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Bacterial Lysates (bacterial + lysate)
Selected AbstractsSystemic Immunization with Unadjuvanted Whole Helicobacter pylori Protects Mice Against Heterologous ChallengeHELICOBACTER, Issue 6 2008Stacey N. Harbour Abstract Background:, Adjuvant-free vaccines have many benefits, including decreased cost and toxicity. We examined the protective effect of systemic vaccination with adjuvant-free formalin-fixed Helicobacter pylori or bacterial lysate and the ability of this vaccine to induce protection against heterologous challenge. Materials and Methods:, Mice were vaccinated subcutaneously with H. pylori 11637 lysate or formalin-fixed bacteria, with or without ISCOMATRIXTM adjuvant, then orally challenged with H. pylori SS1. Serum was taken prior to challenge to examine specific antibody levels induced by the vaccinations, and protection was assessed by colony-forming assay. Results:, Vaccination with H. pylori 11637 lysate or formalin-fixed bacteria delivered systemically induced significantly higher levels of Helicobacter -specific serum IgG than the control, unvaccinated group and orally vaccinated group. After heterologous challenge with H. pylori SS1, all vaccinated groups had significantly lower levels of colonization compared with unvaccinated, control mice, regardless of the addition of adjuvant or route of delivery. Protection induced by systemic vaccination with whole bacterial preparations, without the addition of adjuvants, was only associated with a mild cellular infiltration into the gastric mucosa, with no evidence of atrophy. Conclusions:, Subcutaneous vaccination using unadjuvanted formalin-fixed H. pylori has the potential to be a simple, cost-effective approach to the development of a Helicobacter vaccine. Importantly, this vaccine was able to induce protection against heterologous challenge, a factor that would be crucial in any human Helicobacter vaccine. Further studies are required to determine mechanisms of protection and to improve protective ability. [source] Dual-association of gnotobiotic Il-10,/, mice with 2 nonpathogenic commensal bacteria induces aggressive pancolitisINFLAMMATORY BOWEL DISEASES, Issue 12 2007Sandra C. Kim MD Abstract Background: Monoassociating gnotobiotic IL-10-deficient (,/,) mice with either nonpathogenic Enterococcus faecalis or a nonpathogenic Escherichia coli strain induces T-cell-mediated colitis with different kinetics and anatomical location (E. faecalis: late onset, distal colonic; E. coli: early onset, cecal). Hypothesis: E. faecalis and E. coli act in an additive manner to induce more aggressive colitis than disease induced by each bacterial species independently. Methods: Germ-free (GF) inbred 129S6/SvEv IL-10,/, and wildtype (WT) mice inoculated with nonpathogenic E. faecalis and/or E. coli were killed 3,7 weeks later. Colonic segments were scored histologically for inflammation (0 to 4) or incubated in media overnight to measure spontaneous IL-12/IL-23p40 secretion. Bacterial species were quantified by serial dilution and plated on culture media. Mesenteric lymph node (MLN) CD4+ cells were stimulated with antigen-presenting cells pulsed with bacterial lysate (E. faecalis, E. coli, Bacteroides vulgatus) or KLH (unrelated antigen control). IFN-, and IL-17 levels were measured in the supernatants. Results: Dual-associated IL-10,/, (but not WT) mice developed mild-to-moderate pancolitis by 3 weeks that progressed to severe distal colonic-predominant pancolitis with reactive atypia and duodenal inflammation by 7 weeks. NF-,B was activated in the duodenum and colon in dual-associated IL-10,/, × NF-,BEGFP mice. The aggressiveness of intestinal inflammation and the degree of antigen-specific CD4+ cell activation were greater in dual- versus monoassociated IL-10,/, mice. Conclusion: Two commensal bacteria that individually induce phenotypically distinct colitis in gnotobiotic IL-10,/, mice act additively to induce aggressive pancolitis and duodenal inflammation. (Inflamm Bowel Dis 2007) [source] Production of Polyclonal Antibodies to Potato virus X Using Recombinant Coat ProteinJOURNAL OF PHYTOPATHOLOGY, Issue 1 2010Noemi Cerovska Abstract Polyclonal antibodies to recombinant Potato virus X (PVX) coat protein (PVX-CP) were developed and their effectiveness determined in different ELISA protocols. The PVX-CP gene was amplified by reverse transcription-polymerase chain reaction, cloned and expressed in Escherichia coli. For immunization, the CP fractions from bacterial lysate were purified either by simple fractionation or by excision from sodium dodecyl sulphate gels. The PVX-CP was injected into rabbits for antibody production. The PVX-CP antibodies reacted in an indirect plate trapped antigen enzyme-linked immunosorbent assay and immunoblot assay and were useful for the detection of a broad spectrum of isolates of PVX. [source] Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionationELECTROPHORESIS, Issue 12 2008Matthew R. Richardson Abstract Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOMŽ IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins. [source] Molecular responses of Campylobacter jejuni to cadmium stressFEBS JOURNAL, Issue 20 2008Nadeem O. Kaakoush Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium. [source] Bifidobacterium animalis causes extensive duodenitis and mild colonic inflammation in monoassociated interleukin-10-deficient miceINFLAMMATORY BOWEL DISEASES, Issue 7 2009James P. Moran PhD Abstract Background: We recently showed that Bifidobacterium animalis is more prevalent within the colons of interleukin (IL)-10-deficient (,/,) mice than in wildtype (WT) animals colonized with the same specific pathogen-free (SPF) fecal contents. Here we tested the ability of this organism to cause T-cell-mediated intestinal inflammation by introducing it into germ-free (GF) IL-10,/, mice. Methods: GF IL-10,/, or WT mice were monoassociated with Bifidobacterium animalis subsp. animalis ATCC (American Type Culture Collection, Manassas, VA) 25527T or with B. infantis ATCC 15697T. Inflammation was measured by blinded histologic scores of the duodenum, cecum, and colon and by spontaneous secretion of IL-12/IL-23 p40 from colonic explants. Bacterial antigen-specific CD4+ mesenteric lymph node (MLN) T-cell recall responses were measured in response to antigen-presenting cells (APC) pulsed with bacterial lysates. Results:B. animalis caused marked duodenal inflammation and mild colitis in monoassociated IL-10,/, mice, whereas the intestinal tracts of WT animals remained free of inflammation. B. infantis colonization resulted in mild inflammation in the duodena of IL-10,/, mice. CD4+ MLN T cells from B. animalis monoassociated IL-10,/, mice secreted high levels of IFN-, and IL-17 in response to B. animalis lysate. B. animalis equally colonized the different intestinal regions of WT and IL-10,/, mice. Conclusions:B. animalis, a traditional probiotic species that is expanded in experimental colitis in this model, induces marked duodenal and mild colonic inflammation and TH1/TH17 immune responses when introduced alone into GF IL-10,/, mice. This suggests a potential pathogenic role for this commensal bacterial species in a susceptible host. (Inflamm Bowel Dis 2009) [source] A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite SynthesisADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 13 2009Roland Weis Abstract Innovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self-sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co-solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4,-hydroxydiclofenac and 6-hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s. [source] Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation,pit-stop semi-nested PCR procedureJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2000J. Theron A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml,1 was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples. [source] Innate immune responses of gingival epithelial cells to nonperiodontopathic and periodontopathic bacteriaJOURNAL OF PERIODONTAL RESEARCH, Issue 6 2007S. Ji Background and Objective:, We have previously reported different susceptibilities of periodontopathic and nonperiodontopathic bacteria to antimicrobial peptides and phagocytosis by neutrophils. Differences between the two groups of bacteria may exist also in their ability to induce immune responses from the host. Therefore, we evaluated the effects of various oral bacteria on innate immune responses by gingival epithelial cells. Material and Methods:, HOK-16B cells were cocultured with live or lysed nonperiodontopathic (n = 3) and periodontopathic (n = 5) bacterial species. The levels of human beta defensin-1, -2 and -3, and of the cathelicidin, LL-37, were examined by real-time reverse transcription-polymerase chain reaction, and the accumulated interleukin-8 and interleukin-1, were measured by enzyme-linked immunosorbent assay. Results:, Nonperiodontopathic bacteria up-regulated some antimicrobial peptides without affecting the levels of cytokines. In the periodontopathic group, the orange-complex bacteria induced antimicrobial peptides and interleukin-8 efficiently, but the red-complex bacteria often demonstrated suppressive effects. In contrast to live bacteria, bacterial lysates had no suppressive effects. In addition, some bacterial lysates demonstrated a reduced ability to induce antimicrobial peptides compared with live bacteria. Conclusion:, The nonperiodontopathic, the orange-complex, and the red-complex bacteria had different effects on the innate immune responses from gingival epithelial cells, which may affect the outcome of their host,microbial interaction in gingival sulcus. [source] | ||||||||||||||||