Plasma proteome responses in zebrafish following λ-carrageenan-Induced inflammation are mediated by PMN leukocytes and correlate highly with their human counterparts.

Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant (p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.

Effects of photobiomodulation on sport performance in swimming para-athletes - a case series.

This study evaluates photobiomodulation (PBM) on cardio-respiratory function and swimming performance in parathletes. Ten swimming parathletes were tested before PBM, after PBM and placebo irradiation applied on upper musculature. After warmup, the parathletes rested for 1 min, and heart rate was recorded. Three sessions of 50 m free style swimming at maximum effort, with 5-min interval were performed and time, peak and recovery heart rate were recorded. After 1 week, biceps, deltoid, and trapezius received 108 J of energy from an LED array or sham-irradiation in a crossover study. After another week, the same protocol was repeated. All athletes improved time in 50 ms swimming. On average, time decreased 4 s after PBM and 1.5 s after placebo. Also, the peak heartbeat was 10% lower after PBM. A muscular pre-conditioning using PBM with an infrared LED could modulate upper musculature and cardio-respiratory function, leading tobetter swimming performance in parathletes.

Melanin pigmented gingival tissue impairs red-light lateral scattering for antimicrobial photodynamic therapy.

Gingival melanin pigmentation is present in many African and Oriental descendant people and its occurrence in patients may interfere with the absorption and scattering of therapeutic doses of light. Antimicrobial photodynamic therapy (aPDT) is used as an adjunctive treatment for periodontitis and light irradiation may be impaired by tissue size and its melanin content. The aim of this clinical study was to measure the red-light attenuation in gingival tissue naturally pigmented with melanin. Ten patients with melanized gingival tissue were selected and irradiated by 100 mW red laser. The patients were photographed in frontal and incisal regions with a T2i camera (Canon, Japan) with 100 mm macro lens, 35 mm focal length, aperture f22, 1/100 shutter speed and ISO 200. Three randomly selected sites of each patient were used for evaluations and the irradiation values were assessed in the IMAGEJ software (NIH, Wayne Rasband, USA). Intensity in pixels was quantified in relation to the distance from the light incident point. Data were normalized and the results were presented as relative light intensity as a function of distance. The results demonstrated that red laser light is exponentially attenuated as a function of lateral distance and loses approximately 50 % of its intensity by 2.23 mm. On the other hand, the light travels 3 mm in depth to decay 50 %. In conclusion, our data suggest that melanin presence decreases optical pathway and irradiation protocols for antimicrobial photodynamic therapy in gingival tissue should consider light attenuation and depth of periodontal pockets so that efficient illumination of the target tissue occurs. Periodontal pockets bigger than 6 mm should be irradiated with more than one point.

Safety and Clinical Impact of a Single Red Light Irradiation on Breast Tumor-Bearing Mice.

Low-level light therapy has been used in health care as a therapeutic strategy for different diseases. However, its effects on cancer are controversial. This work evaluated the effects of three energies on breast cancer-bearing mice after a single red light-emitting diode (LED) irradiation. 4T1 cells were inoculated into the mammary fat pad of female BALB/c mice. When tumor volume reached 100 mm3 , animals were irradiated by a LED irradiator (660 ± 11 nm) with energies of 1.2, 3.6, and 6.0 J. Control without irradiation and healthy animals were also evaluated. Mice were monitored regarding tumor volume and total blood count. After euthanasia, their organs were examined. We observed that a single irradiation does not increase tumor volume. All irradiated groups exhibited better clinical conditions than control, which presented a significant decrease in platelet and red blood cell levels compared with healthy mice. The energy of 3.6 J arrested neutrophil-lymphocyte rate besides promoting longer survival and a lower number of metastatic nodules in the lungs. These findings suggest that a single red LED irradiation causes no impact on the course of the disease. Besides, the intermediary dose-effect should be further investigated since it seems to promote better outcomes on breast cancer-bearing mice.

Photodynamic inactivation of Candida ssp. on denture stomatitis. A clinical trial involving palatal mucosa and prosthesis disinfection.

Denture stomatitis (DS) is the most common oral fungal infection in denture wearers. Photodynamic inactivation (PDI) has been showing to be an effective technique in vivo against fungi, including fungal infections in the oral cavity. The disinfection of both oral mucosa and denture may represent a real advantage in terms of fungus control. This clinical study was designed to explore methylene blue (MB)-mediated PDI on oral mucosa and prosthesis of patients with DS. Subjects with DS were divided into two groups. One group received treatment based on the use of oral miconazole gel 2% (MIC). The other group received treatment by PDI using MB at 450 µg/mL and a diode laser (λ = 660 nm) with 100 mW and fluence of 28 J/cm2. Clinical outcome was evaluated regarding the degree of oral mucosa erythema and microbiological reduction of Candida spp. located in both palatal mucosa and prosthesis. Our results showed that PDI was significantly more effective than MIC in ameliorating inflammation after 15 days. Following 30 days, no statistically significant differences were observed between groups. Regarding the fungal burden, although the MIC group has presented more pronounced inactivation than PDI for both mucosa and prosthesis, no statistically significant differences were detected between them. This clinical study suggests that PDI can reduce fungal load and decrease the inflammation degree in patients with Candida-associated denture stomatitis.

Photodynamic inactivation assisted by localized surface plasmon resonance of silver nanoparticles: In vitro evaluation on Escherichia coli and Streptococcus mutans.

Localized surface plasmon resonance (LSPR) of gold nanoparticles has been reported to increase the antimicrobial effect of the photodynamic therapy. Although silver nanoparticles (AgNPs) are an efficient growth inhibitor of microorganisms, no studies exploring LSPR of AgNPs to enhance the photodynamic inactivation (PDI) have been related. In this work, we described the LSPR phenomenon of AgNP sand investigated its interaction with riboflavin, a natural photosensitizer. We evaluated the use of AgNPs coated with pectin (p-AgNP) in riboflavin (Rb)-mediated PDI of Escherichia coli (Gram- bacteria) and Streptococcus mutans (Gram + bacteria) using a blue light-emitting diode (λ = 455 ±â€¯20 nm) of optical power 200 mW. Irradiance was 90 mW/cm2 and radiant exposure varied according to the time exposure. Uptake of Rb and p-AgNP by the cells was evaluated by measuring the supernatant absorption spectra of the samples. We observed that LSPR of p-AgNPs was able to enhance the riboflavin photodynamic action on S. mutans but not on E. coli, probably due to the lower uptake of Rb by E. coli. Taken together, our results provide insights to explore the use of the LPRS promoted by silver nanostructures to optimize antimicrobial PDI protocols.

Photodynamic damage predominates on different targets depending on cell growth phase of Candida albicans.

Photodynamic inactivation (PDI) has been reported to be effective to eradicate a wide variety of pathogens, including antimicrobial-resistant microorganisms. The aim of this study was to identify the potential molecular targets of PDI depending on growth phase of Candida albicans. Fungal cells in lag (6h) and stationary (48h) phases were submitted to PDI mediated by methylene blue (MB) combined with a (662±21) nm-LED, at 360mW of optical power. Pre-irradiation time was 10min and exposure times were 12min, 15min and 18min delivering radiant exposures of 129.6J/cm2, 162J/cm2 and 194.4J/cm2, respectively, on a 24-well plate of about 2cm2 at an irradiance of 180mW/cm2. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force spectroscopy (AFS) and Fourier transform infrared spectroscopy (FT-IR) were employed to evaluate the photodynamic effect in young and old fungal cells following 15min of irradiation. Morphological analysis revealed wrinkled and shrunk fungal cell membrane for both growth phases while extracellular polymeric substance (EPS) removal was only observed for old fungal cells. Damaged intracellular structures were more pronounced in young fungal cells. The surface nanostiffness of young fungal cells decreased after PDI but increased for old fungal cells. Cellular adhesion force was reduced for both growth phases. Fungal cells in lag phase predominantly showed degradation of nucleic acids and proteins, while fungal cells in stationary phase showed more pronounced degradation of polysaccharides and lipids. Taken together, our results indicate different molecular targets for fungal cells in lag and stationary growth phase following PDI.

Urea enhances the photodynamic efficiency of methylene blue.

Methylene blue (MB) is a well-known photosensitizer used mostly for antimicrobial photodynamic therapy (APDT). MB tends to aggregate, interfering negatively with its singlet oxygen generation, because MB aggregates lean towards electron transfer reactions, instead of energy transfer with oxygen. In order to avoid MB aggregation we tested the effect of urea, which destabilizes solute-solute interactions. The antimicrobial efficiency of MB (30 µM) either in water or in 2M aqueous urea solution was tested against a fungus (Candida albicans). Samples were kept in the dark and irradiation was performed with a light emitting diode (λ = 645 nm). Without urea, 9 min of irradiation was needed to achieve complete microbial eradication. In urea solution, complete eradication was obtained with 6 min illumination (light energy of 14.4 J). The higher efficiency of MB/urea solution was correlated with a smaller concentration of dimers, even in the presence of the microorganisms. Monomer to dimer concentration ratios were extracted from the absorption spectra of MB solutions measured as a function of MB concentration at different temperatures and at different concentrations of sodium chloride and urea. Dimerization equilibrium decreased by 3 and 6 times in 1 and 2M urea, respectively, and increased by a factor of 6 in 1M sodium chloride. The destabilization of aggregates by urea seems to be applied to other photosensitizers, since urea also destabilized aggregation of Meso-tetra(4-n-methyl-pyridyl)porphyrin, which is a positively charged porphyrin. We showed that urea destabilizes MB aggregates mainly by causing a decrease in the enthalpic gain of dimerization, which was exactly the opposite of the effect of sodium chloride. In order to understand this phenomenon at the molecular level, we computed the free energy for the dimer association process (ΔG(dimer)) in aqueous solution as well as its enthalpic component in aqueous and in aqueous/urea solutions by molecular dynamics simulations. In 2M-urea solution the atomistic picture revealed a preferential solvation of MB by urea compared with MB dimers while changes in ΔH(dimer) values demonstrated a clear shift favoring MB monomers. Therefore, MB monomers are more stable in urea solutions, which have significantly better photophysics and higher antimicrobial activity. This information can be of use for dental and medical professionals that are using MB based APDT protocols.

Effects of photodynamic therapy on Gram-positive and Gram-negative bacterial biofilms by bioluminescence imaging and scanning electron microscopic analysis.

OBJECTIVE: The aim of this study was to test photodynamic therapy (PDT) as an alternative approach to biofilm disruption on dental hard tissue, We evaluated the effect of methylene blue and a 660 nm diode laser on the viability and architecture of Gram-positive and Gram-negative bacterial biofilms. MATERIALS AND METHODS: Ten human teeth were inoculated with bioluminescent Pseudomonas aeruginosa or Enterococcus faecalis to form 3 day biofilms in prepared root canals. Bioluminescence imaging was used to serially quantify and evaluate the bacterial viability, and scanning electron microscopic (SEM) imaging was used to assess architecture and morphology of bacterial biofilm before and after PDT employing methylene blue and 40 mW, 660 nm diode laser light delivered into the root canal via a 300 µm fiber for 240 sec, resulting in a total energy of 9.6 J. The data were statistically analyzed with analysis of variance (ANOVA) followed by Tukey test. RESULTS: The bacterial reduction showed a dose dependence; as the light energy increased, the bioluminescence decreased in both planktonic suspension and in biofilms. The SEM analysis showed a significant reduction of biofilm on the surface. PDT promoted disruption of the biofilm and the number of adherent bacteria was reduced. CONCLUSIONS: The photodynamic effect seems to disrupt the biofilm by acting both on bacterial cells and on the extracellular matrix.

The use of optical fiber in endodontic photodynamic therapy. Is it really relevant?

This study analyzed the necessity of use of an optical fiber/diffusor when performing antimicrobial photodynamic therapy (PDT) associated with endodontic therapy. Fifty freshly extracted human single-rooted teeth were used. Conventional endodontic treatment was performed using a sequence of ProTaper (Dentsply Maillefer Instruments), the teeth were sterilized, and the canals were contaminated with Enterococcus faecalis 3 days' biofilm. The samples were divided into five groups: group 1--ten roots irradiated with a laser tip (area of 0.04 cm(2)), group 2--ten roots irradiated with a smaller laser tip (area of 0.028 cm(2)), and group 3--ten teeth with the crown, irradiate with the laser tip with 0.04 cm(2) of area. The forth group (G4) followed the same methodology as group 3, but the irradiation was performed with smaller tip (area of 0.028 cm(2)) and G5 ten teeth with crown were irradiated using a 200-mm-diameter fiber/diffusor coupled to diode laser. Microbiological samples were taken after accessing the canal, after endodontic therapy, and after PDT. Groups 1 and 2 showed a reduction of two logs (99%), groups 3 and 4 of one log (85% and 97%, respectively), and group 5 of four logs (99.99%). Results suggest that the use of PDT added to endodontic treatment in roots canals infected with E. faecalis with the optical fiber/diffusor is better than when the laser light is used directed at the access of cavity.

Antimicrobial photodynamic therapy on drug-resistant Pseudomonas aeruginosa-induced infection. An in vivo study.

Pseudomonas aeruginosa is considered one of the most important pathogens that represent life-threatening risk in nosocomial environments, mainly in patients with severe burns. Antimicrobial photodynamic therapy (aPDT) has been effective to kill bacteria. The purpose of this study was to develop a burn wound and bloodstream infection model and verify aPDT effects on it. In vitro, we tested two wavelengths (blue and red LEDs) on a clinical isolate of P. aeruginosa strain with resistance to multiple antibiotics using HB:La(+3) as photosensitizer. Verapamil(®) associated to aPDT was also studied. In vivo, P. aeruginosa-infected burned mice were submitted to aPDT. Bacterial counting was performed on local infection and bloodstream. Survival time of animals was also monitored. In this study, aPDT was effective to reduce P. aeruginosa in vitro. In addition, Verapamil(®) assay showed that HB:La(+3) is not recognized by ATP-binding cassete (ABC) efflux pump mechanism. In the in vivo study, aPDT was able to reduce bacterial load in burn wounds, delay bacteremia and keep the bacterial levels in blood 2-3 logs lower compared with an untreated group. Mice survival was increased on 24 h. Thus, this result suggests that aPDT may also be a novel prophylactic treatment in the care of burned patients.

Photodynamic therapy associated with conventional endodontic treatment in patients with antibiotic-resistant microflora: a preliminary report.

INTRODUCTION: This study reports the antimicrobial effect of photodynamic therapy (PDT) combined with endodontic treatment in patients with necrotic pulp infected with microflora resistant to a previous antibiotic therapy. METHODS: Thirty anterior teeth from 21 patients with periapical lesions that had been treated with conventional endodontic treatment and antibiotic therapy were selected. Microbiological samples were taken (1) after accessing the root canal, (2) after endodontic therapy, and (3) after PDT. RESULTS: All the patients had at least 1 microorganism resistant to antibiotics. PDT used polyethylenimine chlorin(e6) as a photosensitizer and a diode laser as a light source (P = 40 mW, t = 4 minutes, E = 9.6 J). Endodontic therapy alone produced a significant reduction in numbers of microbial species but only 3 teeth were free of bacteria, whereas the combination of endodontic therapy with PDT eliminated all drug-resistant species and all teeth were bacteria-free. CONCLUSIONS: The use of PDT added to conventional endodontic treatment leads to a further major reduction of microbial load. PDT is an efficient treatment to kill multi-drug resistant microorganisms.

Low-intensity red laser on the prevention and treatment of induced-oral mucositis in hamsters.

OBJECTIVE: The purpose of this study was to investigate the effects of laser phototherapy as preventive and therapeutic regime on induced-oral mucositis in hamsters. DESIGN: The animals were divided into four groups: preventive cryotherapy, preventive laser, therapeutic laser and therapeutic control group. Mucositis was induced in hamsters by intraperitoneal injection of 5-fluorouracil (5-FU) and superficial scratching. All preventive treatment was performed on the right cheek pouch mucosa. The left pouch mucosa was used for a spontaneous development of mucositis and did not receive any preventive therapy. Laser parameters were: lambda=660nm, P= 30mW, D=1.2J/cm(2), Deltat=40s, spot size 3mm(2), I=1W/cm(2). Cryotherapy was done positioning ice packs in the hamster mucosa 5min before 5-FU infusion and 10min afterward. To study the healing of mucositis, the left pouch mucosa of each of the hamsters in the TLG received laser irradiation on the injured area. Irradiation parameters were kept the same as abovementioned. The control hamsters in the TCG did not receive any treatment. The mucositis degree and the animal's body mass were evaluated. An assessment of blood vessels was made based on immunohistochemical staining. RESULTS: The CG animals lost 15.16% of theirs initial body mass while the LG animals lost 8.97% during the first 5 days. The laser treated animals had a better clinical outcome with a faster healing, and more granulation tissue. The quantity of blood vessels at both LG and CG were higher than in healthy mucosa. Regarding the therapeutic analysis, the severity of the mucositis in the TLG was always lower than TCG. TLG presented higher organization of the granulation tissue, parallel collagen fibrils, and increased angiogenesis. CONCLUSION: The results suggest that laser phototherapy had a positive effect in reducing mucositis severity, and a more pronounced effect in treating established mucositis.

Effects of a single near-infrared laser treatment on cutaneous wound healing: biometrical and histological study in rats.

BACKGROUND: Low intensity laser therapy has been recommended to support the cutaneous repair; however, so far studies do not have evaluated the tissue response following a single laser treatment. This study investigated the effect of a single laser irradiation on the healing of full-thickness skin lesions in rats. METHODS: Forty-eight male rats were randomly divided into three groups. One surgical lesion was created on the back of rats using a punch of 8mm in diameter. One group was not submitted to any treatment after surgery and it was used as control. Two energy doses from an 830-nm near-infrared diode laser were used immediately post-wounding: 1.3 J cm(-2) and 3 J cm(-2). The laser intensity 53 m W cm(-2) was kept for both groups. Biometrical and histological analyses were accomplished at days 3, 7 and 14 post-wounding. RESULTS: Irradiated lesions presented a more advanced healing process than control group. The dose of 1.3 J cm(-2) leaded to better results. Lesions of the group irradiated with 1.3 J cm(-2) presented faster lesion contraction showing quicker re-epithelization and reformed connective tissue with more organized collagen fibers. CONCLUSIONS: Low-intensity laser therapy may accelerate cutaneous wound healing in a rat model even if a single laser treatment is performed. This finding might broaden current treatment regimens.

Effects of low power red laser on induced-dental caries in rats.

OBJECTIVE: The purpose of this study was to investigate the effects of low power red laser associated with acidulated phosphate fluoride on the development of induced-dental caries in rats. DESIGN: Dental caries were induced in molars of 40 rats divided into five groups: control group (CG), the teeth were not submitted to any treatment; laser group (LG), teeth were irradiated with a low power red laser (LPRL), power of 30 mW and dose of 5 J/cm(2); fluoride group (FG), teeth were treated with topical acidulated phosphate fluoride (APF) 1.23% applied for 4 min; laser+fluoride group (LFG), teeth were irradiated with LPRL followed by APF; fluoride+laser group (FLG), teeth were treated with APF followed by LPRL. The animals were killed after 48 days, and the first and second molars were extracted to analyze the caries lesion area, microhardness, and calcium and phosphorus ratio. RESULTS: There were no statistical differences among FG, LFG, and FLG regarding to caries area and microhardness, although the caries area were smaller in LFG. Ca/P ratio did not show significant differences among all groups. CONCLUSIONS: Although LPRL before APF application appeared to diminish the caries progression, LPRL did not present any additional benefit compared with acidulated phosphate fluoride on the prevention of induced-dental caries in rats.

Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection.

BACKGROUND AND OBJECTIVE: To compare the effectiveness of antimicrobial photodynamic therapy (PDT), standard endodontic treatment and the combined treatment to eliminate bacterial biofilms present in infected root canals. STUDY DESIGN/MATERIALS AND METHODS: Ten single-rooted freshly extracted human teeth were inoculated with stable bioluminescent Gram-negative bacteria, Proteus mirabilis and Pseudomonas aeruginosa to form 3-day biofilms in prepared root canals. Bioluminescence imaging was used to serially quantify bacterial burdens. PDT employed a conjugate between polyethylenimine and chlorin(e6) as the photosensitizer (PS) and 660-nm diode laser light delivered into the root canal via a 200-micro fiber, and this was compared and combined with standard endodontic treatment using mechanical debridement and antiseptic irrigation. RESULTS: Endodontic therapy alone reduced bacterial bioluminescence by 90% while PDT alone reduced bioluminescence by 95%. The combination reduced bioluminescence by >98%, and importantly the bacterial regrowth observed 24 hours after treatment was much less for the combination (P<0.0005) than for either single treatment. CONCLUSIONS: Bioluminescence imaging is an efficient way to monitor endodontic therapy. Antimicrobial PDT may have a role to play in optimized endodontic therapy.