[Preliminary evaluation of immunogenicity and protective effect of multicomponent recombinant protein vaccine EPRHP014 against tuberculosis].

Objective: To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB. Methods: Three full-length Mycobacterium (M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M. tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results: Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance (P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group (P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group (P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group (P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group (P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups (P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant (P>0.05). Conclusion: Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Comparison of toluidine blue-mediated photodynamic therapy and conventional scaling treatment for periodontitis in rats.

BACKGROUND AND OBJECTIVE: Periodontitis is a disease caused by bacterial infection accompanied with the inflammation of connected tissues and resorption of alveolar bone. The aim of this study was to investigate the in vivo photosensitization of periodontal bacteria in rats and to compare its efficacy with that of routine scaling and root planing. MATERIAL AND METHODS: Periodontitis was developed by submerging ligatures at the subgingival region of maxillary molars in 16 rats. Six weeks later, the infection sites were treated either with 1 mg/mL of toluidine blue plus 12 J/cm2 red laser irradiation, or by routine scaling and root planing. The therapeutic efficacy was assessed by evaluating the reduction of total bacterial flora and histological changes of periodontal tissues. RESULTS: Significant reduction of total bacterial flora was achieved by both photodynamic therapy and conventional therapy. The signs of inflammation that accompanied periodontitis, such as redness, increased plaque index and gingival index values, bleeding on probing and inflammatory cell infiltration, were greatly reduced without any obvious detectable injury to host tissues. Both photodynamic therapy and conventional therapy showed similar therapeutic results. CONCLUSION: Toluidine blue-mediated photodynamic therapy could effectively treat periodontitis in vivo and has high potential in clinical application.

Real-time detection of dental calculus by blue-LED-induced fluorescence spectroscopy.

Successful periodontal therapy requires sensitive techniques to discriminate dental calculus from healthy teeth. The aim of the present study was to develop a fluorescence-based procedure to enable real-time detection and quantification of dental calculus. Thirty human teeth--15 teeth with sub- and supragingival calculus and 15 healthy teeth--covered with a layer of physiological saline solution or blood were illuminated by a focused blue LED light source of 405 nm. Autofluorescence spectra recorded along a randomly selected line stretching over the crown-neck-root area of each tooth were utilized to evaluate a so called calculus parameter R, which was selected to define a relationship between the integrated intensities specific for healthy teeth and for calculus in the 477-497 nm (S(A)) and 628-685 nm (S(B)) wavelength regions, respectively. Statistical analysis was performed and a cut-off threshold of R=0.2 was found to distinguish dental calculus from healthy teeth with 100% sensitivity and specificity under various experimental conditions. The results of the spectral evaluation were confirmed by clinical and histological findings. Automated real-time detection and diagnostics for clinical use were implemented by a corresponding software program written in Visual Basic language. The method enables cost-effective and reliable calculus detection, and can be further developed for imaging applications.

Histological evaluation of the safety of toluidine blue-mediated photosensitization to periodontal tissues in mice.

Our previous in vitro study has shown that toluidine blue (TB)-mediated lethal photosensitization of periodontal pathogens (PPs) from periodontal patients is possible. The purpose of this study was to investigate whether TB-mediated photosensitization exerted damaging effects on periodontal tissues in mice. Twenty-four mice were randomly divided into four groups; the experimental photodynamic therapy (PDT) group was treated with 1 mg/ml TB and light irradiation (60 J/cm(2), 635 nm, 337 s). Those in control groups were subjected to 140 J/cm(2) laser irradiation alone or to 2.5 mg/ml TB alone or received neither TB nor light exposure. All the mice were killed 72 h after they had been subjected to PDT, and periodontal tissue samples were taken for histological examination. During the 72 h observation period, no mice showed any distress. No necrotic or inflammatory changes were found in the gingiva, dentin, dental pulp or alveolar bone of the mice in any of the groups in this study. The results suggest that TB-mediated PDT is a safe antimicrobial approach for the treatment of periodontopathy without damaging effects to adjacent normal tissues.