Effect of electron-beam irradiation on antimicrobial, antibiofilm activity, and cytotoxicity of mouth rinses.

BACKGROUND: Oral health diseases are common in all regions of the world. Mouth rinses are widely used generally by population as a port of daily oral care regimen. In addition to antimicrobial activity, mouth rinses possess certain cytotoxic effects. Electron-beam (E-beam) radiation is a form of ionizing energy known to induce structural, physical, and chemical changes in irradiated products. In this study, the modulatory effects of E-beam in irradiated mouth rinses were evaluated for its biological activities. MATERIALS AND METHODS: The antimicrobial activities of nonirradiated and irradiated mouth rinses were evaluated for its antimicrobial and antibiofilm activities against oral pathogens, Enterococcus faecalis, Streptococcus mutans, Staphylococcus aureus, and Candida albicans. The antimicrobial activity was evaluated by disc diffusion method and antibiofilm activity was evaluated by O'Toole method. The cytotoxicity was evaluated against human gingival fibroblast (HGF) cells by 3-(4, 5 Dimethythiazol-yl)-2,5-Diphenyl-tetrazolium bromide assay. RESULTS: Colgate Plax (CP) exhibited the antimicrobial activity against the tested pathogens, and a significant (P< 0.05) increase was observed against S. aureus at 750 Gy irradiation. Further, CP significantly (P< 0.05) suppressed S. mutans, S. aureus, and C. albicans biofilm. Listerine (LS) inhibited S. mutans and C. albicans biofilm. Whereas irradiated CP and LS significantly (P< 0.05) suppressed the biofilm formed by oral pathogens. The suppression of biofilm by irradiated mouth rinses was dose- and species-dependent. There was no significant (P > 0.05) difference in the cytotoxicity of irradiated and nonirradiated mouth rinses on HGF cells. However, an increased percentage viability of HGF cells was observed by mouth rinses irradiated at 750 Gy.xs CONCLUSION: The E-beam irradiation enhanced the antibiofilm activity of mouth rinses without modifying the cytotoxicity.

Antimicrobial activity and stability of electron beam irradiated dental irrigants.

BACKGROUND: The electron beam (e-beam) radiation is considered as an effective means of sterilization of healthcare products as well as to induce the structural changes in the pharmaceutical agents/drug molecules. In addition to structural changes of pharmaceutical it also induces the formation of low molecular weight compounds with altered microbiological, physicochemical and toxicological properties. Among the several known medicaments, sodium hypochlorite (NaOCl) and chlorhexidine digluconate (CHX) are used as irrigants in dentistry to kill the pathogenic microorganisms like Enterococcus faecalis, Staphylococcus aureus, Streptococcus mutans and Candida albicans inhabiting the oral cavity. OBJECTIVES: The aim of this study was to evaluate the antimicrobial activity and stability of e-beam irradiated dental irrigants, NaOCl and CHX. MATERIALS AND METHODS: Two dental irrigants NaOCl (1.25% and 2.5%) and CHX (1% and 2%) were exposed to various doses of e-beam radiation. The antimicrobial activities of e-beam irradiated irrigants were compared with the non-irradiated (control) irrigants against E. faecalis, S. aureus, S. mutans and C. albicans by disc diffusion method. Following the storage, physico-chemical properties of the irrigants were recorded and the cytotoxic effect was evaluated on human gingival fibroblast cells. RESULT: The irrigants, 1.25% NaOCl and 1% CHX showed significantly increased antimicrobial activity against both E. faecalis, (16+0.0) and S. aureus (25+0.0) after irradiation with 1 kGy e-beam. Whereas, 2.5% NaOCl and 2% CHX showed slightly increased antimicrobial activity only against S. aureus (28+0.0). The significant difference was noticed in the antimicrobial activity and cytotoxicity of irradiated and non-irradiated irrigants following the storage for 180 d at 4(0)C. CONCLUSION: The e-beam irradiation increased the antimicrobial activity of irrigants without altering the biocompatibility.

Melatonin plus exercise-based neurorehabilitative therapy for spinal cord injury.

Spinal cord injury (SCI) is damage to the spinal cord caused by the trauma or disease that results in compromised or loss of body function. Subsequent to SCI in humans, many individuals have residual motor and sensory deficits that impair functional performance and quality of life. The available treatments for SCI are rehabilitation therapy, activity-based therapies, and pharmacological treatment using antioxidants and their agonists. Among pharmacological treatments, the most efficient and commonly used antioxidant for experimental SCI treatment is melatonin, an indolamine secreted by pineal gland at night. Melatonin's receptor-independent free radical scavenging action and its broad-spectrum antioxidant activity makes it an ideal antioxidant to protect tissue from oxidative stress-induced secondary damage after SCI. Owing to the limitations of an activity-based therapy and antioxidant treatment singly on the functional recovery and oxidative stress-induced secondary damages after SCI, a melatonin plus exercise treatment may be a more effective therapy for SCI. As suggested herein, supplementation with melatonin in conjunction with exercise not only would improve the functional recovery by enhancing the beneficial effects of exercise but would reduce the secondary tissue damage simultaneously. Finally, melatonin may protect against exercise-induced fatigue and impairments. In this review, based on the documented evidence regarding the beneficial effects of melatonin, activity-based therapy and the combination of both on functional recovery, as well as reduction of secondary damage caused by oxidative stress after SCI, we suggest the melatonin combined with exercise would be a novel neurorehabilitative strategy for the faster recovery after SCI.

Evaluation of an immunodot test to manage white spot syndrome virus (WSSV) during cultivation of the giant tiger shrimp Penaeus monodon.

A monoclonal antibody-based immunodot test was compared to a polymerase chain reaction (PCR) assay for managing white spot syndrome virus (WSSV) on shrimp farms at Kundapur and Kumta situated in Udupi and Uttar Kannada Districts, respectively, of Karnataka on the west coast of India. Of 12 grow-out farms in Kundapur, 6 (F1 to F6) yielded shrimp samples that were negative for WSSV by both immunodot test and 1-step PCR from stocking to successful harvest. Samples from the other 6 farms (F7 to F12) were positive for WSSV by both immunodot test and 1-step PCR at various times post stocking, and their crops failed. In the 2 farms at Kumta (F13, F14), immunodot and 1-step PCR results were both negative, and harvests were successful. In contrast to 1-step PCR results, farms F5, F6, F13, and F14 gave positive results for WSSV by 2-step PCR, and they were successfully harvested at 105 d post stocking. Our results indicate that an inexpensive immunodot assay can be used to replace the more expensive 1-step PCR assay for disease monitoring.

Evaluation of non-specific immune components from the skin mucus of olive flounder (Paralichthys olivaceus).

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish under intensive aquaculture and at prolonged low temperatures from relevant infections due to inadequate adaptive immune responses. In the present study we aimed to understand the mucosal immunity of an economically important mariculture fish, olive flounder (Paralichthys olivaceus) by evaluating the immune components from its skin mucus. The activities of lysozyme (233.33+/-171.82 units mg(-1)), trypsin-like protease (42.84+/-1.249 units mg(-1)), alkaline phosphatase (0.376+/-0.005 units mg(-1)) and esterase (0.170+/-0.006 units mg(-1)) were detected in the skin mucus. Transferrin was identified by MALDI-TOF/MS analysis. ELISA and immunoblot assays using anti-flounder IgM monoclonal antibody showed the presence of a significant level (1.80+/-0.001, n=3) of monomer immunoglobulin M (IgM) with approximate molecular weight of 160 and 25 kDa under non-denaturing and denaturing states, respectively. Skin mucus showed strong antibacterial activity against tested fish pathogenic bacteria. In addition, skin mucus successfully agglutinated (HA titre 2(8)), but completely failed to haemolyse, rabbit erythrocytes. In conclusion, the major immune components of the skin mucus, identified in the present study, are possibly involved in the broad spectrum non-specific immunity of olive flounder.

Immunoproteomic analysis of capsulate and non-capsulate strains of Lactococcus garvieae.

A comparative immunoproteomic study was carried out to investigate the immunogenicity of capsulate (KG9408) and non-capsulate (NSS9310) strains of Lactococcus garvieae. Immunoblot assays, following two-dimensional gel electrophoresis (2-DE) for L. garvieae strains, revealed a significant difference between anti-capsulate and anti-non-capsulate rabbit sera with respect to the number and antigenicity of antigenic spots. Anti-capsulate and anti-non-capsulate rabbit sera reacted with an average of 72 and 127 antigenic spots, respectively. The strong reaction of anti-non-capsulate sera with elongation factor (EF)-G and -Tu, and GMP synthase, of the L. garvieae strains identifies these as specific major antigens. This study clearly demonstrates the differences in 2-DE immunoblot profiles between the capsulate and non-capsulate strains of L. garvieae. These differences may be the reason for variations in immunogenicity between capsulate and non-capsulate strains. Glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, arginine deaminase and ornithine carbamoyltransferase were identified from the 2-DE immunoblot profiles of both strains. Therefore, these common antigens are potential markers for the development of vaccines against L. garvieae, irrespective of strain. Immunoproteomics, a powerful tool for studying antigens at the proteomic level, allowed a comparative investigation of the immunogenicity of capsulate and non-capsulate strains of L. garvieae for vaccine development.

Application of immunoproteomics in developing a Streptococcus iniae vaccine for olive flounder (Paralichthys olivaceus).

Streptococcus iniae is the major etiological agent of streptococcosis, which is responsible for hemorrhagic septicemia in fish, particularly olive flounder (Paralichthys olivaceus). In the present study, we sought to understand the pathogenicity and immunogenicity of S. iniae in order to develop a vaccine for streptococcosis. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting, was employed to investigate the pathogenicity and immunogenicity of two S. iniae isolates, Jeju-13 and Jeju-45, in olive flounder. The virulence of Jeju-13 was moderate whereas that of Jeju-45 was high. A vaccination trial with formalin-killed Jeju-45 demonstrated relatively low protection against the homologous isolate compared with the heterologous isolate. A significant difference in the secretion of extracellular products (ECPs) was noticed between the two S. iniae isolates. ECP antigens were highly immunogenic compared to those from whole cell lysates as determined by 2-DE immunoblot assay of Jeju-13 and Jeju-45 anti-sera collected from post-challenge survival fish. Furthermore, there were differences in the appearance of antigenic spots on 2-DE immunoblot profiles of ECPs of the respective sera. Interestingly, the mixture of killed-cells and concentrated ECPs from Jeju-45 led to significant protection against the homologous isolate of S. iniae in olive flounder. The present study demonstrates the usefulness of immunoproteomics in understanding the pathogenicity of S. iniae to aid the development of a vaccine for fish streptococcosis.

Partial two-dimensional gel electrophoresis (2-DE) maps of Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408.

To examine the proteomes of 2 important causative agents of fish streptococcosis, Streptococcus iniae ATCC29178 and Lactococcus garvieae KG9408, we used 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry to generate 2-DE maps of these type strains. Silver-stained 2-DE gels of S. iniae ATCC29178 and L. garvieae KG9408 revealed approximately 320 and 300 spots, respectively, and immobilized pH gradient strips (13 cm, pH 4 to 7) revealed that the majority of the detected spots were concentrated in the pH range of 4.5 to 5.5. The spots were randomly selected from the 2-DE profiles and identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry. The majority of the identified proteins were functionally related to energy and carbohydrate metabolism (e.g. enolase ATPase, glyceraldehyde-3-phosphate dehydrogenase) or translation and translocation (e.g. elongation factor G, elongation factor Tu, DNA-directed RNA polymerase alpha chain). These data, along with our partial 2-DE maps of S. iniae ATCC29178 and L. garvieae KG9408, may help suggest antigenic proteins for the development of effective diagnostic tools and vaccines against S. iniae and L. garvieae.

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf).

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.

Efficacy of protein A-HRP in an immunological study of black rockfish (Sebastes schlegeli Higendorf) humoral immune responses.

The efficacy of protein A-horse radish peroxidase (HRP), as compared to that of mouse polyclonal antibody raised against purified Ig, in detection of black rockfish (Sebastes schlegeli Higendorf) immunoglobulin (Ig) was examined. Protein A affinity chromatography successfully purified Ig from black rockfish serum; the purified-Ig could be visualised as two protein bands (MW 70 and 25kDa) following resolution with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. In SDS-PAGE immunoblot profiles of the purified-Ig, the mouse polyclonal antibody recognised both the light chain and heavy chains of rockfish Ig, whereas protein A-HRP immunostained only the heavy chain of rockfish Ig. These results suggest that protein A-HRP may be used to detect rockfish antibody-antigen complexes in immunoassays. In a 2-DE immunoblot assay for exploring antigenic profiles of Lactococcus garvieae KG9408, protein A-HRP successfully detected specific antibodies to antigenic proteins of L. garvieae in the rockfish Ig. In addition, enzyme linked immunosorbent assay (ELISA) showed a high correlation between the results obtained for positivity of L. garvieae when protein A-HRP and the mouse polyclonal antibody-was used to analyse samples from 25 diseased rockfish. These results collectively indicate that protein A-HRP has a high affinity for Ig, and may be useful for new investigations into the humoral immune responses of rockfish.

Comparison of proteome and antigenic proteome between two Neospora caninum isolates.

This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.

Application of proteomics for comparison of proteome of Neospora caninum and Toxoplasma gondii tachyzoites.

Protein profiles of two isolates of Neospora caninum (KBA-2 and JPA1) and Toxoplasma gondii RH strain were investigated by proteomic approach. Approximately, 78% of protein spots on two-dimensional gel electrophoresis (2-DE) profiles and 80% of antigen spots on 2-DE immunoblotting profiles were exhibited to share the same pI and M(r) between KBA-2 and JPA1 of N. caninum. On the other hand, a total of 30 antigen spots of T. gondii were recognized on 2-DE immunoblotting profile using rabbit antiserum against N. caninum KBA-2. A number of homologue proteins, such as heat shock protein 70, tubulin alpha- and beta-chain, putative protein disulfide isomerase, actin, enolase and 14-3-3 protein homologue are believed as the conserved proteins in both N. caninum and T. gondii. On the contrary, NcSUB1, NcGRA2 and NCDG1 (NcGRA7) might be the species-specific proteins for N. caninum tachyzoites. The present study showed that the high degree of similarity between N. caninum isolates (KBA-2 and JPA1), whereas large differences between N. caninum and T. gondii were noticed by proteome comparisons.