In vitro assessment of Thimerosal cytotoxicity and antimicrobial activity.

BACKGROUND: Thimerosal (Merthiolate) is a well-known preservative used in pharmaceutical products, the safety of which was a matter of controversy for decades. Thimerosal is a mercury compound, and there is a debate as to whether Thimerosal exposure from vaccination can contribute to the incidence of mercury-driven disorders. To date, there is no consensus on Thimerosal safety in Vaccines. In 1977, a maximum safe dose of 200 µg/ml (0.5 mM) was recommended for Thimerosal by the WHO experts committee on biological standardization. Up-to-date guidelines, however, urge national control authorities to establish their own standards for the concentration of vaccine preservatives. We believe such safety limits must be studied at the cellular level first. The present study seeks a safe yet efficient dose of Thimerosal exposure for human and animal cells and control microorganism strains. METHODS: The safety of Thimerosal exposure on cells was analyzed through an MTT cell toxicity assay. The viability of four cell types, including HepG2, C2C12, Vero Cells, and Peripheral blood mononuclear cells (PBMCs), was examined in the presence of different Thimerosal concentrations and the maximum tolerable dose (MTD) and the half maximal inhibitory concentration (IC50) values for each cell line were determined. The antimicrobial effectiveness of Thimerosal was evaluated on four control strains, including Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Aspergillus brasiliensis, to obtain the minimum inhibitory concentration (MIC) of Thimerosal. The MIC test was performed in culture media and under optimal growth conditions of microorganisms in the presence of different Thimerosal concentrations. RESULTS: The viability of all examined cell lines was suppressed entirely in the presence of 4.6 µg/ml (12.5 µM) of Thimerosal. The MTD for HepG2, C2C12, PBMC, and Vero cells was 2, 1.6, 1, and 0.29 µg/ml (5.5, 4.3, 2.7 and 0.8 µM), respectively. The IC50 of Thimerosal exposure for HepG2, C2C12, PBMC, and Vero cells was 2.62, 3.17, 1.27, and 0.86 µg/ml (7.1, 8.5, 3.5 and 2.4 µM), respectively. As for antimicrobial effectiveness, the growth capability of Candida albicans and Staphylococcus aureus was suppressed entirely in the presence of 6.25 µg/ml (17 µM) Thimerosal. The complete growth inhibition of Pseudomonas aeruginosa in culture media was achieved in 100 µg/ml (250 µM) Thimerosal concentration. This value was 12.5 µg/ml (30 µM) for Aspergillus brasiliensis. CONCLUSION: According to our results Thimerosal should be present in culture media at 100 µg/ml (250 µM) concentration to achieve an effective antimicrobial activity. We showed that this amount of Thimerosal is toxic for human and animal cells in vitro since the viability of all examined cell lines was suppressed in the presence of less than 5 µg/ml (12.5 µM) of Thimerosal. Overall, our study revealed Thimerosal was 333-fold more cytotoxic to human and animal cells as compared to bacterial and fungal cells. Our results promote more study on Thimerosal toxicity and its antimicrobial effectiveness to obtain more safe concentrations in biopharmaceuticals.

Diphtheria toxoid nanoparticles improve learning and memory impairment in animal model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder involving memory. The present study aimed at evaluating the effects of encapsulated diphtheria toxoid (DT) on behavioral learning impairment, and XBP1 mRNA splicing in AD. METHODS: A DT-loaded nanoparticle (NP) carrier was prepared using the ionic gelation method. Sixty-three rats were divided into nine groups: (1) healthy, (2-4) sham, and (5-9) AD models: (5) AD was induced by intracerebroventricular injection of amyloid beta (Aß) 1-42. (6) The rats received a subcutaneous diphtheria vaccine only 28 days before Aß injection. (7) The rats received an intranasal diphtheria vaccine, in group 8, induced by administering empty chitosan NPs. 9) it was induced by administering chitosan NPs carrying DT. Morris water maze (MWM) test was used to examine the animals' learning and memory. Also, X-box binding protein 1 (XBP-1) mRNA gene splicing was studied in the hippocampus by reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: For the first time, chitosan NPs were prepared with an average diameter size of 40 nm and the effectiveness of approximately 70% during DT encapsulation. In comparison with the healthy group, the AD models exhibited significant impairment of learning and memory (P < 0.05), while DT-administrated animals showed significant improvements in learning and memory impairment (P < 0.05). XBP-1 mRNA gene splicing was only detected in an untreated AD group, while encapsulated DT completely inhibited splicing. CONCLUSION: The therapeutic effects of DT chitosan NPs against learning and memory impairment were observed in this study, and XBP1 mRNA splicing was reported in the animal models.

A new triple chimeric protein as a high immunogenic antigen against anthrax toxins: theoretical and experimental analyses.

Background: Anthrax is a zoonotic disease caused by Bacillus anthracis and it can be deadly in 6 days. Considerable efforts have been conducted toward developing more effective veterinary and human anthrax vaccines because these common vaccines have several limitations. B. anthracis secretes a tripartite toxin, comprising protective antigen (PA), edema factor (EF), and lethal factor (LF). Several studies have shown important role of PA in protection of anthrax. LF and EF induce production of toxin neutralizing antibodies too. PA in fusion form with LF/EF has synergistic effects as a potential subunit vaccine. Methods: In this study, for the first time, a triple chimeric protein called ELP was modeled by fusing three different domains of anthrax toxic antigens, the N-terminal domains of EF and LF, and the C-terminal domain of PA as a high immunogenic antigen using Modeller 9.19 software. Immunogenicity of the ELP was assessed in guinea pigs using enzyme-linked immunosorbent assay (ELISA) test and MTT assay. Results: Theoretical studies and molecular dynamics (MD) simulation results suggest that the ELP model had acceptable quality and stability. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified ELP, its domains, and PA were matched with their molecular size and confirmed by western blotting analysis. In the immune guinea pigs, antibody was produced against all of the ELP domains. It was observed that ELP induced strong humoral response and could protect murine macrophage cell line (RAW 264.7 cells) against anthrax lethal toxin (LeTx). Conclusions: ELP chimeric antigen could be considered as a high immunogenic antigen.

In vitro evaluation of cross-strain inhibitory effects of IgY polyclonal antibody against H. pylori.

The present study aimed to evaluate in vitro cross-strain inhibitory effects of IgY polyclonal antibody on both growth and urease enzyme of four local strains of H. pylori. Leghorn chickens were immunized with whole cells of four different strains of H. pylori, separately. Rising of specific IgY was detected by ELISA. The IgY purified using polyethylene glycol method and the purity was evaluated by SDS-PAGE and Western blotting. Each strain was treated with its own-specific and also other strain-specific IgYs. The strain-specific IgY could inhibit the growth of specific strains by 49-72% and also other different strains of H. pylori by 29-86%. Our findings revealed that strain-specific IgY could inhibit urease activity of its own by 64-72% and other different strains by 49-79%. These findings confirmed strain-specific and also cross-strain inhibitory effects of the IgY polyclonal antibody on both growth and urease activity of H. pylori.

Cloning and Expression of S1 Subunit of Pertussis Toxin in Escherichia coli.

Bordetella pertussis is a gram negative bacterium that causes respiratory tract infection in human (whooping cough). Pertussis toxin (PT) is the main component of current acellular pertussis vaccine and the S1 (subunit1) is the main immunogenic part of it. Thus, S1 has been the target of many studies as a potent candidate of acellular vaccine against Bordetella pertussis, lacking the side effects of whole cell based ones. S1 gene was amplified and inserted in three expression vectors including pET-14b, pET-22b(+) and pAED4. The possibility and level of expression of these constructs were investigated in BL21 (DE3) strain of Escherichia coli (E.coli) as expression host. The highest expression was in pET-22b(+)-S1. Best expression achieved 6 hr post induction with 0.2 mM IPTG in LB broth containing ampicillin, at 30°C with shaking (250 rpm). Recombinant S1 protein was observed in two distinct separated proteins with 28 and 31 kDa estimated molecular weight. In spite of toxicity of PT and S1 in the E.coli, considerable amount of S1 was expressed in E.coli. Two rS1 bands were detected on SDS-PAGE. Both were confirmed as S1 in western blot with specific monoclonal and polyclonal antibodies against pertussis toxin. Appearance of two distinct bands could be the result of leader peptidase activity or nonspecific peptidase from E.coli on recombinant S1. As the recombinant S1 is a suitable antigen for studies as a candidate acellular vaccine or development of ELISA for detection of Bordetella pertussis, further studies are underway.