Morphological aspects and the effectiveness of photodynamic inactivation against Rhizopus oryzae in different life cycles.

Mucormycosis is an extremely aggressive fungal disease with a high mortality rate, especially in people with compromised immune systems. Most cases of mucormycosis are caused by the fungus Rhizopus oryzae. The treatments used are based on high doses of antifungals, associated with surgical resections, when it is possible. However, even with this aggressive treatment, the estimated attributable mortality rate is high. There is therefore a need to develop adjuvant treatments. Photodynamic Inactivation (PDI) may be an auxiliary therapeutic option for mucormycosis. Due to the lack of reports in the literature on the morphology and photodynamic inactivation of R. oryzae, characterization of the fungus using Confocal Microscopy and Transmission Electron Microscopy, and different protocols using Photodithazine® (PDZ), a chlorin e6 compound, as a photosensitizer, were performed. The fungus growth rate under different concentrations and incubation times of the photosensitizer and its association with the surfactant Sodium Dodecyl Sulphate (SDS) was evaluated. For the hyphae, both in the light and dark phases, in the protocols using only PDZ, no effective photodynamic response was observed. Meanwhile with the combination of SDS 0.05% and PDZ, inhibition growth rates of 98% and 72% were achieved for the white and black phase, respectively. In the conidia phase, only a 1.7 log10 reduction of the infective spores was observed. High concentration of melanin and the complex and resistant structures, especially at the black phase, results in a high limitation of the PDI inactivation response. The combined use of the SDS resulted in an improved response, when compared to the one obtained with the amphotericin B treatment.

A proof-of-principle for decontamination of transplantation kidney through UV-C exposition of the perfusate solution.

Kidney transplantation is a common yet highly demanding medical procedure worldwide, enhancing the quality of life for patients with chronic kidney disease. Despite its prevalence, the procedure faces a shortage of available organs, partly due to contamination by microorganisms, leading to significant organ disposal. This study proposes utilizing photonic techniques associated with organ support machines to prevent patient contamination during kidney transplantation. We implemented a decontamination system using ultraviolet-C (UV-C) irradiation on the preservation solution circulating through pigs' kidneys between harvest and implant. UV-C irradiation, alone or combined with ultrasound (US) and Ps80 detergent during ex-vivo swine organ perfusion in a Lifeport® Kidney Transporter machine, aimed to reduce microbiological load in both fluid and organ. Results show rapid fluid decontamination compared to microorganism release from the organ, with notable retention. By including Ps80 detergent at 0.5% during UV-C irradiation 3 log10 (CFU mL-1) of Staphylococcus aureus bacteria previously retained in the organ were successfully removed, indicating the technique's feasibility and effectiveness.

Evaluation of Antimicrobial Photodynamic Therapy against Streptococcus mutans Biofilm in situ.

AIM: This study investigated the effect of antimicrobial photo-dynamic therapy (aPDT) over Streptococcus mutans biofilm. MATERIALS AND METHODS: Eighteen (n = 18) patients were selected and one palatine device with dental blocks was used. The biofilm was treated by curcumin and Photogem® with a LED and the effect was analyzed by CFU/ml. RESULTS: Although, statistical analysis showed significant reductions for aPDT mainly with Photogem® (p = 0.02), these were low. CONCLUSION: The results suggest a low antimicrobial effect of aPDT over S. mutans biofilm. Some parameters used need to be improved. CLINICAL SIGNIFICANCE: This technique can be a promising in Dentistry.

Identification of skin lesions through aminolaevulinic acid-mediated photodynamic detection.

Non-melanoma skin cancer is the most common cancer lesion worldwide. In Brazil, it represents 95% of all skin cancer lesions, and 25% of all tumor types. Early diagnosis allows treatment at initial stages of the disease, improving patient's prognosis. Thus, it is of great importance the development of techniques to aid diagnosis, such as marked fluorescence, which we propose here for early detection of skin cancer lesions. In this study, we use a photosensitive substance, aminolaevulinic acid (ALA), as biomarkers, and analyze its in situ fluorescence response to light excitation. The use of ALA as a biomarker precursor is interesting because it shows selectivity for protoporphyrin IX production/concentration in abnormal cells. Protoporphyrin IX shows high fluorescence yield when excited with UV-blue light. In this study, ALA solutions (at 5% and 10% concentrations) were applied to malignant (basal cell carcinoma) and potentially malignant skin lesions (actinic and seborrheic keratoses), aiming to investigate our ability in detecting and distinguishing them by using this technique. At regular time intervals (15, 30, 45 and 60min), fluorescence images were collected with a prototype system for widefield fluorescence imaging. ALA has provided a marked fluorescence that allowed significant discrimination of normal and tumor. Potentially malignant and benign lesions were all well-identified by their autofluorescence; photodynamic detection did not improve diagnostics. This technique also provided a better delineation of the lesion margins, which is very important for an effective treatment of malignant, potentially malignant and benign skin lesions.

Evaluation of vascular effect of Photodynamic Therapy in chorioallantoic membrane using different photosensitizers.

Photodynamic Therapy (PDT) is a local treatment that requires a photosensitizing agent, light and molecular oxygen. With appropriate illumination, the photosensitizer is excited and produces singlet oxygen that is highly reactive and cytotoxic. Tumor vascular network is essential for the tumor growth and the understanding of vascular response mechanisms enables an improvement in the PDT protocol for cancer treatment. Compounds of porphyrin (Photogem®) and chlorin (Photodithazine®) were the photosensitizers tested. The incubation times varied from 20 to 80 min and the concentration ranged between 0.1 and 100 µg/cm(2). Different light doses were used between 4.8 and 40 J/cm(2) with irradiance varying between 80 and 100 mW/cm(2). The light dose of 30 J/cm(2) was used in the intravenous photosensitizer application. The membrane images were made from 0 to 300 min after treatment. The vascular response was evaluated by the average vessel area. Different responses was observed depending on the photosensitizer concentration and administration form. Intravenous application has been more efficient to produce vessel constriction and the most pronounced effect was observed for the chlorin.

Effects of low-level laser therapy on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid.

Low-level laser therapy (LLLT) has been indicated as an adjuvant therapy for bisphosphonate-induced osteonecrosis. However, the effects of LLLT on bisphosphonate-treated cells are not yet clear. This study evaluated the effects of LLLT on the proliferation and apoptosis of gingival fibroblasts treated with zoledronic acid (ZA). Cells were exposed to ZA at 5µM for 48h. Irradiation was performed using a laser diode prototype (LaserTABLE, InGaAsP; 780nm±3nm, 25mW) at 0.5 or 3J/cm(2), three times every 24h. Cell proliferation and apoptosis were evaluated by fluorescence microscopy. Data were analyzed by Mann-Whitney test at the 5% level of significance. ZA decreased cell proliferation to 47.62% (interquartile range (IQR) 23.80-57.14%; P=0.007) and increased apoptosis of gingival fibroblasts to 27.7% (IQR 20.9-33.4%; P=0.0001). LLLT increased cell proliferation compared with non-irradiated cells, at 0.5J/cm(2) (57.14%, IQR 57.14-71.43%; P=0.003) and at 3J/cm(2) (76.19%, IQR 61.90-76.19%; P=0.0001), but did not increase cell proliferation in ZA-treated cells. Irradiated fibroblasts presented lower apoptosis rates than the ZA-treated cells, but apoptosis was no different in ZA-treated cells compared to those that were ZA-treated and also irradiated.

Phototherapy during treadmill training improves quadriceps performance in postmenopausal women.

OBJECTIVE: To evaluate the effects of infrared-light-emitting diode (LED) during treadmill training on functional performance. METHODS: Thirty postmenopausal women aged 50-60 years were randomly assigned to one of three groups and successfully completed the full study. The three groups were: (1) the LED group, which performed treadmill training associated with phototherapy (n = 10); (2) the exercise group, which carried out treadmill training only (n = 10); and (3) the sedentary group, which neither performed physical training nor underwent phototherapy (n = 10). Training was performed over a period of 6 months, twice a week for 45 min per session at 85-90% of maximal heart rate, which was obtained during progressive exercise testing. The irradiation parameters were 100 mW, 39 mW/cm(2) and 108 J/cm(2) for 45 min. Quadriceps performance was measured during isokinetic exercise testing at 60°/s and 300°/s. RESULTS: Peak torque did not differ amongst the groups. However, the results showed significantly higher values of power and total work for the LED group (∆ = 21 ± 6 W and ∆ = 634 ± 156 J, p < 0.05) when compared to both the exercise group (∆ = 13 ± 10 W and = 410 ± 270 J) and the sedentary group (∆ = 10 ± 9 W and ∆ = 357 ± 327 J). Fatigue was also significantly lower in the LED group (∆ = -7 ± 4%, p < 0.05) compared to both the exercise group (∆ = 3 ± 8%) and the sedentary group (∆ = -2 ± 6%). CONCLUSIONS: Infrared-LED during treadmill training may improve quadriceps power and reduce peripheral fatigue in postmenopausal women.

Long-term follow-up of topical 5-aminolaevulinic acid photodynamic therapy diode laser single session for non-melanoma skin cancer.

Photodynamic therapy (PDT) is based on the association of a light source and light sensitive agents in order to cause the selective death of tumor cells. To evaluate topical 5-aminolaevulinic acid (5-ALA) and diode laser photodynamic single session therapy single session for non-melanoma skin cancer (NMSC), a long-term follow-up was performed. Nineteen Bowen's disease (BD) and 15 basal cell carcinoma (BCC) lesions were submitted to 6-h topical and occlusive 20% 5-ALA plus DMSO and EDTA, and later were exposed to 630 nm diode laser, 100 or 300 J cm(-2) dose. At 3 months tumor-free rate was 91.2% (31/34) whereas at 60 months, 57.7% (15/26), slightly higher in BCC (63.6%; 7/11). The relation between the reduction of the clinical response and the increase of tumor dimension observed at 18 months was lost at 60 months. The sBCC recurrence was earlier compared to the nBCC one. ALA-PDT offered important advantages: it is minimally invasive, an option for patients under risk of surgical complications; clinical feasibility; treatment of multiple lesions in only one session or lesions in poor healing sites and superior esthetical results. However, the recurrence rate increase after ALA-PDT diode laser single session can be observed at long-term follow-up, and the repetitive sessions, an additional advantage of the method, is strongly recommended. The clinical response and recurrence time seem to be related to the laser light dose and NMSC types/sub-types, thickness and dimension, which must be considered for the choice of the ALA-PDT.

Renal ischemia in rats: mitochondria function and laser autofluorescence.

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser- (442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r2 = 0.39, P < .001) and 532 nm, (r2 = 0.18, P = .003); reflecting laser/fluorescence-induced, 442 nm (r2 = 0.20, P = .002) and 532 nm (r2 = 0.004, P = .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r2 = 0.43, P = .0004), state 4 respiration (r2 = 0.03, P = 0.38), RCR (r2 = 0.28, P = .007), and membrane swelling (r2 = 0.02, P = .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.

Autofluorescence spectroscopy in liver transplantation: preliminary results from a pilot clinical study.

The evaluation of graft function at various stages after transplantation is relevant, particularly at the moment of organ harvest, when a decision must be made whether to use the organ. Autofluorescence spectroscopy is noninvasive technique to monitor the metabolic condition of a liver graft throughout its course, from an initial evaluation in the donor, through cold ischemia transportation, to reperfusion and reoxygenation in the recipient. Preliminary results are presented in six liver transplantations spanning the periods from liver harvest to implant. The laser-induced fluorescence spectrum at 532-nm excitation was investigated before cold perfusion (autofluorescence), during cold ischemia, at the back table procedure, as well as 5 and 60 minutes after reperfusion. The results showed that the fluorescence analysis was sensitive to changes during the transplantation procedure. Fluorescence spectroscopy potentially provides a real-time, noninvasive technique to monitor liver graft function. The information could potentially be valuable for surgical decisions and transplant success.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats.

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70%) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm(2). A group irradiated with laser, 590 nm (N = 7, 15 J/cm(2)) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean +/- SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm(2)). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.

The use of light-emitting diodes to stimulate mitochondrial function and liver regeneration of partially hepatectomized rats

The biostimulating effect of laser radiation has been observed in many areas of Medicine. However, there are still several questions to be answered, among them the importance of light coherence in the stimulatory process. In the present study, we used light-emitting diodes (LED) to promote the stimulation of liver regeneration after partial hepatectomy in rats. Fourteen male Wistar rats weighing 200-250 g were submitted to partial hepatectomy (70 percent) followed by LED light irradiation (630 nm) of the remaining part of the liver at two doses, i.e., 10 (N = 7) and 140 (N = 7) J/cm². A group irradiated with laser, 590 nm (N = 7, 15 J/cm²) was performed for the study of proliferating cell nuclear antigen-labeling index. Data are reported as mean ± SEM. Statistical comparisons of the groups were performed by analysis of variance for parametric measurements followed by the Bonferroni post-test, with the level of significance set at P < 0.05. Respiratory mitochondrial activity was increased in the irradiated groups (states 3 and 4; P < 0.05), with better results for the group exposed to the lower LED dose (10 J/cm²). The proliferating cell nuclear antigen-labeling index, by immunohistochemical staining, was similar for both LED-exposed groups (P > 0.05) and higher than for the control group (P < 0.05). The cell proliferation index obtained with LED and laser were similar (P > 0.05). In conclusion, the present results suggest that LED irradiation promotes biological stimulatory effects during the early stage of liver regeneration and that LED is as effective as laser light, independent of the coherence, divergence and cromaticity.

Comparison of fluorescence detection of carious dentin for different excitation wavelengths.

This study compared the fluorescence spectra obtained at three laser excitation wavelengths from intact enamel, sound dentin and carious dentin. Results were analyzed using a figure of merit (ratio of backscattered peak to maximum fluorescence amplitude). The results showed that the best excitation wavelengths for caries detection, which offered the greatest contrast between sound and carious tissue, were 442 and 632 nm. A discussion of the results between these two ends of the visible spectrum is provided. The results confirmed the choice of red light as a good excitation source for fluorescence detection of caries.

PDT experience in Brazil: A regional profile.

The success of PDT and its establishment into the existent hall of therapeutic modalities depends on the collection of reported experiences from around the world. In that sense, it is important to report approaches taken by different countries and what their views are on the future of PDT. Following this idea, we present our clinical experience in photodynamic therapy (PDT) in Brazil, as well as the experimental advances coming up in parallel with clinical implementation. This report is a consequence of pioneering work in a collaborative program involving the Physics Institute in São Carlos, São Paulo State (SP), Brazil, the Medical School of the University of São Paulo, Ribeirão Preto, SP, Brazil and the Cancer Hospital Amaral Carvalho, Jaú, SP, Brazil. This collaborative program, begun in 1997, with the first patient treated in 1999, has treated over 400 patients by late 2004. About 80% of lesions were located in the head and neck or skin, but experience is being built in esophagus, bladder, gynecology, and cutaneous recurrence of breast cancer, among others. The overall results have shown to be compatible with previously reported data. Modifications, whose goal is to improve patient benefit and optimize results, are being implemented as we gain experience. In parallel with the clinical development, several laboratories have started studying experimental whose purpose is to analyze the clinical results and to contribute to the worldwide effort to bring PDT to the forefront of therapies offered to patients. We present the overall results of our 5 years experience as well as the whole implementation process.

Pharmacokinetics of Photogem using fluorescence monitoring in Wistar rats.

In this study we investigated the pharmacokinetics of a hematoporphyrin derivative (Photogem) in Wistar rats using the fluorescence spectroscopy to evaluate the drug distribution in liver, kidney and skin tissues. The detection system is composed of a 532 nm exciting laser, a Y-type catheter for light delivery and collection, a monochromator and a computer for data acquisition. The analysis of the fluorescence spectra was based on the intensity of porphyrin emission bands from specific tissues of the investigated organ. A simple transport model is proposed to determine the accumulation and elimination times for each type of investigated tissue. The obtained results show the viability of the fluorescence spectroscopic technique for the drug concentration monitoring in different target tissues and related pharmacokinetics. These effects should be considered before any in vivo study of Photodynamic Therapy using Photogem.

Spectral response for laser enhancement in hepatic regeneration for hepatectomized rats.

BACKGROUND AND OBJECTIVES: The low intensity laser therapy (LILT) has been widely used in all medical fields due to its therapeutic effects in reparative process, pain relief, and biostimulation. Even though there is a therapeutic window of wavelengths for clinical application, little has been done concerning the frequency spectrum response to biological effects. In this work, we investigate the dependence of different wavelengths irradiation in the enhancement of the tissue regeneration after partial hepatectomy in Wistar rats. STUDY DESIGN/MATERIALS AND METHODS: The proliferating cell nuclear antigen (PCNA) labeling index and the respiratory control (oxygen consumption in extracted mitochondria) were the tests used to evaluate the liver regeneration after laser irradiation with different wavelengths. RESULTS AND CONCLUSIONS: The results show a correlated spectral response that can be explained based on the combined effect of light penetration on biological tissues and the biomolecular excitation efficiency for each wavelength used.

Hardness evaluation of a dental composite polymerized with experimental LED-based devices.

OBJECTIVE: The main goal of this study was the hardness evaluation of a composite resin cured by five LED (Light Emitting Diodes) based devices and a comparison with a conventional curing unit. The hardness test was used to compare the efficacy of both types of light source. METHODS: The LED-based devices were made employing an array of LEDs (Nichia Chem. Ind., Japan) emitting light peaked at 470nm. Composite resin (Z100, shade A3) was cured for 20, 40, 60, 120 and 180s with each LED-based device and for 40s with the halogen lamp. The composite samples were prepared with 0.35, 1.25 and 1.8mm of thickness. Five samples of each set of parameters were done. The hardness evaluation was performed at the non-illuminate surface with three indentations for each sample. RESULTS: All the samples cured by the LED-based devices showed inferior hardness values when compared with the halogen lamp at the typical curing time (40s). The L6 (device composed of six LEDs) was the most efficient one of the LED-based devices. Its obtained irradiance was 79mW/cm(2), whereas the halogen lamp irradiance was of 475mW/cm(2). For the L6 device here presented, longer exposure times or a thinner resin layer are required to achieve reasonable hardness values. SIGNIFICANCE: Besides the difference of irradiance when compared with halogen lamps, LED-based devices show to be a promising alternative curing instrument. Further development in instrumentation may result in devices even more efficient than conventional lamps.