter strains are opportunistic pathogens often responsible for serious enteric as well as extra-intestinal diseases, and therefore the O-antigenic scheme, still in use in diagnostic identification, should be set for proper serotyping. The structures of more than 30 different Citrobacter O-antigens (O-polysaccharide chains of the lipopolysaccharides) of 43 Citrobacter O-serogroups have been elucidated so far. However, relationships between strains in several heterogeneous serogroups still need to be clarified by immunochemical studies. These include complex serogroups O3 and O8, represented by 20 and 7 strains, respectively, which are the subject of the present work. Earlier, the O-polysaccharide structures have been determined for Citrobacter O3 strain Be35/57 (PCM 1508) and Citrobacter O8 strain Be64/57 (PCM 1536). RESULTS: Serological studies (immunoblotting) carried out on Citrobacter lipopolysaccharides from different strains ascribed to serogroups O3 and O8 showed that each of these serogroups should be divided into non-cross-reacting subgroups. Based on the results of chemical analyses and 1H and 13C NMR spectroscopy the structure of Citrobacter O-antigens from strains PCM 1504 (O6) and PCM 1573 (O2) have been established. Chemical data combined with serological analyses showed that several Citrobacter strains should be reclassified into other serogroups. CONCLUSIONS: Immunochemical studies carried out on Citrobacter LPS, described in this paper, showed the expediency of reclassification of: 1) strains PCM 1504 and PCM 1573 from serogroups O6 and O2 to serogroups O3 and O8, respectively, 2) strains PCM 1503 and PCM 1505 from serogroups O3 and O8 to new serogroups O3a and O8a, respectively.

Midkine is up-regulated in both cancerous and inflamed bowel, reflecting lymph node metastasis in colorectal cancer and clinical activity of ulcerative colitis.

Midkine is a multifunctional cytokine and growth factor displaying proinflammatory and pro-tumorigenic activity. Its association with bowel diseases has not been fully elucidated. Our purpose was to delineate midkine expression pattern by RT-qPCR in inflamed/cancerous bowel (n=208) and whole blood (n=150) in colorectal cancer (CRC), Crohn's disease (CD), and ulcerative colitis (UC) and to evaluate midkine dynamics in early postoperative period following colorectal surgery. The expression of midkine was significantly up-regulated in stage III CRC and independently associated with lymph node metastasis. The expression of midkine in whole blood was up-regulated solely in N1 CRC. Midkine expression in cancer-free tissue (CRC) was also elevated and dependent on CRC advancement. In IBD, inflammation increased the bowel expression of midkine solely in UC, in a manner proportional to the disease clinical activity. Large and small bowel differed with respect to the expression of midkine in quiescent tissue (higher in small bowel) and to its correlation pattern with chemokines (in a large bowel) and angiogenic factors and cell cycle regulators (in a small bowel). Circulating midkine and its expression in whole blood dropped directly following colorectal surgery; however, the concentration of midkine in serum was restored on postoperative day three. Midkine is involved in bowel inflammation in UC and lymph node metastasis in CRC, rendering midkine an attractive target for their treatment. Owing to midkine elevation in early postoperative period and its overexpression in tumor-adjacent tissue, targeting midkine might be considered also as a prevention of CRC recurrence following curative tumor resection.

Pathogenic factors of Pseudomonas aeruginosa - the role of biofilm in pathogenicity and as a target for phage therapy.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause several acute and chronic infections in humans, and it has become an important cause of nosocomial infections and antibiotic resistance. Biofilm represents an important virulence factor for these bacteria, plays a role in P. aeruginosa infections and avoidance of immune defence mechanisms, and has the ability to protect the bacteria from antibiotics. Alginate, Psl and Pel, three exopolysaccharides, are the main components in biofilm matrix, with many biological functions attributed to them, especially with respect to the protection of the bacterial cell from antibiotics and the immune system. Pseudomonas infections, biofilm formation and development of resistance to antibiotics all require better understanding to achieve the best results using alternative treatment with phage therapy. This review describes the P. aeruginosa pathogenicity and virulence factors with a special focus on the biofilm and its role in infection and resistance to antibiotics and summarizes phage therapy as an alternative approach in treatment of P. aeruginosa infections.

Chemical characterization and immunomodulatory properties of polysaccharides isolated from probiotic Lactobacillus casei LOCK 0919.

The Lactobacillus casei strain, LOCK 0919, is intended for the dietary management of food allergies and atopic dermatitis (LATOPIC® BIOMED). The use of a probiotic to modulate immune responses is an interesting strategy for solving imbalance problems of gut microflora that may lead to various disorders. However, the exact bacterial signaling mechanisms underlying such modulations are still far from being understood. Here, we investigated variations in the chemical compositions and immunomodulatory properties of the polysaccharides (PS), L919/A and L919/B, which are produced by L. casei LOCK 0919. By virtue of their chemical features, such PS can modulate the immune responses to third-party antigens. Our results revealed that L919/A and L919/B could both modulate the immune response to Lactobacillus planatarum WCFS1, but only L919/B could alter the response of THP-1 cells (in terms of tumor necrosis factor alpha production) to L. planatarum WCFS1 and Escherichia coli Nissle 1917. The comprehensive immunochemical characterization is crucial for the understanding of the biological function as well as of the bacteria-host and bacteria-bacteria cross-talk.

Identification of Lactobacillus proteins with different recognition patterns between immune rabbit sera and nonimmune mice or human sera.

BACKGROUND: The genus Lactobacillus belongs to a large heterogeneous group of low G + C Gram-positive anaerobic bacteria, which are frequently used as probiotics. The health-beneficial effects, in particular the immunomodulation effect, of probiotics depend on the strain and dose used. Strain variations may be related to diversity of the cell surface architecture of bacteria and the ability to express specific antigens or secrete compounds. The use of Lactobacillus as probiotic requires a comprehensive understanding of its effect on host immune system. To evaluate the potential immunoreactive properties of proteins isolated from four Lactobacillus strains: L. johnsonii 142 and L. johnsonii 151, L. rhamnosus LOCK 0900 and L. casei LOCK 0919, the polyclonal sera obtained from mouse and human have been tested as well as with sera from rabbits immunized with whole lactobacilli cells. RESULTS: The reactivity of isolated proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and sequenced, in particular the fractions were identified as phosphoglycerate kinase (L. johnsonii 142), glyceraldehyde 3-phosphate dehydrogenase (L. johnosnii 142, L. rhamnosus LOCK 0900), hypothetic protein JDM1_1307 (L. johnsonii 151) and fructose/tagatose-bisphosphate-aldolase (L. casei LOCK 0919). CONCLUSION: The different prevalence of reactions against tested antigens in rabbit, mouse and human sera may indicate significant differences in immune system and commensal cross-talk in these groups. The identification of immunoreactive lactobacilli proteins opens the possibility to use them as an antigens for development of vaccines.

[Selected aspects of Clostridium difficile infection]. / Wybrane aspekty zakazen Clostridium difficile.

Clostridium difficile pathogen is a cause of the most frequent nosocomial infection, which is antibiotic-associated diarrhea. Antibiotic treatment causes disruption of the microbiome balance, which makes the gut a friendly environment for the pathogen. It leads to pseudomembranous colitis, toxic megacolon and even death. Clostridium difficile infection (CDI) is particularly dangerous to elderly patients, leading to the highest mortality rate. C. difficile is equipped with many virulence factors such as toxin A and B, binary toxin CDT, flagellum, S-layer proteins, Cwp66 and GroEL proteins, protease Cwp84, fibronectin-binding protein and the ability to form biofilm and spores. Problems with anti-CDI therapy prompt researchers and clinicians to seek alternative ways of therapy. Identification of immunological epitopes in outer layer proteins and the use of them as antigens for anti-CDI vaccines would be a rational approach to prevent the disease, but unfortunately such vaccines are not available yet. In this article we review the course of the disease, virulence and risk factors. We summarize briefly epidemiological data and the latest achievements in CDI treatment.

Distinct immunomodulation of bone marrow-derived dendritic cell responses to Lactobacillus plantarum WCFS1 by two different polysaccharides isolated from Lactobacillus rhamnosus LOCK 0900.

The structures of polysaccharides (PS) isolated from Lactobacillus rhamnosus LOCK 0900 and results from stimulation of mouse bone marrow-derived dendritic cells (BM-DC) and human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR) upon exposure to these antigens were studied. L. rhamnosus LOCK 0900 produces PS that differ greatly in their structure. The polymer L900/2, with a high average molecular mass of 830 kDa, is a branched heteropolysaccharide with a unique repeating unit consisting of seven sugar residues and pyruvic acid, whereas L900/3 has a low average molecular mass of 18 kDa and contains a pentasaccharide repeating unit and phosphorus. Furthermore, we found that both described PS neither induce cytokine production and maturation of mouse BM-DC nor induce signaling through TLR2/TLR4 receptors. However, they differ profoundly in their abilities to modulate the BM-DC immune response to the well-characterized human isolate Lactobacillus plantarum WCFS1. Exposure to L900/2 enhanced interleukin-10 (IL-10) production induced by L. plantarum WCFS1, while in contrast, L900/3 enhanced the production of IL-12p70. We conclude that PS, probably due to their chemical features, are able to modulate the immune responses to third-party antigens. The ability to induce regulatory IL-10 by L900/2 opens up the possibility to use this PS in therapy of inflammatory conditions, such as inflammatory bowel disease, whereas L900/3 might be useful in reverting the antigen-dependent Th2-skewed immune responses in allergies.