A novel formulation of Mtb72F DNA vaccine for immunization against tuberculosis.

OBJECTIVES: Mycobacterium tuberculosis (M. tuberculosis), an intracellular pathogen, causes 1.5 million deaths globally. Bacilli Calmette-Guérin (BCG) is commonly administered to protect people against M. tuberculosis infection; however, there are some obstacles with this first-generation vaccine. DNA vaccines, the third generation vaccines, can induce cellular immune responses for tuberculosis (TB) protection. In this study, optimized DNA vaccine (pcDNA3.1-Mtb72F) entrapped in poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) was used to achieve higher immunogenicity. MATERIALS AND METHODS: Plasmid Mtb72F was formulated in PLGA NPs using double emulsion method in the presence of TB10.4 and/or CpG as an adjuvant. Female BALB/c mice were immunized either with NP-encapsulated Mtb72F or naked Mtb72F with or without each adjuvant, using the BCG-prime DNA boost regimen. RESULTS: These NPs were approximately 250 nm in diameter and the nucleic acid and protein encapsulation efficiency were 80% and 25%, respectively. The NPs smaller than 200 nm are able to promote cellular rather than humoral responses. The immunization with the formulation consisting of Mtb72F DNA vaccine and TB10.4 entrapped in PLGA NPs showed significant immunogenicity and induced predominantly interferon-É£ (IFN-É£) production and higher INF-É£/interleukin-4 (IL-4) ratio in the cultured spleen cells supernatant. CONCLUSION: PLGA NPs loaded with Mtb72F DNA-based vaccine with TB10.4 could be considered as a promising candidate for vaccination against TB. These results represent an excellent initial step toward development of novel vaccine for TB protection.

Construction of Mtb72F Plasmid as a DNA Vaccine Candidate for Mycobacterium tuberculosis.

BACKGROUND: With one-third of the world's population infected, tuberculosis (TB) is one of the most common infectious diseases and a major public health problem, especially in developing countries. The efficacy of the BCG vaccine for controlling the disease in adults is poor. The development of an effective TB vaccine is a global objective. An effective tuberculosis vaccine should stimulate cellular immunity. DNA vaccines are a new generation of vaccines with the potential to achieve this goal. The aim of this study was to produce a DNA vaccine of Mtb72F. METHODS: mtb32C, mtb39, and mtb32N were cloned into pcDNA3.1 using restriction enzyme digestion and T4 DNA ligase. Colony-PCR and restriction enzyme digestion were performed to detect transformed bacteria. DNA sequencing confirmed the desired gene insertion into the vector. A Chinese hamster ovary (CHO) cell line was transfected with the recombinant plasmid and RT-PCR was performed to assess gene expression. RESULTS: Gel electrophoresis showed the expected amplified gene fragments of 429, 614, and 1200 base pairs (bps) for mtb32C, mtb32N, and mtb39, respectively. Enzyme digestion and gel electrophoresis showed the expected fragments, indicating the desired gene position and orientation in the recombinant plasmid. This finding was verified by DNA sequencing, and RT-PCR demonstrated gene expression in the CHO cell line. CONCLUSION: An Mtb72F DNA plasmid was successfully constructed. This plasmid may be a candidate for animal immunizations.

Study on imipenem resistance and prevalence of blaVIM1 and blaVIM2 metallo-beta lactamases among clinical isolates of Pseudomonas aeruginosa from Mashhad, Northeast of Iran.

BACKGROUND AND OBJECTIVES: The main cause of serious nosocomial infections is a Gram-negative pathogen known as Pseudomonas aeruginosa (P. aeruginosa). Carbapenems are widely used as an appropriate treatment for these infections, however resistance to these agents has been observed and is increasing. Metallo beta-lactamase (MBLs) enzyme is one of the main causes of resistance to carbapenem. In the current study the frequency and production of VIM1 and VIM2 by imipenem-resistant P. aeruginosa isolates of patients hospitalized in Imam Reza hospital were evaluated. MATERIALS AND METHODS: In this study, 131 clinical samples were collected from patients hospitalized in Imam Reza hospital in Mashhad during a 15-month period from May 2011 to November 2012. After verification of P. aeruginosa isolates, antibiotic resistance patterns of isolates were determined for 14 antibiotics by Kirby-Bauer standard disk diffusion according to the CLSI guidelines. Combined-disk test was used for phenotypic determination of MBLs-producing isolates and after DNA extraction, genotypic determination of VIM1 and VIM2 metallo beta-lactamase genes was carried out using Multiplex-PCR. RESULTS: Of 63 imipenem-resistant isolates (48.5%), 56 (88.8%) were MBL-producing in phenotypic assessments. Also amongst imipenem-resistant isolates, the frequency of VIM1 and VIM2 genes were 58.7 and 3.17%, respectively. CONCLUSION: The results of the current study along with the results of the other conducted studies in Iran in recent years demonstrate that the average resistance to imipenem in P. aeruginosa isolates was 51.3% which has increased in comparison with the results in 2006 (32.9%). It was also determined that the frequency of VIM1 gene was more than VIM2 gene. In phenotypic assessment by using CD method, 49.6% of isolates were determined as MBLs-producing. The sensitivity and specificity of this method were verified in comparison with the results of PCR test.

Shear bond strength and antibacterial effects of orthodontic composite containing TiO2 nanoparticles.

Nanofillers can reduce enamel demineralization without compromising physical properties of the composite. The aim of this study was to evaluate shear bond strength (SBS) and antibacterial effects of an orthodontic composite after adding titanium oxide (TiO2) nanoparticles. Light cure orthodontic composite paste (Transbond XT) was blended with TiO2 nanoparticles. A total of 30 extracted premolars were randomly allocated into two groups of 15. In order to bond brackets, Transbond XT adhesive and nanocomposite were used in each group, respectively. SBS of two groups were determined, and the adhesive remnant index (ARI) scores were assessed. A total of 45 composite discs specimen were prepared. Of the 45 discs, 30 discs were made from nanocomposite and tested for antibacterial properties immediately and 30 days after curing by direct contact test. The antibacterial properties of the remaining 15 discs that were made from the conventional composite were tested immediately after curing as control group. Student's t-test and chi-square tests were used to analyse the data with the significance level of 0.05. No significant difference was found between SBS of conventional and nanocomposites, 24 hours after curing (P = 0.58). Chi-square test showed that ARI scores of two groups were not significantly different after debonding (P = 0.69). Comparison of antibacterial effects between conventional and nanocomposite demonstrated significant difference between two groups, with nanocomposites having a higher antibacterial activity (P = 0.03). Colony count revealed no significant difference in bacterial growth immediately and 30 days after curing in nanocomposite group. Adding TiO2 nanoparticles to orthodontic composite enhances its antibacterial effects without compromising the SBS.

Comparative study of the antimicrobial effect of three irrigant solutions (chlorhexidine, sodium hypochlorite and chlorhexidinated MUMS).

AIM: To compare the antimicrobial effect of 2% chlorhexidine, 2.5% sodium hypochlorite and MUMS containing 2% chlorhexidine. MATERIALS AND METHODS: All of the above irrigants were examined on Enterococcus faecalis, Streptococcus mutans, Candida albicans, Lactobacillus casei and E. coli. A total of 0.5 CC of each solution and 0.5 CC of McFarland solution bacterium were added to each examination tube. After 15, 30 and 45 minutes, colony count was performed for each tube. The difference in the number of bacteria indicated the effect taken by disinfectant material. RESULTS: MUMS containing chlorhexidine showed the antimicrobial properties just like chlorhexidine's effect against E. coli, Streptococcus mutans, Candida albicans, Enterococcus faecalis and Lactobacillus casei in preventing these entire microorganisms to incubate. Sodium hypochlorite was not effective against Enterococcus faecalis and Candida albicans incubated in 15, 30 and 45 minutes and Enterococcus faecalis in 15 minutes. CONCLUSION: MUMS has antimicrobial properties similar to chlorhexidine. CLINICAL SIGNIFICANCE: As MUMS containing chlorhexidine can transfer chlorhexidine through its own surfactant around apical area and it can open the dentinal tubules by its own chelator for more penetration of chlorhexidine, it may be a choice for canal irrigation.