Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR4

<p>Abstract</p> <p>Background</p> <p>Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. <it>Lactobacillus </it>is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic <it>Lactobacillus rhamnosus </it>GR-1 on epithelial immune activation.</p> <p>Results</p> <p>Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed <it>Escherichia coli </it>and this was further potentiated when cells were co-cultured with live <it>L. rhamnosus </it>GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with <it>E. coli </it>and <it>L. rhamnosus </it>GR-1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, <it>L. rhamnosus </it>GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR-1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with <it>E. coli </it>and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB.</p> <p>Conclusions</p> <p>The immunological outcome of <it>E. coli </it>challenge of bladder cells was influenced by probiotic <it>L. rhamnosus </it>GR-1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic <it>L. rhamnosus </it>GR-1 modulated the activation of the NF-kappaB through increased levels of TLR4 on the bladder cells and altered subsequent release of cytokines from urothelial cells. By influencing immunological factors such as TLR4, important in the process of fighting pathogens, lactobacilli could facilitate pathogen recognition and infection clearance.</p

Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus)

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks

Development and analysis of prospective anti-HIV probiotic vaccines

Major improvements have been made in the treatment and prevention of HIV/AIDS. However, a prophylactic vaccine is still unavailable, and several vaccine-candidate trials yielded less than favourable results. Given that the HIV pandemic has not slowed down significantly, there is an urgent need for the development of an effective vaccine. The HIV-1 Gag protein, a key player in HIV particle assembly, is a suitable antigen for use in HIV vaccine development since antibodies targeting HIV-1 Gag will interfere with the replication of the virus. In our vaccine development strategy, it was important for us to develop a candidate for mucosal administration. This is because the mucosal route is the major site for HIV transmission and early viral replication, which is associated with extensive and rapid depletion of CD4+ T-cells in the Gut-Associated Lymphoid Tissue (GALT). Here, we transformed probiotic strains of Lactobacillus plantarum and Lactobacillus fermentum with the recombinant plasmid vectors pSIP409 and pSIP411 harbouring the HIV-1 GagM gene. Following electroporation, HIV-1 GagM expression was induced in the probiotics using peptide pheromone. Via PCR and sequencing, the presence of GagM was confirmed in the L. plantarum+ pSIP409-GagM and L. fermentum+ pSIP411-GagM clones. Protein expression was induced with peptide pheromone. Then, protein expression was confirmed by western blotting with goat anti-HIV p24 primary antibody and anti-goat secondary antibody. ELISA was also performed to confirm the antigenicity of the HIV-1 Gag antigen and to also quantify the antigen in the two Lactobacilli clones. Our results show that 1.5×109 CFU of L. plantarum+ pSIP409-GagM expressed 125μg of HIV-1 Gag and 1.9×109 CFU of L. fermentum+ pSIP411-GagM clones expressed 125μg of HIV-1 Gag respectively. In vitro digestion with pepsin, pancreatin and bile salts suggested that partial digestion of the probiotic vaccine candidates may occur when administered orally. Taken together, our probiotic HIV-1 vaccine candidates showed good prospects for further immunological analysis via animal trial

Development and analysis of prospective anti-HIV probiotic vaccines

Major improvements have been made in the treatment and prevention of HIV/AIDS. However, a prophylactic vaccine is still unavailable, and several vaccine-candidate trials yielded less than favourable results. Given that the HIV pandemic has not slowed down significantly, there is an urgent need for the development of an effective vaccine. The HIV-1 Gag protein, a key player in HIV particle assembly, is a suitable antigen for use in HIV vaccine development since antibodies targeting HIV-1 Gag will interfere with the replication of the virus. In our vaccine development strategy, it was important for us to develop a candidate for mucosal administration. This is because the mucosal route is the major site for HIV transmission and early viral replication, which is associated with extensive and rapid depletion of CD4+ T-cells in the Gut-Associated Lymphoid Tissue (GALT). Here, we transformed probiotic strains of Lactobacillus plantarum and Lactobacillus fermentum with the recombinant plasmid vectors pSIP409 and pSIP411 harbouring the HIV-1 GagM gene. Following electroporation, HIV-1 GagM expression was induced in the probiotics using peptide pheromone. Via PCR and sequencing, the presence of GagM was confirmed in the L. plantarum+ pSIP409-GagM and L. fermentum+ pSIP411-GagM clones. Protein expression was induced with peptide pheromone. Then, protein expression was confirmed by western blotting with goat anti-HIV p24 primary antibody and anti-goat secondary antibody. ELISA was also performed to confirm the antigenicity of the HIV-1 Gag antigen and to also quantify the antigen in the two Lactobacilli clones. Our results show that 1.5×109 CFU of L. plantarum+ pSIP409-GagM expressed 125μg of HIV-1 Gag and 1.9×109 CFU of L. fermentum+ pSIP411-GagM clones expressed 125μg of HIV-1 Gag respectively. In vitro digestion with pepsin, pancreatin and bile salts suggested that partial digestion of the probiotic vaccine candidates may occur when administered orally. Taken together, our probiotic HIV-1 vaccine candidates showed good prospects for further immunological analysis via animal trial

Reference gene selection for qPCR Is dependent on cell type rather than treatment in colonic and vaginal human epithelial cell lines

The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ΔCq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines after bacterial challenge

Anti-inflammatory effects of probiotic yogurt in inflammatory bowel disease patients

Background: Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation. Results: Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR-1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR-1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB. Conclusions: The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR-1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1 modulated the activation of the NF-kappaB through increased levels of TLR4 on the bladder cells and altered subsequent release of cytokines from urothelial cells. By influencing immunological factors such as TLR4, important in the process of fighting pathogens, lactobacilli could facilitate pathogen recognition and infection clearance. © 2012 Karlsson et al; licensee BioMed Central Ltd

The Pisum sativum SAD short-chain dehydrogenase/reductase: quinone reduction, tissue distribution, and heterologous expression

The pea (Pisum sativum) tetrameric short-chain alcohol dehydrogenase-like protein (SAD) family consists of at least three highly similar members (SAD-A, -B, and -C). According to mRNA data, environmental stimuli induce SAD expression (Brosché and Strid (1999) Plant Physiol 121: 479-487). The aim of this study was to characterize the SAD proteins by examining their catalytic function, distribution in pea, and induction in different tissues. In enzyme activity assays using a range of potential substrates, the SAD-C enzyme was shown to reduce one- or two-ring membered quinones lacking long hydrophobic hydrocarbon tails. Immunological assays using a specific antiserum against the protein, demonstrated that different tissues and cell types were shown to contain small amounts of SAD protein that was predominantly located within epidermal or sub-epidermal cells and around vascular tissue. Particularly high local concentrations were observed in the protoderm of the seed cotyledonary axis. Two bow-shaped rows of cells in the ovary and the placental surface facing the ovule also exhibited considerable SAD staining. UV-B irradiation led to increased staining in epidermal and sub-epidermal cells of leaves and stems. The different localization patterns of SAD suggest functions in both development and in responses to environmental stimuli. Finally, the pea SAD-C promoter was shown to confer heterologous wound-induced expression in Arabidopsis thaliana, which confirmed that the inducibility of its expression is regulated at the transcriptional level

Production of anti-viral vaccines using probiotic bacteria

The mucosal surfaces throughout the body are constantly exposed to microorganisms. The mucosal surfaces accommodate a large part of the body’s immune system. For effective vaccination, it is believed that direct immunization on mucosal surfaces will be more effective than the more conventional systemic immunization. Certain probiotic bacteria provide significant adjuvant effects that may be utilized for the desired immune response. Virus-like particles (VLPs) are complexes of viral proteins that without being infectious are efficient in mimicking the natural viral structures. While presenting the patterns of viral antigens, the VLPs have been shown to efficiently interact with dendritic cells and be effective in triggering the B and T-cell immunity. HIV-1 Gag is one of the most highly conserved structural antigens and contains several immunodominant T- and B-cell epitopes. Mosaic Gag protein matches 74% of 9-amino-acid potential epitopes in global Gag sequences thus maximizing the coverage of potential T-cell epitopes for a viral population.The current study aimed to develop probiotic strains of Lactobacillus plantarum NC8 and E. coli Nissle 1917 that produced recombinant mosaic HIV-1 Gag protein as VLPs.For the transient expression of the mosaic HIV-1 Gag protein (GagM), the gene-carrying expression vectors were transformed into the L. plantarum and in probiotic E.coli Nissle 1917. Protein expression of the GagM was shown to lead to the formation of the VLPs of the recombinant HIV-1 Gag proteins. This was confirmed through immunoblotting and transmission electron microscopy (TEM).Our study shows that probiotic lactobacteria can be developed to express HIV-1 Gag VLPs, which may be used for VLP production and potentially for vaccine delivery. Further evaluation of the concept is merited.Developvaccines@or