Activation of toll-like receptors 2 by high-mobility group box 1 in monocytes from patients with ischemic stroke

Background: Stroke is a leading cause of death all around the world, and ischemic stroke is considered to be the most common stroke type. Toll-like receptors [TLRs] are important molecules for detection of both pathogen invasion and tissue damage. In this regard, the purpose of this study was to assess the expression level of TLR2 on monocytes in patients with ischemic stroke and to evaluate the expression change profile following high-mobility group box 1 [HMGB1] stimulation

Methods: A total of 30 patients with ischemic stroke were enrolled from November 2013 to September 2014. The real-time PCR and ELISA assays were applied to detect the concentrations of TLR2 mRNAs

Results: TLR2 expression was found to be increased in patients with ischemic stroke, as compared to the healthy control group [P<0.001]. Also, anti-TLR2 antibodies were able to decrease the expression levels of IL-17, IL-6 and IL-33. This result implies that the enhanced TLR2 pathway and Th17 cell polarization may be due to HMGB1 stimulation in ischemic stroke

Conclusion: Further clinical studies are needed for development of a new treatment strategy to inhibit the HMGB1 pathway, thus preventing the inflammation in ischemic stroke patients

Protective properties of nontoxic recombinant exotoxin A [domain I-II] against pseudomonas aeruginosa infection

Antibiotic resistance and the need for long-term treatments especially for chronic infections necessitate the development of a vaccine against Pseudomonas aeruginosa infection. In this study, recombinant exotoxin A [domains I and II], [ExoA I-II] protein was expressed, purified and its immunological characteristics were evaluated in a mouse model. The genomic DNA was extracted from P. aeruginosa strain PAO1. The DNA encoding for domains I and II of exotoxin A was amplified by PCR and cloned into the pET22b expression vector. The construct was then transformed into E. coli BL21 and the protein expression was evaluated by the SDS-PAGE method. The Ni-NTA affinity chromatography was used for recombinant protein purification. Mice were then immunized subcutaneously on day 0, 21, 42 and 72 with exotoxin A [Domains I, II]. Antibody production was evaluated by the ELISA method. The immunized and control group mice were exposed to an approximate 2 x LD50 [7.5 x 10[7] CFU] of clinical strain of mucoid P. aeruginosa. Sequencing of the cloned gene showed that the sequence of ExoA one-two gene was in accordance with ExoA I-II from P. aeruginosa PAO1. SDS-PAGE analysis indicated the expression of recombinant protein with a molecular weight of 45 KDa. Vaccination with ExoA I-II produced a significant amount of specific IgG antibodies in mice. Also immunization of mice with ExoA one-two increased survival times against intra-peritoneal challenge with an approximate 7.5 x 10[7] CFU [2 x LD50] of clinical strain of P. aeruginosa. Results of this study suggested that recombinant ExoA I-II is a highly immunogenic protein which can be used as a new vaccine candidate against P. aeruginosa

Cloning, expression and characterization of recombinant exotoxin A-flagellin fusion protein as a new vaccine candidate against pseudomonas aeruginosa infections

Background: infections due to Pseudomonas aeruginosa are among the leading causes of morbidity and mortality in patients who suffer from impaired immune responses and chronic diseases such as cystic fibrosis. At present, aggressive antibiotic therapy is the only choice for management of P. aeruginosa infections, but emergence of highly resistant strains necessitated the development of novel alternative therapeutics including an effective vaccine. Several P. aeruginosa antigens have been tested for vaccine development, including lipopolysaccharide alone, polysaccharides alginate, extracellular proteins, exotoxin A [exo A] and killed whole cell. However, none of them are currently available clinically

Methods: in this research, recombinant exoA-flagellin [fliC] fusion protein as a cocktail antigen was expressed and purified and its antigenic characteristics were evaluated

Results: expression of recombinant fusion protein by E. coli using pET22b vector resulted in production of exoA-fliC fusion protein in high concentration. Based on Western-blotting results, recombinant fusion protein showed a good antigenic interaction with sera from patients with various P. aeruginosa infections

Conclusion: these results suggested that recombinant exoA-fliC fusion protein can be produced in the laboratory, and tested as a candidate vaccine in P. aeruginosa infections. Iran. Biomed. J. 17 [1]: 1-7, 2013