Mast cells in oral lichen planus and oral lichenoid lesions related to dental amalgam contact.

The aim of this study was to analyze the expression of mast cell markers toluidine blue, c-kit, and tryptase and presence of mononuclear inflammatory cells in oral lichen planus (OLP) and oral lichenoid lesions related to dental amalgam. Nineteen specimens of OLP, OLLC, and healthy oral mucosa were selected. Mononuclear inflammatory cells were analyzed. Histochemical and immunohistochemical analyses were performed using toluidine blue, anti-c-kit and anti-tryptase reagents, and the results were quantified in areas A and B of connective tissue. Mast cells of all OLP and OLLC samples were positive for toluidine blue, c-kit, and tryptase. The density of toluidine blue+, c-kit+ and tryptase+ mast cells was higher in tissue with OLP and OLLC compared with healthy controls (p < 0.05). No difference was noted in mast cells density between OLP and OLLC (p > 0.05). The density of tryptase+ mast cells was higher in the subepithelial region (area A) than the region below it (Area B) in OLLC (p = 0.047). The mononuclear inflammatory cell density was higher in OLLC compared to OLP, but without statistical significance (p > 0.05). A positive statistical correlation was found between mononuclear immune cells and density of c-kit+ and tryptase+ mast cells in OLP (r = 0.943 and r = 0.886, respectively). Our data demonstrate that the etiopathogenesis process of OLP and OLLC modulates the expansion and degranulation of mast cells; mast cells density, however, was similar between OLP and OLLC. The distribution of mast cells appears to vary along the lamina propria.

DPP4 Inhibitor Sitagliptin Reduces Inflammatory Responses and Mast Cell Activation in Allergic Rhinitis.

INTRODUCTION: DPP4 is thought to be involved in certain immune processes and plays an important role in allergic reactions in the lungs. The effect of the DPP4 inhibitor sitagliptin on the effector phase of allergic rhinitis (AR) in ovalbumin (OVA)-sensitized mice and on mast cell degranulation in vitro was assessed. METHODS: The AR mouse model was established by intraperitoneal injection combined with OVA intranasal method. OVA was injected intraperitoneally 3 times for the first 2 weeks, and the mice were subsequently given DPP4 inhibitors by oral gavage, accompanied by an OVA intranasal challenge. The impacts of DPP4 inhibitors on DPP4 levels in mouse model were determined. Nasal mucosa tissue was collected for H&E staining and toluidine blue staining. Immunoglobulin E (IgE) levels and histamine levels were analyzed, and IL-4, IL-5, and IL-12 as well as IFN-γ levels were assessed. Following the treatment of dinitrophenol (DNP)-IgE or DNP-IgE plus sitagliptin in RBL-2H3 cells, ß-hexosaminidase activity was analyzed and toluidine blue staining was performed. RESULTS: DPP4 level was reduced in AR patients, as well as in AR mouse models. Nasal allergic symptoms such as sneezing and nose-scratching showed high frequency in OVA-induced mice. Sitagliptin treatment during the intranasal challenge of OVA decreased DPP4 levels, suppressed allergic symptoms, eosinophil infiltration, IgE levels, mast cell infiltration, as well as the levels of inflammatory cytokines. We further found that sitagliptin inhibited mast cell activation and histamine levels in vitro. CONCLUSION: Sitagliptin suppresses the effector phase of AR, and this mechanism is partly attributed to the suppression of inflammatory response and mast cell degranulation.

Abnormal hyperplasia of chondrocytes in a rat model of glucocorticoid-induced osteonecrosis of the femoral head.

OBJECTIVE: The aim of our study was to determine whether abnormal hyperplasia of chondrocytes occurs in glucocorticoid-induced osteonecrosis of the femoral head (GC-ONFH) using a well-established rat model. MATERIALS AND METHODS: Rats were injected with lipopolysaccharide and methylprednisolone to induce GC-ONFH, while control animals were injected with saline (12 animals per group). Establishment of the disease model was confirmed using micro-computed tomography and hematoxylin-eosin (HE) staining of femoral head tissue sections. Chondrocyte hyperplasia was detected using HE staining and semi-quantitated using toluidine blue and saffron O staining. Expression of the autophagy marker LC3B was assessed in cartilage tissues of femoral head using immunohistochemistry. RESULTS: GC-ONFH animals showed significantly greater area of abnormal chondrocyte hyperplasia in femoral head tissue sections than control animals. They also showed significantly higher expression of LC3B in articular cartilage of the femoral head. CONCLUSIONS: GC-ONFH may be associated with abnormal chondrocyte hyperplasia in articular surface cartilage, which may be related to glucocorticoid-induced overactivation of autophagy.

Light Energy Dose and Photosensitizer Concentration Are Determinants of Effective Photo-Killing against Caries-Related Biofilms.

Caries-related biofilms and associated complications are significant threats in dentistry, especially when biofilms grow over dental restorations. The inhibition of cariogenic biofilm associated with the onset of carious lesions is crucial for preventing disease recurrence after treatment. This in vitro study defined optimized parameters for using a photosensitizer, toluidine blue O (TBO), activated via a red light-emitting diode (LED)-based wireless device to control the growth of cariogenic biofilms. The effect of TBO concentrations (50, 100, 150, and 200 µg/mL) exposed to light or incubated in the dark was investigated in successive cytotoxicity assays. Then, a mature Streptococcus mutans biofilm model under sucrose challenge was treated with different TBO concentrations (50, 100, and 150 µg/mL), different light energy doses (36, 108, and 180 J/cm2), and different incubation times before irradiation (1, 3, and 5 min). The untreated biofilm, irradiation with no TBO, and TBO incubation with no activation represented the controls. After treatments, biofilms were analyzed via S. mutans colony-forming units (CFUs) and live/dead assay. The percentage of cell viability was within the normal range compared to the control when 50 and 100 µg/mL of TBO were used. Increasing the TBO concentration and energy dose was associated with biofilm inhibition (p < 0.001), while increasing incubation time did not contribute to bacterial elimination (p > 0.05). Irradiating the S. mutans biofilm via 100 µg/mL of TBO and ≈180 J/cm2 energy dose resulted in ≈3-log reduction and a higher amount of dead/compromised S. mutans colonies in live/dead assay compared to the control (p < 0.001). The light energy dose and TBO concentration optimized the bacterial elimination of S. mutans biofilms. These results provide a perspective on the determining parameters for highly effective photo-killing of caries-related biofilms and display the limitations imposed by the toxicity of the antibacterial photodynamic therapy's chemical components. Future studies should support investigations on new approaches to improve or overcome the constraints of opportunities offered by photodynamic inactivation of caries-related biofilms.

In vitro evaluation of adhesion/proliferation of human gingival fibroblasts on demineralized root surfaces by toluidine blue O in antimicrobial photodynamic therapy.

BACKGROUND: Antimicrobial photodynamic therapy (aPDT) in Dentistry has important effects as bacterial destruction in areas with periodontal disease. Some dyes applied in aPDT could present low pH and, consequently, result in tooth demineralization. This study evaluated demineralization produced by aPDT with toluidine blue O (TBO) at low pH and analyzed adhesion/proliferation of human gingival fibroblasts (HGF). METHODS: In the 1st phase, bovine enamel and root dentin fragments received 2 treatments: PDT4 group (TBO-100 µg/ml-pH 4-60s) plus laser (660 nm, 45 J/cm(2), 1.08 J, 30 mW, 30 s, spot 0.024 cm(2), 1.25 W/cm(2), sweeping, non-contact) and CA group (citric acid plus tetracycline-pH 1-180 s). Surface hardness loss and tooth wear were statistically analyzed (Student's t test, ANOVA/Tukey, p<0.05). In the 2nd phase, human dentin fragments were divided in C (control group-scaling and root planing), PDT4 and CA. HGF (10(4), 5th passage) were cultured on these fragments for 24, 48 and 72 h and counted in scanning electron microscopy photographs. Number of HGF was analyzed using repeated-measures ANOVA and Tukey (p<0.05). RESULTS: Percentage of surface hardness loss was similar in dentin for PDT4 (71.5%) and CA (76.1%) (p>0.05) and higher in enamel for CA (68.0%) compared to PDT4 (34.1%) (p<0.05). In respect to wear, no difference was found between PDT4 (dentin: 12.58 µm, enamel: 12.19 µm respectively) and CA (dentin: 11.74 µm and enamel: 11.03 µm) (p>0.05). Number of HGF was higher after 72 h in CA group (2.66, p<0.05) compared to PDT4 (2.2) and C (1.33). CONCLUSION: PDT4 is not as aggressive as CA for enamel. However, dentin demineralized promoted by PDT4 does not stimulate HGF adhesion and proliferation as CA.

Toluidine Blue 0.05% Vital Staining for the Diagnosis of Ocular Surface Squamous Neoplasia in Kenya.

IMPORTANCE: Clinical features are unreliable for distinguishing ocular surface squamous neoplasia (OSSN) from benign conjunctival lesions. OBJECTIVE: To evaluate the adverse effects, accuracy, and interobserver variation of toluidine blue 0.05% vital staining in distinguishing OSSN, confirmed by histopathology, from other conjunctival lesions. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study in Kenya from July 2012 through July 2014 of 419 adults with suspicious conjunctival lesions. Pregnant and breastfeeding women were excluded. EXPOSURES: Comprehensive ophthalmic slitlamp examination was conducted. Vital staining with toluidine blue 0.05% aqueous solution was performed before surgery. Initial safety testing was conducted on large tumors scheduled for exenteration looking for corneal toxicity on histology before testing smaller tumors. We asked about pain or discomfort after staining and evaluated the cornea at the slitlamp for epithelial defects. Lesions were photographed before and after staining. MAIN OUTCOMES AND MEASURES: Diagnosis was confirmed by histopathology. Six examiners assessed photographs from a subset of 100 consecutive participants for staining and made a diagnosis of OSSN vs non-OSSN. Staining was compared with histopathology to estimate sensitivity, specificity, and predictive values. Adverse effects were enumerated. Interobserver agreement was estimated using the κ statistic. RESULTS: A total of 143 of 419 participants (34%) had OSSN by histopathology. The median age of all participants was 37 years (interquartile range, 32-45 years) and 278 (66%) were female. A total of 322 of the 419 participants had positive staining while 2 of 419 were equivocal. There was no histological evidence of corneal toxicity. Mild discomfort was reported by 88 (21%) and mild superficial punctate keratopathy seen in 7 (1.7%). For detecting OSSN, toluidine blue had a sensitivity of 92% (95% CI, 87%-96%), specificity of 31% (95% CI, 25%-36%), positive predictive value of 41% (95% CI, 35%-46%), and negative predictive value of 88% (95% CI, 80%-94%). Interobserver agreement was substantial for staining (κ = 0.76) and moderate for diagnosis (κ = 0.40). CONCLUSIONS AND RELEVANCE: With the high sensitivity and low specificity for OSSN compared with histopathology among patients with conjunctival lesions, toluidine blue 0.05% vital staining is a good screening tool. However, it is not a good diagnostic tool owing to a high frequency of false-positives. The high negative predictive value suggests that a negative staining result indicates that OSSN is relatively unlikely.

Tooth discoloration induced by endodontic phenothiazine dyes in photodynamic therapy.

OBJECTIVE: This study sought to assess if discoloration of tooth structures occurs after photodynamic therapy (PDT) and to determine the efficacy of a protocol to remove the photosensitizers. BACKGROUND DATA: PDT has been used in root canal treatment to enhance cleaning and disinfection of the root canal system. PDT uses a low power laser in association with a dye as a photosensitizer. Photosensitizers can induce staining of the dental structures, resulting in an unaesthetic appearance. METHODS: Forty teeth were randomly divided into four groups according to the photosensitizer used and pre-irradiation time: 0.01% methylene blue for 5 min (MB5); 0.01% methylene blue for 10 min (MB10); 0.01% toluidine blue for 5 min (TB5); and 0.01% toluidine blue for 10 min (TB10). Specimens were irradiated with a 660 nm diode laser with a 300 µm diameter optical fiber, at 40 mW power setting for 3 min. Immediately after, the photosensitizers were removed with Endo-PTC cream+2.5% sodium hypochlorite (NaOCl). The shade was measured by a Vita Easyshade spectrophotometer based on the CIELAB color system (L*a*b* values) at three different experimental times: before PDT (T0), immediately after PDT (T1), and after removal of the photosensitizer (T2). RESULTS: The results showed a decrease in the averages of the L*a*b* coordinate values after PDT (T1) in all the groups, when compared with the number at T0, with a significant statistical difference in group MB10. After photosensitizer removal (T2), all the values of the coordinates increased with significant statistical differences (p<0.05) between T1 and T2 in L* and a*. CONCLUSIONS: It can be concluded that both methylene blue and toluidine blue dyes cause tooth discoloration, and that Endo-PTC cream associated with 2.5% NaOCl effectively remove these dyes, regardless of the pre-irradiation time used for PDT.

Hemolytic anemia and methemoglobinemia in a preterm baby as a complication of antenatal intraamnial injection of toluidine blue.

A late preterm infant was born 4.5 h after intraamniotic injection of 90 mg of Toluidine blue to confirm premature rupture of membranes. Due to the fetal exposition to the dye, the entire body of the patient was blue stained and the baby suffered from methemoglobinemia, Heinz' body positive hemolytic anemia and hyperbilirubinaemia requiring exchange transfusion. These complications underline that antenatal exposition of toluidine blue may result in considerable postnatal infant morbidity. Therefore intraamniotic application of toluidine blue should be discouraged.

Toluidine blue for the intraoperative staining of the ureters. Studies on the safe administration in rats.

PURPOSE: Acute cardiovascular events have repeatedly been reported to occur during the intraoperative presentation of the urinary tract with toluidine blue (TB). We here assessed the minimum TB dose required, and its safest and most suitable form of intravenous administration for the intraoperative staining of the ureters in rats. METHODS: TB (0.13, 0.4, 1.3, or 4.0 mg/kg) was administered to anesthetized rats either by intravenous injection within 1 min or by infusion within 10 min. During the experiments,biomonitoring parameters such as electrocardiograms (ECGs)and mean arterial blood pressure (MAP) were recorded,blood gas analysis was performed, and methemoglobin measured. Tissue injury was assessed from released plasma enzyme activities and histopathologically. The intraoperative staining of the ureters was documented photographically,and total urinary excretion and final urine/plasma TB concentrations were determined. RESULTS: Parameters of blood gas analysis, methemoglobin concentrations, and markers of tissue injury were slightly affected by the two highest TB doses but not at all by the lower ones. At doses of ≥0.4 mg/kg, ureters were stained sufficiently. Staining was more intense, and urine excretion of TB higher on average when the dye was injected.The 1-min injection of ≥1.3 mg TB/kg strongly and temporarily decreased the MAP, while the infusions caused lesser effects. Mean ECG parameters were not affected by any TB administration, but one animal developed a temporary bundle branch block after the 1-min injection of 4.0 mg/kg. CONCLUSIONS: In rats, intravenous injection of 0.4 mg TB/kg was sufficient for the intraoperative staining of the urinary tract without the risk of severe cardiovascular and hemodynamic side effects. Provided our results are transferable to humans, the administration of low TB doses could allow its safer clinical use for the intraoperative visualization of the ureters.

[Ventricular fibrillation with tolonium chloride: axillary brachial plexus anesthesia led to cessation of circulation]. / Kammerflimmern nach Toluidinblau : Axilläre Plexusanästhesie führte zum Kreislaufstillstand.

A 22-year-old patient underwent surgery for a glass wound incision of the hand and anesthesia was carried out using an axillary brachial plexus block with prilocaine. Following surgery the patient developed methemoglobinemia which was treated with tolonium chloride. After administration of the drug the sinus rhythm changed into ventricular fibrillation. The current treatment options of methemoglobinemia will be discussed.

Avaliação da citologia e do teste do azul de toluidina no diagnóstico dos tumores malignos da mucosa oral / Evaluation of the cytology and the toluidine blue test in the diagnosis of oral malignant tumors

Introdução: a morte causada pelas neoplasias malignas está dentre as três mais comuns no Brasil (acidentes, doenças cardiovasculares e câncer). A maioria é diagnosticada em fases avançadas (estádios III e IV) e apenas 7% como carcinoma in situ. O diagnóstico precoce aumenta de maneira diretamente proporcional a fatores como: formação e informaçãodos profissionais da saúde, sistema de saúde amplo e a educação profissional da população. Objetivo: fornecer informa;áo para os profissionais no que diz respeito aos exames de citologia exfoliativa e teste do azul de toluidina para o diagnóstico do carcinoma oral, tendo com padrão-ouro o exame histopatológico. Pacientes e métodos: a pesquisa foi prospectiva, coletando-se dados referentes a 100 pacientes atendidos no ambulatório do Serviço de Cirurgia da Cabeça e Pescoço do Hospital Erasto Gaetner, em Curitiba, Paraná. Procedeu-se a coleta de material para exame citológico a Papanicolaou (das áreas de declive da boca, lavado da cavidade bucal e o raspado da lesão, nessa seqüência), aplicou-se o teste do azul de toluidina e realizou-se a biópsia ("saca-bocado") das áreas suspeitas (teste positivo ou não). Resultados: o teste do azul de toluidina teve sensibilidade de 80.9% e valor preditivo negativo de 70.30%. A citologia do raspado da lesão teve uma especificidade de 80.7%. Conclusão: a utilização do teste do azul de toluidina e do exame citológico do raspado da lesão em paralelo pode ser de valia para a detecçao de lesões malignas.

Introduction: the deaths caused by malignant neoplasms are among the three commonest causes in Brazil (trauma, cardiovascular diseases and cancer). The majority of cancer cases are diagnosesd in advanced stages (clinical stages III and IV) and only 7% are in situ carcinoma. The early diagnosis is directly related to factors such as: formation and information of health staff, wide health system and people education. Objective: to provide information to professionals regarding the exfoliative cytology and the toluidine blue stain test fro the oral cancer diagnosis, compared to the gold standard 0 the histopathological evaluation. Patients and methods: a prospective study was developed at the Erasto Gaertner hospital, in the ambulatory of the Head and Neck Surgery Service (Curitiba-Paraná), a hundred patients were included. , the cytologic exam (Papanicolaou staining) of the material collected (from the lower parts of the mouth and the lesion exfoliations, and from a mothwashing technic) was performed. The toluidine blue staining test was applied and the biopsy was collected from the suspicious areas (posistive test or not). Results: the toluidine blue test presented a 80,9% of sensibility and a negative predictive value of 70,3%, whereas the lesion exfoliative cytology presented a specificity of 80,7%. Conclusion: the association (at same time) of the toluidine blue test and the exfoliative cytologic exam can be effective and useful concerning the oral malignant lesions detection

Jejunal atresia related to the use of toluidine blue in genetic amniocentesis in twins.

Since 1990 avoidance of methylene blue as a dye in diagnostic amniocentesis is recommended. This is the result of the observation that a high incidence of jejuno-ileal atresia appeared in twin pregnancies following intraamniotic injection of methylene blue. We report a case of jejunal atresia in twins after injection of toluidine blue. We describe the clinical course, discuss possible teratogenic mechanisms and emphasize that no synthetic dyes should be used in second trimester amniocentesis.

Fluorescence biodistribution and photosensitising activity of toluidine blue o on rat buccal mucosa.

The antimicrobial activity of toluidine blue O (TBO) in the presence of red light has been demonstrated for a wide range of microorganisms. The response of tissues to TBO-induced photosensitisation is an important factor in assessing the clinical usefulness of this technique for the treatment of infectious diseases. The aims of this study were to determine the effect of TBO-mediated photosensitisation on rat buccal mucosa and the biodistribution of the photosensitiser in this tissue. An aqueous solution of TBO was applied to one side of the buccal mucosa of the animals. A 6 mm diameter area was then exposed to light (633 nm) from a copper vapour pumped-dye laser. The opposite, untreated, side of the buccal mucosa served as a control. TBO concentrations of 25, 50 and 200 microg/ml, laser light doses of 110, 170 and 340 J/cm(2) were assessed. Control groups of animals were subjected to 340 J/cm(2) laser light alone or to 200 microg/ml TBO alone. Serial sacrifices were performed after 72 h to obtain mucosal tissue samples for histological examination. For the determination of TBO biodistribution, additional groups received the same TBO doses and were sacrificed after 1 min or 10 min. Specimens were removed and frozen immediately for digital fluorescence imaging. No necrotic or inflammatory changes were found in the buccal mucosa of the animals with any of the treatments (using up to 200 microg/ml TBO and 340 J/cm(2) laser light). A high TBO fluorescence in the epithelium, particularly in the keratinised layer, with almost no fluorescence in the underlying connective tissue was demonstrated by the digital imaging. The results of this study suggest that TBO-mediated PDT (within the concentrations and light doses tested) could be a safe antimicrobial approach for the oral infections without damaging the adjacent normal tissue.