Anti-tumor therapy with macroencapsulated endostatin producer cells

Background: Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors. Results: Mice were inoculated subcutaneously with melanoma (B16F10 cells) or Ehrlich tumor cells at the foot pads. Treatment began when tumor thickness had reached 0.5 mm, by subcutaneous implantation of 10(7) recombinant encapsulated or non-encapsulated endostatin producer cells. Similar melanoma growth inhibition was obtained for mice treated with encapsulated or non-encapsulated endostatin-expressing cells. The treatment of mice bearing melanoma tumor with encapsulated endostatin-expressing cells was decreased by 50.0%, whereas a decrease of 56.7% in tumor thickness was obtained for mice treated with non-encapsulated cells. Treatment of Ehrlich tumor-bearing mice with non-encapsulated endostatin-expressing cells reduced tumor thickness by 52.4%, whereas lower tumor growth inhibition was obtained for mice treated with encapsulated endostatin-expressing cells: 24.2%. Encapsulated endostatin-secreting fibroblasts failed to survive until the end of the treatment. However, endostatin release from the devices to the surrounding tissues was confirmed by immunostaining. Decrease in vascular structures, functional vessels and extension of the vascular area were observed in melanoma microenvironments. Conclusions: This study indicates that immunoisolation devices containing endostatin-expressing cells are effective for the inhibition of the growth of melanoma and Ehrlich tumors. Macroencapsulation of engineered cells is therefore a reliable platform for the refinement of innovative therapeutic strategies against tumors.

Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease. Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 mu g of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c(+), CD11b(+) and CD19(+) cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7). Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Apoptosis in Glioma Cells Treated with PDT

Background: Despite significant advances in neurosurgical techniques, the median survival time of patients with glioblastoma has improved little over the past 50 years and remains less than one year. Photodynamic therapy (PDT) is presently established as a widely accepted modality for the treatment of a variety of solid tumors. Objectives: This study evaluated the effect of PDT-Photogem (R) on five glioma cell lines (U87, U138, U251, U343, and T98G). Methods: The experiments were carried out in 25-cm(3) flasks with different groups of cells seeded at a density of 1 x 10(5) cells per flask. After 3 h, the medium was removed, and the cells were incubated for 4 h with Photogem (5 mu g/mL). After the incubation time, the photosensitizer-containing medium was removed and the cells were irradiated with LED (630 nm, 25 mW/cm(2), 25 J/cm(2)) devices for 17 min. For the final steps of the PDT, the cells were returned to the incubator and kept at 37 degrees C with 5% CO(2) for 24 h, the cell viability assay was assessed using the trypan blue method, and the expression of Caspase 3 mRNA levels was assessed by real-time quantitative PCR. Results: Upon PDT-Photogem (R) treatment, viable cells, as evaluated by the trypan blue dye-exclusion method, decreased in two cell lines (U87 and U138) but not in the other three. Apoptosis, as assessed by the expression of caspase-3 mRNA levels, was at least partly involved in the death mechanism of the cell lines. Conclusions: Collectively, our results indicated that PDT-Photogem (R) can act in glioma cells, thus encouraging new experiments in this field.

Effects of Er:YAG Laser Irradiation on the Microtensile Bond Strength to Bleached Enamel

Objective: The objective of this study was to evaluate the influence of different Er:YAG laser (lambda = 2.94 mu m) energy parameters on the microtensile bond strength (mu TBS) and superficial morphology of bovine enamel bleached with 16% carbamide peroxide. Background: Laser irradiation could improve adhesion to bleached enamel surfaces. Methods: Sixty bovine enamel blocks (7x3x3 mm(3)) were randomly assigned to six groups according to enamel preparation procedures (n = 10): G1-bleaching and Er:YAG laser irradiation with 25.52 J/cm(2) (laser A, LA); G2-bleaching and Er:YAG laser irradiation with 4.42J/cm(2) (laser B, LB); G3-bleaching; G4-Er:YAG laser irradiation with 25.52 J/cm(2); G5-Er:YAG laser irradiation with 4.42J/cm(2); G6-control, no treatment. G1 to G3 were bleached for 6 h during 21 days. Afterwards, enamel surfaces in all groups were slightly abraded with 600-grit SiC papers and G1, G2, G4 and G5 were irradiated according to each protocol. Enamel blocks were then restored with an etch-and-rinse adhesive system and a 4-mm thick composite buildup was made in two increments (n = 9). After 24 h, restored blocks were serially sectioned with a cross-section area of similar to 1 mm(2) at the bonded interface and tested in tension in a universal testing machine (1 mm/min). Failure mode was determined at a magnification of x100 using a stereomicroscope. One treated block of each group was selected for scanning electron microscopy (SEM) analysis. mu TBS data were analyzed by two-way ANOVA and no statistical differences were observed among groups. Results: Mean bond strengths (SD) in MPa were: G1-30.4(6.2); G2-27.9(8.5); G3-32.3(3.9); G4-23.7(5.8); G5-29.3(6.0); G6-29.1(6.1). A large number of adhesive failures was recorded for bleached and irradiated enamel surfaces. Conclusions: Bleached enamel surfaces mu TBS values were not significantly different from those of unbleached enamel. Even though Er:YAG laser irradiation with both parameters had no influence on mu TBS for bleached and unbleached enamel, SEM analysis revealed that Er:YAG laser irradiation with 25.52J/cm(2) should not be recommended, as enamel ablation was observed, whereas irradiation with 4.42J/cm(2) did not promote any remarkable changes on enamel surface.

Nd:YAG Laser Improves Biocompatibility of Human Dental Root Surfaces

Objective: Our goal was to compare the in vivo biocompatibility of dental root surfaces submitted to four different treatments after tooth avulsion followed by implantation into rat subcutaneous tissue. Background Data: Dental root surface preparation prior to replanting teeth remains a challenge for endodontists. Root surface changes made by Nd:YAG irradiation could be an alternative preparation. Methods: Forty-eight freshly extracted human dental roots were randomly divided into four treatment groups prior to implantation into rat subcutaneous tissue: G1, dry root, left in the environment up to 3 h; G2, the same treatment as G1, followed by a soaking treatment in a 2.4% sodium fluoride solution (pH 5.5); G3, root soaked in physiologic saline after avulsion for 72 h; G4, the same treatment as G1, followed by Nd:YAG laser irradiation (2.0 W, 20 Hz, 100 mJ, and 124.34 J/cm(2)). The animals were sacrificed 1, 7, and 45 d later. Histological and scanning electron microscopy analyses were done. Results: All dental roots were involved and in intimate contact with connective tissue capsules of variable thicknesses. Differences were observed in the degree of inflammation and in connective tissue maturation. In G3 the inflammatory infiltrate was maintained for 45 d, whereas the Nd:YAG laser irradiation (G4) led to milder responses. The overall aspects of the root surfaces were similar, except by the irradiated roots, where fusion and resolidification of the root surface covering the dentinal tubules were observed. Conclusion: Nd:YAG laser irradiation improves the biocompatibility of dental root and thus could be an alternative treatment of dental root prior to replantation.

LED Phototherapy to Prevent Mucositis: A Case Report

Objective: The purpose of this case report was to evaluate the efficacy of phototherapy using light-emitting diodes (LEDs) to prevent oral mucositis in a Hodgkin's disease patient treated with the ABVD ( doxorubicin [Adriamycin], bleomycin, vinblastine, and dacarbazine) chemotherapy regimen. Background Data: Mucositis is a common dose-limiting complication of cancer treatment, and if severe it can lead to alterations in treatment planning or suspension of cancer therapy, with serious consequences for tumor response and survival. Therefore, low-power lasers and more recently LEDs, have been used for oral mucositis prevention and management, with good results. Materials and Methods: In this study, a 34-year-old man received intraoral irradiation with an infrared LED array (880 nm, 3.6 J/cm(2), 74 mW) for five consecutive days, starting on chemotherapy day 1. In each chemotherapy cycle, he received the ABVD protocol on days 1 and 15, and received LED treatment for 5 d during each cycle. To analyze the results, the World Health Organization (WHO) scale was used to grade his mucositis, and a visual analogue scale (VAS) was used for pain evaluation, on days 1, 3, 7, 10, and 13 post-chemotherapy. Results: The results showed that the patient did not develop oral mucositis during the five chemotherapy cycles, and he had no pain symptoms. Conclusion: LED therapy was a safe and effective method for preventing oral mucositis in this case report. However, further randomized studies with more patients are needed to prove the efficacy of this method.

Morphologic changes and removal of debris on apical dentin surfaces after Nd : YAG laser and diode laser irradiation

Objective: To verify the effects of laser energy on intracanal dentin surfaces, by analyzing the morphologic changes and removal of debris in the apical third of 30 extracted human teeth, prepared and irradiated with the Nd:YAG laser and diode laser. Background Data: Lasers have been widely used in endodontics. The morphologic changes in dentin walls caused by Nd: YAG and diode laser irradiation could improve apical seals and cleanliness. Materials and Methods: The protocol used for Nd: YAG laser irradiation was 1.5 W, 100 mJ, and 15 Hz, in pulsed mode, and for diode laser was 2.5 W in continuous mode. Each specimen was irradiated four times at a speed of 2 mm/sec with a 20-sec interval between applications. Five calibrated examiners scored the morphologic changes and debris removal on a 4-point scale. Results: In analyzing the scores, there were no statistically significant differences between the two types of laser for either parameter, according to Kruskal-Wallis testing at p = 0.05. The SEM images showed fusion and resolidification of the dentin surface, with partial removal of debris on the specimens irradiated with the Nd: YAG laser and the diode laser, compared with controls. Conclusion: Both lasers promote morphologic changes and debris removal. These alterations of the dentin surface appeared to be more evident in the Nd: YAG laser group, but the diode laser group showed more uniform changes.

Evaluation of the Bond Strength Between a Composite Resin and Enamel Submitted to Bleaching Treatment and Etched with Er:YAG Laser

Background: The use of laser irradiation for dental surface treatment may increase tooth-composite bond strength. Its use on bleached teeth may decrease the waiting time between bleaching and restorative procedures. Objective: This study aimed to evaluate the bond strength between a composite resin and bovine enamel bleached with 35% hydrogen peroxide and etched with Er:YAG laser. Materials and Methods: Thirty bovine teeth were randomly divided into six groups (n = 5): G1, unbleached and restored 24 h after storage in artificial saliva, etching with 35% phosphoric acid (PA) (control); G2, unbleached and restored 24 h after storage in artificial saliva, etching with Er:YAG laser and 35% PA; G3, bleached and restored immediately afterward, etching with 35% PA; G4, bleached and restored 24 h after bleaching, etching with 35% PA; G5, bleached and restored immediately afterward, etching with Er:YAG and 35% PA laser; G6, bleached and restored 24 h after bleaching, etching with Er:YAG laser and 35% PA. Bond strength was quantitatively evaluated by microtensile test (1.0 mm/min). Data were submitted to statistical analysis using ANOVA and Tukey tests (alpha - 0.05). Results: Bond strength values (MPa) were G1, 26.17 +/- 4.44; G2, 28.87 +/- 3.94; G3, 17.25 +/- 4.58; G4, 21.93 +/- 5.02; G5, 16.69 +/- 2.31; and G6, 29.06 +/- 8.31. There was no statistically significant difference among groups G1, G2, and G6 (p - 0.119), which presented higher bond strength than group G4, followed by groups G3 and G5. Conclusion: Er:YAG irradiation of bleached surfaces may favor bonding procedures when performed 24 h after bleaching.

Venous Lake of the Lips Treated Using Photocoagulation with High-Intensity Diode Laser

Objective: To evaluate the effectiveness of photocoagulation with high-intensity diode laser in the treatment of venous lake (VL) lesions. Background Data: VL is a common vascular lesion characterized by elevated, usually dome-shaped papules, ranging in color from dark blue to dark purple, seen more frequently in elderly patients. They often occur as single lesions on the ears, face, lips, or neck. Once formed, lesions persist throughout life. Although these lesions are usually asymptomatic, they can bleed if injured. Methods: Seventeen patients (7 men and 10 women) with VL on the lip were treated using a noncontact diode laser (wavelength 808 nm, power output 2-3 W in continuous wave). Results: After only one irradiation exposure, all lesions were successfully treated. Healing was completed in approximately 2 to 3 weeks, and none of the patients experienced complications. Postoperative discomfort and scarring were not present or were minimal. Conclusion: Photocoagulation with high-intensity diode laser is an effective, bloodless procedure for the treatment of VL.

Analysis of Permeability and Morphology of Root Canal Dentin After Er,Cr:YSGG Laser Irradiation

Objective: The aim of this study was to evaluate the morphology and permeability of root canal walls irradiated with Er,Cr:YSGG laser after conventional endodontic treatment. Background: Laser irradiation can be used for dentinal tubule exposure, smear layer removal, and disinfection. Another potential, interesting application is as an adjunct to endodontic treatment, especially in the intracanal medication phase. Methods: Fifty-two single-rooted teeth had their crowns sectioned at the cementoenamel junction and were randomly divided into four groups (n = 13): G1: conventional preparation (CP) + irrigation with EDTA-T+rhodamine B dye solution associated with NDP (dexamethasone phosphate, paramonochlorophenol, polyethylenoglycol) (Rhod-NDP); G2: CP+EDTA-T + Er,Cr:YSGG laser irradiation 0.75W+Rhod-NDP; G3: CP + EDTA-T + Er,Cr:YSGG 1.5W+Rhod-NDP; G4: CP + EDTA-T + Er,Cr:YSGG 2.5W + Rhod-NDP. For the permeability analysis (n = 9), teeth were transversely cut and two slices of each third were selected. The images were analyzed by ImageLab software (Softium Informatica Ltda., Sao Paulo, SP, Brazil). Additional samples (n = 4) were examined by scanning electron microscopy. Results: Data were analyzed statistically using the Kruskal-Wallis and Student-Newman-Keuls tests for the following areas: apical third (H = 23.4651): G1 (14.25)(a), G2 (17.66)(ab), G3 (26.50)(b), G4 (39.58)(c); medium (H = 23.1611): G1 (14.16)(a), G2 (16.66)(ab), G3 (28.83)(b), G4 (38.33)(b); and cervical (H = 32.4810): G1 (9.66)(a), G2 (20.00)(ab), G3 (27.00)(b), G4 (41.33)(c), (p<0.01). Despite the irregular aspect of laser irradiation along the canal walls, the parameters of 1.5W and 2.5W allowed morphologic modifications that increased dentinal permeability. Conclusions: Irradiation with Er, Cr: YSGG laser could be effective in endodontic treatment for increasing dentinal permeability.

Low- and High-Intensity Lasers in the Treatment of Herpes Simplex Virus 1 Infection

Herpes simplex virus (HSV) is one of the most common viral infections of the human being. Although most of the seropositive persons do not manifest symptoms, infected individuals may present recurrent infections, characterized by cold sores. HSV-1 infection can result in potentially harmful complications in some patients, especially in those with compromised immunity. We report a clinical case of a patient with severe oral HSV-1 infection in the lower lip. The treatment of the lesions with the association of high-intensity (erbium-doped yttrium aluminum garnet, 2.94 mu m, 80 mJ/pulse, 2-4 Hz) and low-intensity (indium gallium aluminum phosphide, 660 nm, 3.8 J/cm(2), 10mW) lasers has not been reported in the literature. During treatment, no systemic or topical medication was used. Pain sensitivity was completely gone after the first irradiation with the low-intensity laser. During the healing process, lesions were traumatized twice, on the days 4 and 7. Even though the lesions were completely healed within 10 days.

Apical Leakage of Three Resin-Based Endodontic Sealers after 810-nm-Diode Laser Irradiation

Objective: To evaluate the influence of 810-nm-diode laser irradiation, applied before root canal filling, on apical sealing ability of three different resin-based sealers (AH Plus, EndoRez, and RealSeal). Background: Lasers have been widely used in endodontics. The dentin wall changes caused by laser irradiation could improve the sealing ability of endodontic cements. Methods: Sixty single-rooted human teeth were divided into six groups, according to the endodontic sealer used and previous 810-nm-diode laser irradiation. The protocol for laser irradiation was 2.5W in a continuous wave, in scanning mode, with four irradiations per tooth. After sample preparation, they were analyzed according to apical leakage with silver nitrate impregnation. Results: The RealSeal sealer achieved minimum leakage rates (1.24 mm), with significant differences at the 1% level (Tukey's test, p < 0.01) from AH Plus (1.84 mm) in nonirradiated groups. When the laser was used, there were also significant differences at the 5% level (p < 0.05) between irradiated groups (1.31 and 1.78 mm, respectively). Conclusion: The 810-nm-diode laser irradiation did not promote significant differences in apical leakage.

Effect of Nd:YAG Laser and Acidulated Phosphate Fluoride on Bovine and Human Enamel Submitted to Erosion/Abrasion or Erosion Only: An in Vitro Preliminary Study

Objective: The aim of the present in vitro study was to evaluate, using two different methodologies, the effectiveness of pulsed Nd:YAG laser irradiation associated with topical acidulated phosphate fluoride (APF) for preventing enamel erosion and structure loss under regimes of erosion and abrasion or erosion only. Background Data: An increased incidence of noncarious lesions (erosion and abrasion) has been observed, consequently new preventative therapies have been proposed. Materials and Methods: Two different methodologies were performed. For the first, 100 bovine crowns were submitted to four different treatments (n = 25): no treatment (control), 4 min application of APF, Nd:YAG laser irradiation (1 W, 100 mJ, 10 Hz, 141.5 J/cm(2)), and Nd:YAG laser irradiation+4 min of APF. After the specimens were exposed to citric acid (2% w/v; 30 min), they were submitted to 5000 brushing cycles. Specimen mass was measured before and after the treatments. For the second methodology, 20 human crowns were embedded in acrylic resin and cut surfaces were exposed and polished. The specimens were divided into four groups (n = 10): no treatment (control), APF for 4 min, Nd:YAG laser irradiation (1 W, 100 mJ, 10 Hz, 125 J/cm(2)), and Nd:YAG laser irradiation+APF. The samples were then immersed in citric acid (2% w/v; 90 min). Vickers hardness was obtained before and after the treatments. Results: The Nd:YAG laser irradiation+APF (bovine and human enamel) was more effective and yielded statistically significant results for surface microhardness and enamel wear. Conclusion: Nd:YAG laser irradiation associated with APF reduced bovine enamel wear and human enamel softening when samples were submitted to a regime of erosion and abrasion or erosion only in vitro.

Diode Laser Decreases the Activity of Catalase on Submandibular Glands of Diabetic Rats

Objective: The aim of this study was to evaluate the effect of laser irradiation on the amylase and the antioxidant enzyme activities, as well as on the total protein concentration of submandibular glands (SMG) of diabetic and non-diabetic rats. Background: Laser has been used aiming to improve some biochemical alterations observed in salivary glands of streptozotocin-induced diabetic rats. Materials and Methods: Ninety-six female rats were divided into eight groups: D0, D5, D10, and D20 (diabetic animals), and C0, C5, C10, and C20 (non-diabetic animals), respectively. Diabetes was induced by administering streptozotocin and confirmed later by the glycemia results. Twenty-nine days after diabetes induction, the SMG of groups D5 and C5, D10 and C10, and D20 and C20 were irradiated with 5, 10, and 20 J/cm(2), respectively. A diode laser (660nm/100mW) was used. On the day after irradiation, the rats were euthanized and the SMG were removed. Catalase, peroxidase, and amylase activities, as well as protein concentration, were assayed. Results: Diabetic rats without irradiation (D0) showed higher catalase activity (p<0.05) when compared to C0 (0.16 +/- 0.05 and 0.07 +/- 0.01 U/mg protein, respectively). However, laser irradiation of 5, 10, and 20 J/cm(2) reduced the catalase activity of diabetic groups (D5 and D20) to non-diabetic values (p>0.05). Conclusion: Based on the results of this study, laser irradiation decreased catalase activity in diabetic rats' SMG.

New methodology to assess activity status of occlusal caries in primary teeth using laser fluorescence device

An in vivo study was conducted to verify the ability of laser fluorescence (LF) to assess the activity status of occlusal caries in primary teeth, using different air-drying times. Occlusal sites (707) were examined using LF (DIAGNOdent) after air-drying for 3 s and 15 s, and the difference between readings (DIF15 s-3 s) was calculated. For concurrent validation of LF, visual criteria-Nyvad (NY) and Lesion Activity Assessment associated with the International Caries Detection and Assessment System (LAA-ICDAS)-were the reference standards for lesion activity. Histological exam using a pH-indicator dye (0.1% methyl red) was performed in 46 exfoliated/extracted teeth for criterion validation. LF readings and DIF15 s-3 s were compared using Kruskall-Wallis and Mann-Whitney tests. Receiver operating characteristic analyses were performed and validity parameters calculated, considering the caries activity assessment. Using NY, active lesions (3 s: 30.0 +/- 29.3; 15 s: 34.2 +/- 30.6) presented higher LF readings than inactive lesions (3 s: 17.0 +/- 16.3; 15 s: 19.2 +/- 17.3; p <0.05), different from LAA-ICDAS. Active cavitated caries resulted in higher LF readings (3 s: 50.3 +/- 3.5; 15 s: 54.7 +/- 30.2) than inactive cavitated caries (3 s: 19.9 +/- 16.3; 15 s: 22.8 +/- 16.8). Therefore, LF can distinguish cavitated active and inactive lesions classified by NY, but not by LAA-ICDAS; however, this difference might be related to the visual system rather than to LF. The air-drying time could be an alternative to improve the caries activity assessment; however, longer air-drying time is suggested to be tested subsequently. (C) 2010 Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.3463007]