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Objectives The objective of this study was to determine the effects of antimicrobial photodynamic therapy (aPDT) with indocyanine green (ICG) and toluidine blue (TB) on protease activity (matrix-bound cathepsin K and matrix metalloproteinase (MMP) and dentin bond strength. Methods Caries-free human third molars were assigned to five groups: 1—control group, 2—application of ICG with activation using an 810 nm diode (aPDT), 3—application of ICG, 4—application of TB with activation using a 660 nm diode (aPDT), and 5—application of TB. For the enzymatic investigation, dentin beams were incubated for either 3 days or 3 weeks. Aliquots of the incubation media were analyzed by ELISA for CTX (C-terminal cross-linked telopeptide of type I Collagen) and ICTP (cross-linked carboxy-terminal telopeptide of type I collagen). For microtensile bond strength testing (μTBS), composite resins were layered onto the tooth surface; the samples were then subjected to μTBS. Kruskall–Wallis and Mann–Whitney U tests were applied for statistical analysis of CTX and ICTP, one way-ANOVA and Tukey’s test were applied for statistical analysis of μTBS. Results Pretreating the dentin matrices with aPDT decreased the endogenous protease activity. ICG with laser activation resulted in the highest μTBS. Therefore, aPDT should be considered as a treatment method because it can reduce MMP-mediated dentin degradation and increase the μTBS. Significance Inhibiting endogenous protease activity improves the stability of the dentin–adhesive bond and the durability of the bond strength.

Atualmente os exames de sêmen bovino fornecem um grau limitado de informação de prognóstica e diagnóstica da fertilidade, o que demostra a necessidade de testes de função espermática, incluindo análises da integridade da cromatina. Neste contexto, foi proposto avaliar o desempenho de diferentes técnicas de identificação de defeitos de cromatina espermática de touros (Bos taurus) e correlacionar os resultados com a taxa de clivagem e a formação de blastocisto nas rotinas de produção in vitro de embriões (PIVE), com intuito de se identificar os métodos mais eficientes, de menor custo e de fácil aplicação no campo e em laboratórios de reprodução animal. Foram utilizadas quatro amostras de sêmen congeladas em meio TRIS-gema de quatro touros (Bos taurus) Girolandos com diferentes graus de fertilidade. Essas amostras foram submetidas à avaliação da cromatina espermática por diferentes técnicas: avaliação visual e computacional de esfregaços corados com azul de toluidina (AT) e com reação de Feulgen (RF), avaliação visual de esfregaços corados com laranja de acridina (LA) em microscopia de fluorescência e microscopia confocal de varredura a laser, avaliação em microscopia eletrônica de transmissão (MET) e SCSA (sperm chromatin structure assay). As mesmas amostras foram utilizadas em rotinas de produção in vitro de embriões, das quais foram calculadas as taxas de clivagem e blastocisto. Os resultados obtidos em cada técnica foram correlacionados entre eles e com as taxas de clivagem e blastocisto. De maneira geral, todos as alterações de cromatina identificadas por qualquer que seja o método, apresentaram maiores correlações com a taxa de blastocisto do que com a de clivagem, possivelmente porque as alterações cromatínicas interferem mais no desenvolvimento embrionário inicial do que na capacidade fecundante dos espermatozoides. Dentre os métodos testados, a avaliação de alterações de cromatina por microscopia eletrônica de transmissão (MET) e a coloração com laranja de acridina (LA) avaliado em microscopia de florescência e microscopia confocal de varredura a laser foram os mais eficazes para identificar alterações de cromatina que interferem na eficiência da produção in vitro de embriões bovinos. No entanto, a MET é de altíssimo custo. As técnicas que utilizam esfregaços corados com AT e Feulgen, mesmo sendo menos eficazes que MET e LA, são as de menor custo e podem ser utilizadas com relativa eficácia. Currently, bovine semen tests provide a limited degree of prognostic and diagnostic information on fertility, which demonstrates the need for sperm function tests, including analyses of chromatin integrity. In this context, it is proposed to evaluate the performance of different techniques for identifying sperm chromatin defects in bulls (Bos taurus) and to correlate the results with the cleavage rate and blastocyst formation in routines of in vitro embryo production (IVP), with the aim of identifying the most efficient, lowest-cost and easy-to-apply methods in the field and in animal reproduction laboratories. Four samples of semen frozen in TRIS-yolk medium from four Girolando bulls (Bos taurus) with different degrees of fertility were used. These samples were submitted to sperm chromatin evaluation by different techniques: visual and computational evaluation of smears stained with toluidine blue (TB) and with Feulgen reaction (FR), visual evaluation of smears stained with acridine orange (AO) under fluorescence microscopy and confocal laser scanning microscopy, evaluation in transmission electron microscopy (TEM) and SCSA (sperm chromatin structure assay). The same samples were used in routine in vitro embryo production, from which cleavage and blastocyst rates were calculated. The results obtained in each technique were correlated with each other and with the cleavage and blastocyst rates. In general, all the chromatin alterations identified by any method presented greater correlations with the blastocyst rate than with the cleavage rate, possibly because the chromatin alterations interfere more in the early embryonic development than in the fertilizing capacity of the embryos. sperm. Among the methods tested, the evaluation of chromatin alterations by transmission electron microscopy (TEM) and staining with acridine orange (AO) evaluated by fluorescence microscopy and confocal laser scanning microscopy were the most effective to identifyin identifying chromatin alterations. that interfere with the efficiency of in vitro bovine embryo production. However, TEM is very expensive. The techniques that use smears stained with AT and Feulgen, despite being less effective than TEM and AO, are the least expensive and can be used with relative effectiveness. Dissertação (Mestrado)

The expansion of wheat crops (Triticum spp.) to the Brazilian Cerrado highlights the need to use pathogen-free seeds. This study aimed at evaluating the efficacy of nanoparticles (NPs) and photodynamic therapy (PhT) in inhibiting the in vitro growth of the fungus Alternaria alternata, in its treatment and control in naturally contaminated wheat seeds, and in the physiological quality of the seeds. The efficacy of NPs (ZnOCl, ZnOCl:1Cu, ZnOCl:0.1Ag; ZnO:1Cu, ZnO, and ZnO:1Ag) and PhT using methylene blue (MB) and toluidine blue (TB) dyes was evaluated in inhibiting the mycelial growth of A. alternata and in the treatment and control of the pathogen in wheat seeds by evaluating germination, emergence, GSI, ESI, accelerated ageing, and health. All NPs at 2.5 and 5 mg mL-1 concentrations and the dyes MB, TB, MB + TB at 50 and 100 µmol L-1 inhibited mycelial growth and reduced the incidence of A. alternata in the seeds. The NP ZnO:1Ag at 5 mg mL-1 and the MB + TB dye at 100 µmol L-1 were the most effective in inhibiting mycelial growth. NPs and PhT did not affect the physiological quality of seeds and controlled A. alternata in wheat seeds, demonstrating potential use in the treatment and control of the pathogen in wheat seeds. RESUMO Com a expansão da cultura do trigo (Triticum spp.) para o Cerrado brasileiro, evidenciando-se a necessidade da utilização de sementes livres de patógenos. O presente trabalho teve como o objetivo avaliar a eficácia de nanopartículas (NPs) e da terapia fotodinâmica (PhT) na inibição do crescimento de Alternaria alternata in vitro, no tratamento, no controle do fungo em sementes de trigo naturalmente contaminadas e na qualidade fisiológica das sementes. A eficácia das NPs (ZnOCl, ZnOCl:1Cu, ZnOCl:0,1Ag; ZnO:1Cu, ZnO e ZnO:1Ag) e da PhT com uso dos corantes azul de metileno (MB) e azul de toluidina (TB) foram avaliadas na inibição do crescimento micelial de A. alternata, no tratamento, no controle do patógeno em sementes de trigo, avaliando-se a germinação, emergência, IVG, IVE, envelhecimento acelerado e a sanidade das sementes. Todas as NPs nas concentrações 2,5 e 5 mg mL-1 e os corantes MB, TB, MB+TB a 50 e 100 µmol L-1 inibiram o crescimento micelial e reduziram a incidência de A. alternata nas sementes. A NP de ZnO:1Ag a 5 mg mL-1 e os corantes MB+TB a 100 µmol L-1 foram os mais eficazes na inibição do crescimento micelial. As NPs e PhT não afetaram a qualidade fisiológica das sementes e controlaram A. alternata nas sementes de trigo, demonstrando potencial de uso no tratamento e no controle do patógeno em sementes de trigo.

Background and Aim: Mast cell tumors (MCTs) are malignant neoplasms that are common in dogs. Their biological behavior is variable and unpredictable. The aim of the present study was to analyze the histological classification and expression of markers of canine MCTs. Materials and Methods: Thirty samples of canine MCTs were graded according to the histological classification methods of Patnaik and those of Kiupel. The expression of phosphoprotein 53 (p53) and c-kit proteins was quantified by immunohistochemistry using image processing software, ImageJ - a public domain computer program, developed at the National Institutes of Health. Results: It was possible to determine the grade of 100% of the samples. According to Patnaik's classification, 20.00% of the samples were Grade 1, 43.30% were Grade 2, and 36.70% were Grade 3. According to Kiupel's classification, 56.67% of the samples were of high intensity and 43.33% were of low intensity. Grade 1 tumors had the highest expression of p53 and c-kit, and Grade 2 had the lowest expression. The results showed that it is necessary to perform both histological grading methods. The classification into high and low intensity may provide more consistent results than the three-level grading system. However, a smaller number of categories, although it facilitates the classification, may not be sufficient for the prognosis. Conclusion: Quantitative evaluation of p-53 and c-kit expression is a useful tool to increase the accuracy of the analysis and to aid in choosing the treatment method for canine MCTs. Histological grading should be combined with other diagnostic methods.

Snail shells (Anadora Fulica) calcined at different temperatures were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, thermal analyses (TG-DTG), scanning electron microscopy (SEM) and N2 adsorption–desorption experiments (surface area measurements were found using the coupled BET/BJH method). The principal objective was to identify different forms of calcium carbonate and calcium hydroxide in snail shells as raw materials. The calcium hydroxide thus obtained was used in the synthesis of the hydroxyapatite/L-lysine (HA/Lys) composite. The composite used to chemically modify a glassy carbon electrode (GCE) was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). It appeared that the developed sensor Lys/HA/GCE facilitated electronic transfer compared to the pristine electrode. In a strongly acid medium, this surface protonated and therefore became positively charged, which allowed it to have a good affinity with [Fe(CN)6]3-. An application in toluidine blue (TB) electroanalysis in the phosphate buffer was carried out. Optimal sensor performances were obtained using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The performance of the sensor was determined in the concentration range 1 to 10 µM of TB, and the limit of detection (LOD) obtained by the S/N = 3 method was 2.78 × 10−7 M. The sensor was also used to detect the TB in spring water at 96.79% recovery.

Background: Any oral potentially malignant disorders (OPMDs) must be regularly monitored through clinical examination to detect any possible malignant transformation. Conventional intraoral exams, however, can be difficult because these conditions may resemble benign lesions. For this reason, several non-invasive diagnostic technologies have been developed to help the clinician in detecting and distinguishing between cancerous and benign lesions. Epithelial dysplasia can be considered the most important predictor of malignant evolution. Therefore, in this study we aim to evaluate the ability of an optical filter for autofluorescence Glasses for Oral Cancer Curing Light Exposed (GOCCLES®) and of toluidine blue staining in identifying dysplastic areas in patients with OPMDs. Methods: In this retrospective study, medical records, photographs and videos of 25 patients with oral lesions were analyzed. Forty-two biopsy samples in 25 patients with OPMDs and at least one suspicious oral mucosa lesion that were evaluated in white light, autofluorescence with optical filter GOCCLES®, toluidine blue staining and then biopsied with histopathological analysis were analyzed. Results: The sensitivity and specificity for the autofluorescence evaluation with GOCCLES® for identifying dysplasia or carcinoma were 66% and 48%, respectively. The positive and negative predictive values were 34% and 77%, respectively, and the accuracy was 53%. The sensitivity and specificity for toluidine blue staining were 91% and 68%, respectively. The positive and negative predictive values were 55% and 95%, respectively, and the accuracy was 75%. Conclusions: The optical filter for autofluorescence (GOCCLES®) and toluidine blue staining are simple, inexpensive, rapid and non-invasive procedures that can assist the clinician in distinguishing OPMDs from healthy mucosa but they are not able to distinguish benign and malignant lesions.

Introduction: Cervical cancer is the leading cancer in Indian women. Mast cell distribution and density support a diagnosis in addition to routine histopathological examination. Aim: To study the variation of mast cell distribution and density in different uterine cervical lesions. Materials and Methods: A total of 352 cases of cervical lesions were studied cross-sectionally from June 2018 to May 2020, in the Department of Pathology, Subharti Medical College, and associated Chhatrapati Shivaji Hospital, Meerut, Uttar Pradesh, India. These included both hysterectomy (296 cases) and cervical (56 cases) biopsies. Routine histopathological examination was done with Haematoxylin and Eosin stain (H&E) to categorise the lesions into non-neoplastic and Neoplastic lesions. For identification of mast cells 1% toluidine blue stain was applied to evaluate mast cell distribution and density. Data analysis was done using analysis of variance (ANOVA): one-way t-test was applied. A p-value of ≤0.05 was taken as significant. R-Software was used for analysis of data. Results: Out of 352 cases, 302 (85.7%) were non-Neoplastic and 14% 48 (13.6%) were neoplastic. Two cases were of unremarkable pathology. Chronic cervicitis was observed as the most common lesion 293 (97%) out of 302 cases. Age of the patients ranged from 21-78 years. Most of the patients were in the reproductive age group: 21-49 years of age 274 (77.8%) cases. Mean mast cell count decreased from premalignant cases to malignant cases. In case of cervical intraepithelial lesion, mean mast cell count was found to be 26.5/10 High Per Field (HPF) and in cases of malignancy mean mast cell count of 17.5/10 HPF was observed. Mean mast cell count showed an inverse relationship with the grade of dysplasia and from dysplasia to cases of malignancy. Conclusion: Mast cell distribution and density support a diagnosis in addition to the routine histopathology, especially in cases of small cervical biopsies which are difficult to interpret.

Background: Ocular surface squamous neoplasia (OSSN) is the most common ocular tumour. The diagnosis of OSSN is based on clinical suspicion and confirmed by various diagnostic modalities, of which histology is the gold standard. With the move to less invasive management options such as topical chemo- or immunotherapy, less invasive diagnostic options have come to the fore. Aim: The purpose of this article was to review the current staging and diagnostic modalities for OSSN with a focus on less invasive modalities. Method: A literature review was performed for publications on ocular surface neoplasia and diagnostic modalities. Results: Histology is the gold standard for diagnosing OSSN. Cytology has been shown to be a simple, repeatable and minimally invasive diagnostic modality, which also allows for additional testing such as polymerase chain reaction. Anterior segment optical coherence tomography provides a non-contact method of evaluating the ocular surface, with OSSN showing a thickened hyper-reflective epithelium, abrupt transition zone and demarcation line. Vital dyes are used less commonly with high sensitivity, but lower specificity for OSSN. Finally, confocal microscopy provides en-face images of the ocular surface, with OSSN showing a classic ‘starry night’ appearance. Conclusion: Histology remains the gold standard for diagnosis; however, with the increasing use of topical therapy for OSSN, there has been an increase in the uptake of less invasive diagnostic modalities.

Background Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide accounting for 90% of all malignant oral lesions with high mortality and a five-year survival rate of about 50%. Various studies have shown mast cells regulate carcinogenesis by immunosuppression, angiogenesis enhancement, and promotion of tumor cell mitosis. Aim Hence, the present study was aimed to compare mast cell counts in normal oral mucosa with histological grades of oral squamous cell carcinoma by using toluidine blue stain. Methodology Sixty formalin-fixed, paraffin-embedded tissue samples included 15 well-differentiated, 15 moderately differentiated, and 15 poorly differentiated OSCC, as well as 15 cases of the normal oral mucosa (control), were sectioned and stained with 1% toluidine blue. Results We observed that the mean mast cell (MMC) count was comparatively more in normal mucosa than in various grades of OSCC. It was higher in low-grade OSCC. However, the differences between grades were not statistically significant. Conclusion In the present study, according to the results obtained, the MMC count was significantly decreased in OSCC in comparison with normal oral mucosa. Therefore, it can be assumed that mast cells could serve as an indicator of tumor progression.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A tropicalização da canola (Brassica napus) e sua recente introdução no Triângulo Mineiro e Alto Paranaíba evidenciam a importância de que as sementes a serem utilizadas estejam livres de patógenos. A bactéria Xanthomonas campestris pv. campestris (Xcc), agente causal da podridão negra das brássicas, é disseminada principalmente pelas sementes. Dessa forma, métodos alternativos de controle da bactéria nas sementes devem ser avaliados. O objetivo deste trabalho foi avaliar a eficácia dos métodos terapia fotodinâmica e o uso de nanopartículas no controle de X. campestris pv. campestris in vitro e no tratamento de sementes de canola. Na terapia fotodinâmica foram utilizados os corantes fotossensíveis Azul de Metileno, Azul de Toluidina e a combinação de ambos, em diferentes concentrações in vitro e no tratamento de sementes. Diferentes nanopartículas, em diferentes concentrações foram avaliadas no controle de Xcc in vitro e no tratamento de sementes. Nas sementes tratadas foram avaliadas a porcentagem de germinação, a emergência, o índice de velocidade de emergência, o teste de sanidade e a detecção da bactéria nas sementes. Os corantes Azul de Metileno, Azul de Toluidina e a associação deles nas concentrações 100, 50 e 25 µmol L-1 respectivamente, quando submetidos à irradiação, foram eficazes no controle de Xcc in vitro. Os nanocristais de ZnO dopados com Ag, Au, Cu, Fe, Mn e Ni inibiram o crescimento bacteriano in vitro em diferentes concentrações, no entanto, o nanocristal de ZnO:11Ag apresentou o maior halo de inibição. Não foi possível estimar a eficiência dos corantes e das nanopartículas no controle da bactéria nas sementes, devido à baixa taxa de infecção das mesmas. Ensaios deverão ser conduzidos para avaliar diferentes concentrações dos corantes e das nanopartículas, no controle da bactéria em sementes, visto que estes apresentaram toxicidade às sementes de canola. Canola (Brassica napus) has been introduced in the Triângulo Mineiro and Alto Paranaíba in the recent years, but to keeping it up growing the seeds used must be free of pathogens. One of the most important bacterium disseminated through the canola seeds is Xanthomonas campestris pv. campestris (Xcc) causal agent of black rot in brassicas. Therefore, the objective of this work was to evaluate the efficacy of photodynamic therapy methods and the use of nanoparticles for the control of X. campestris pv. campestris in vitro and in the canola seed treatment. In the experiment of photodynamic therapy were used the photosensitive dyes, Methylene Blue, Toluidine Blue and the combination of both in different concentrations in vitro and in the seed treatment. In the second experiment were used different nanoparticles and its concentrations to the control of Xcc in vitro and in the seed treatment. Germination percentage, emergence rate, emergence speed index, sanity test and detection of the bacterium in the seeds were evaluated. Methylene Blue, Toluidine Blue and their association at concentrations 100, 50 and 25 μmol L-1 respectively, under irradiation were effectives to the Xcc control in vitro. The nanocrystals from ZnO doped with Ag, Au, Cu, Fe, Mn and Ni inhibited the bacterial growth in vitro using different concentrations, however, the nanocrystal ZnO:11Ag showed the larger bacterial inhibition halo. Therefore, it was not possible to estimate the effectiveness of the dyes and nanoparticles in the control of the bacteria in the seeds due to the low infection rate. Thus, new assays should be conducted to evaluate the concentration of dyes and nanoparticles in the control of the bacteria in seeds mainly because it presented toxicity to the canola seeds. Dissertação (Mestrado)

*Erdönmez, Demet ( Aksaray, Yazar ), Background: Traditional chemomechanical treatment procedures are an indispensable part of endodontic treatment, however, additional treatment approaches such as antimicrobial photodynamic therapy (aPDT) may also be recommended for the elimination of residual microorganisms. In this study, the disinfection efficiency of aPDT performed using methylene blue (MB), toluidine blue (TB), and tetra 2-mercaptopyridine substituted zinc phthalocyanine (TM-ZnPc) was compared in the roots contaminated with Enterococcus faecalis (E. faecalis). Materials and methods: Forty-nine teeth with a single root and canal were included in this study. The roots were sterilized, and inoculated with E. faecalis. The roots were kept in an incubator for 30 days to form the biofilm. Forty-five teeth were prepared up to the F3 file of the ProTaperNext system under 2.5 % NaOCL irrigation. The samples were divided into three groups according to the type of used photosensitizer (PS) (n = 15); MB (313 μM), TB (327 μM), and TM-ZnPc (6μM). All PSs were irradiation with a light-emitting diode (LED) lamp (630 nm, 2–4 mW/cm²) for the 60 s. Two microbiological samples of the intracanal content were taken (one before and one immediately after additional aPDT in all groups) using sterile paper points. The colony-forming units per milliliter (CFU/mL) were calculated after 24 h of incubation at 37 °C. Results: After all aPDT protocols, intracanal bacterial load decreased significantly compared to the amount after chemomechanical preparation (P < 0.05). No significant difference in the reduction in intracanal bacterial load was found between the PSs (P < 0.05). Conclusions: In the current study, the aPDT protocol performed with TM-ZnPc provided similar antimicrobial efficacy, although it was used at a lower concentration compared to MB and TB. Therefore, the use of TM-ZnPc in intra-canal disinfection in endodontics seems promising.

Background When the resected specimen is sent for intraoperative margin assessment, all margins are grossly checked, and selected margins undergo a frozen section (FS) examination. Therefore, there is a possibility of sampling error. This study evaluated the effectiveness of using toluidine blue (TB) as an intraoperative triage screening tool to detect positive mucosal margins of the resected specimens of oral squamous cell carcinoma (OSCC) and serve as a guide for FS sampling. Methods Surgical samples of 30 consecutive patients with biopsy-proven OSCC were included in the study. A total of 140 mucosal margins were analyzed intraoperatively by TB and FS, the results were compared with the final histopathology. Results Of the 140 examined mucosal tumor margins, 14 stained positives with TB, six were true-positives, eight were false-positives, and there were no false-negatives, as confirmed by final histopathology of the same margins. The diagnostic performance measures were sensitivity 100.0%; specificity 94.0%; positive predictive value (PPV) 42.9%; negative predictive value (NPV) 100.0%; and accuracy 94.3% (95% CI: 89.0–97.5%). For FS, there were three true-positives, three false-negatives, and no false-positives. The diagnostic performance measures were sensitivity 50.0%; specificity 100.0%; PPV 100.0%; NPV 97.8%; and accuracy 97.9% (95% CI: 93.9–99.6%). Conclusion TB is less specific but more sensitive than FS for detecting positive mucosal margins of resected OSCC. Screening the tumor mucosal margins with TB before FS sampling may help identify more tumor-bearing margins. Trial registration This trial was registered at ClinicalTrials.gov. Registration number: NCT03554967. Registration date: June 13, 2018.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais Diversos estudos têm demonstrado que durante a passagem do espermatozoide através do epidídimo a cromatina espermática sofre a formação de pontes de dissulfureto entre as protaminas, aumentando a compactação do material genético, etapa fundamental para a proteção e manutenção da estabilidade do DNA. O presente estudo tem por objetivo avaliar o processo de compactação da cromatina espermática durante a maturação do espermatozoide no epidídimo de camundongos. Para isso foram colhidos espermatozoides das regiões da cabeça, corpo e cauda do epidídimo de seis camundongos. Os espermatozoides foram avaliados por análise de imagem computacional de esfregaços corados com azul de toluidina, sendo analisada a distribuição espacial do processo de compactação da cromatina e a descompactação e heterogeneidade cromatínicas médias das cabeças dos espermatozoides. Os espermatozoides também foram avaliados por microscopia eletrônica de transmissão (MET), sendo definidos quatro níveis de baixa compactação cromatínica. A proporção de espermatozoides com alta compactação cromatínica (maduros) foi menor na cabeça quando comparada as demais regiões epididimárias, em ambas as metodologias utilizadas. Além disso, os níveis de baixa compactação na metade basal (BMB) e baixa compactação total (BCT) foram prevalentes nesta região. No corpo, houve o predomínio da baixa compactação na base (BB) e na região central (BRC). Na cauda, todos os níveis de baixa compactação diminuíram consideravelmente, sendo praticamente inexistentes, exceto na região central (BRC), que ainda prevaleceu em pequena quantidade. Baseado nesses dados foi proposta uma sequência do processo de compactação cromatínica no espermatozoide murino. A compactação teria início na extremidade apical em direção a base e das laterais para o centro da cabeça do espermatozoide, compactando-se até próximo da base. Posteriormente, a compactação se daria no sentido da inserção da cauda (base) para o centro e por fim, na região central da cabeça. A análise da descompactação e heterogeneidade médias da cromatina feitas por análise de imagem de esfregaços corados com AT e a realizada por MET demonstraram que o processo de compactação cromatínica do espermatozoide de camundongos iniciado durante a espermiogênese, continua durante o trânsito pelo epidídimo. Several studies have shown that during the passage of the spermatozoid through the epididymis, the spermatic chromatin undergoes the formation of disulfide bridges between the protamines increasing the condensation of the genetic material, a fundamental step for the protection and maintenance of DNA stability. The present study aims to evaluate the process of sperm condensation of chromatin during spermatozoid maturation in the epididymis of mice. For this, spermatozoa were collected from the head, body and tail regions of the epididymis of six mice. These spermatozoa were evaluated by computational image analysis of smears stained with toluidine blue, analyzing the spatial distribution of the condensation of chromatin process and, the mean decondensation and heterogeneity of the sperm heads. Spermatozoa were also evaluated by transmission electron microscopy (TEM), and four levels of non-condensation were defined. The proportion of spermatozoa with high chromatin (mature) condensation was lower in the head when compared to the other epididymal regions, in both methodologies used. In addition, low condensation levels in the basal half (LBH) and the entire length of the head (LCT) were prevalent in this region. In the body, there was a predominance of low condensation of the base (LB) and in the central region (LCR). In the tail, all levels of low condensation decreased considerably, being practically non-existent, except in the central region (LCR), which still were prevailed in small numbers. Based on these data, a sequence of the condensation process in the murine spermatozoa was proposed. The condensation would start at the apical region towards the base and from the lateral sides to the center of the spermatozoa's head, and compaction process would finish near the base. Subsequently, the compaction would occur in the direction of the insertion of the tail (base) to the center and finally, in the central region of the head. The analysis of the means of decondensation and heterogeneity of the chromatin made by image analysis of TB stained smears and the ones which were performed by TEM demonstrated that the process of chromatin compression of mice spermatozoa started during spermiogenesis continues during transit in the epididymis. Dissertação (Mestrado)

CAPS - Centro de Atenção Psicosocial Evidências sugerem prejuízos no desempenho reprodutivo de touros causados pelo estresse calórico, sendo que touros zebuínos são considerados mais resistentes a este tipo de estresse. No entanto, não se sabe se o manejo correto de touros pode minimizar este efeito deletério durante períodos mais quentes do ano. Objetiva-se com o presente trabalho comparar a qualidade de doses de sêmen de touros zebuínos e taurinos produzidos no inverno e no verão na microrregião de Uberaba, MG, Brasil. As amostras foram avaliadas quanto à concentração, motilidade, morfologia dos espermatozoides, morfometria da cabeça dos espermatozoides, descompactação e heterogeneidade médias da cromatina espermática e quanto a localização da descompactação da cromatina espermática. As avaliações de concentração, motilidade e morfologia foram realizadas conforme preconizado pelo Colégio Brasileiro de Reprodução Animal. As avaliações morfométricas e da cromatina espermática foram feitas utilizando técnicas de análises computacionais de imagens de esfregaços de sêmen, hidrolisados com ácido clorídrico e corados com azul de toluidina. As análises morfométricas demonstraram que em taurinos as células possuem área maior em doses produzidas no verão do que no inverno. Quando comparada à morfometria das doses entre zebuínos e taurinos, independente da estação, os taurinos demonstraram área maior em suas células, em comparação aos touros zebuínos. A harmônica 2 da transformada de Fourier, simetria latero-lateral, descompactação e heterogeneidade da cromatina também foram maiores no grupo de taurinos. Ao comparar a região da cabeça com descompactação de cromatina, o grupo de taurinos apresentou maior número de células normais (sem alterações de cromatina) durante o verão e maior descompactação na metade basal (DMB) das cabeças durante o inverno. DMB também foi maior no grupo de taurinos quando comparados com os zebuínos, sem levar em conta a estação do ano. Quando comparado todas as doses produzidas no verão com o inverno sem levar em consideração a raça, as doses do inverno apresentaram maior número de DMB. Ao comparar a morfologia, as doses produzidas no inverno, independente da raça, apresentaram maior número de células com morfologia normal do que nas doses do verão, que apresentaram maior número de defeitos menores. As doses de touros zebuínos apresentaram uma tendência de maior concentração durante o verão do que no inverno. Já quando comparadas todas as doses, independente da raça, as de verão apresentaram maior concentração do que as doses do inverno. Em geral não houve muitas diferenças entre as doses produzidas durante o inverno e o verão, independente das raças. Essas poucas diferenças podem estar ligadas ao manejo dos touros na central em que as doses foram produzidas. O sombreamento dos piquetes e banhos frequentes dos animais durante os dias quentes podem ter amenizado as interferências do estresse térmico nesses animais, uniformizando a qualidade das doses durante o ano. Evidences suggests impaired reproductive performance of bulls caused by caloric stress, and Bos taurus indicus bulls are considered more resistant to this type of stress. However, it is not known whether correct management of bulls can minimize this deleterious effect during warmer periods of the year. The objective of the present study was to compare the quality of sêmen’s strows of B. taurus and B. indicus produced in winter and summer in the microregion of Uberaba, MG, Brazil. Samples were evaluated for sperm concentration, motility, sperm morphology, sperm head morphometry, decondensation and heterogeneity of sperm chromatin and the location of sperm chromatin decondensation. Concentration, motility and morphology assessments were performed as recommended by the Brazilian College of Animal Reproduction. Morphometric and sperm chromatin evaluations were performed using computational image analysis techniques of semen hydrolyzed with hydrochloric acid and stained with toluidine blue. The morphometric analyzes showed that in B. taurus the cells have larger area in strows produced in summer than in winter. When compared the morphometry of the doses between B. indicus and B.taurus, regardless of the season, the taurines showed larger area in their cells, compared to the zebu bulls. Fourier transform harmonic 2, side symmetry, decondensation and chromatin heterogeneity were also higher in the B. taurus group. When comparing the head region with chromatin decondensation, the B. tourus group presented higher number of normal cells (without chromatin changes) during the summer and higher decondensation in the basal half (DMB) of the heads during the winter. DMB was also higher in the B. taurus group compared to B. indicus, regardless of the season. And when compared all doses produced in summer with winter without considering clattle, winter doses showed higher number of DMB. When comparing the morphology, the strows produced in winter, regardless of race, presented higher number of cells with normal morphology than in summer doses, which presented higher number of minor defects. B. indicus’s strows tended to be more concentrated during summer than in winter. When compared all doses, regardless of cattle, summer presented higher concentration than strows produced during winter. In general there were not many differences between the strows produced during winter and summer, regardless of the races. These few differences may be linked to the management of bulls in the place where the strows were produced. The shading of paddocks and frequent bathing of animals during hot days may have softened the interference of heat stress in these animals, uniforming the quality of doses during the year. Dissertação (Mestrado)

SUMMARY: This study was aimed at comparing the commonly used metachromatic stains viz., Papanicolaou stain, WrightGiemsa, Toluidine blue and Methylene blue in the assessment of cell types of the oestrous cycle in rats. Eight female Sprague-Dawley rats aged 8-9 weeks were used for this assessment. Cotton Swabs were gently inserted in the animals vagina to obtain cells from which they were then transferred to glass slides for staining and evaluation under microscopy. The different cell types were compared for their morphological features and clarity of cellular detail under all four stains. The application, advantages and limitations of all stains were then discussed. It was concluded that the selection of the most effective stain in the assessment of vaginal cytology depends on their application to clinical or research which was based on the cellular detail of interest, time, cost and availability of each staining procedure.

Objective: Oral cancer is a significant threat to public health all over the world, especially in Southeast Asia. At the present time, screening of oral cancer, its premalignant stages as well as its early detection, is still largely based on visual examination of the mouth. Visual examination is highly subjective and hence lacks the specificity and sensitivity. The objective of this study was to determine the usefulness of toluidine blue in marking a biopsy site in potentially malignant disorders. Methods: In this study, a total of 500 patients were screened. The study was a case–control study which included 17 lesion cases and 23 normal controls. Toluidine blue staining was taken into consideration to identify clinically doubtful oral potentially malignant lesions and to compare the clinical evaluation with toluidine blue stain followed by a punch biopsy and histological evaluation. SPSS Statistics version 16.0 and Chi-square test were used for statistical analyses. Results: The most common site for potentially malignant lesions was found to be the buccal mucosa. The sensitivity of toluidine blue was found to be 88.89%, while specificity was found to be 74.19%. The positive predictive and negative predictive values were 50% and 97.83%, respectively. P = 0.000672 was considered statistically significant. Conclusions: The results seem to be promising, but many such studies have to be done at larger scales to exactly help us in identifying the capability of toluidine blue in the long run.

In the existing study, a new vortex-assisted cloud point extraction (VA-CPE) method was developed for determination of low levels of thiosulfate in environmental waters at 632 nm by spectrophotometry. The method is selectively based on charge-transfer-sensitive ion-pair complex formation of Ag(S2O3)2 3−, which is produced by the reaction of thiosulfate with excess Ag+ ions with toluidine blue (3-amino-7-dimethylamino-2-methylphenazathionium chloride, TB+) and then its extraction into micellar phase of polyethylene glycol 4-tert-octylphenyl ether (Triton X-45) in presence of Na2SO4 as salting-out agent at pH 7.0. All the factors affecting complex formation and VA-CPE efficiency were optimized in detail. Under the optimized conditions, the linear calibration curves for thiosulfate were in the range of 0.2–120 and 5–180 µg L−1 with sensitivity improvement of 81-folds and 15-folds, respectively, as a result of efficient mass transfer obtained by CPE with and without vortex, while it changed in the range of 260–3600 µg L−1 without preconcentration at 642 nm. The limits of detection and quantification of the method for VA-CPE were found to be 0.05 and 0.22 µg L−1, respectively. The precision (expressed as the percent relative standard deviation) was in range of 2.5–4.8% (5, 10 and 25 µg L−1, n: 5). The method accuracy was validated by comparing the results to those of an independent 5,5′-dithiobis(2-aminobenzoic acid) (DTNB) method as well as recovery studies from spiked samples. It has been observed that the results are statistically in a good agreement with those obtained by DTNB method. Finally, the method developed was successfully applied to the preconcentration and determination of trace thiosulfate from environmental waters.

Systemic mastocytosis (SM) characterized by focal or diffuse infiltrates of neoplastic mast cells can range from indolent cutaneous disease to aggressive systemic disease. Skin involvement is usual in indolent systemic mastocytosis, less frequent in aggressive SM, and rare in mast cell leukemia. Here, we present a rare coexistence of cutaneous mastocytosis and SM with an associated hematologic neoplasm SM-associated hematologic neoplasm [AHN] (atypical chronic myeloid leukemia [CML]-BCR/ABL negative), the first case being reported in the literature so far to the best of our knowledge. An elderly male with leukocytosis, myeloid hyperplasia, and mast cell infiltrates in the marrow was diagnosed with atypical CML and cutaneous mastocytosis. Approximately 40% of cases of SM are associated with a hematologic neoplasm (SM-AHN) encompassing all defined myeloid neoplasms, such as myelodysplastic syndrome, myeloproliferative neoplasm, acute myeloid leukemia, CML, chronic myelomonocytic leukemia, or plasma cell myeloma. The prognosis of SM-AHN is dependent on the associated hematologic neoplasm (AHN).

The extent of clinical applications of oxidative stress-based therapies such as photodynamic therapy (PDT) or respiratory chain disruptors are increasing rapidly, with cold physical plasma (CPP) emerging as a further option. According to the current knowledge, the biological effects of CPP base on reactive oxygen and nitrogen species (RONS) relevant in cell signaling. To monitor the safety and the biological impact of the CPP, determining the local generation of RONS in the same environment in which they are going to be applied is desirable. Here, for the first time, the development of an electrochemical sensor for the simple, quick, and parallel determination of plasma-generated reactive species is described. The proposed sensor consists of a toluidine blue redox system that is covalently attached to a gold electrode surface. By recording chronoamperometry at different potentials, it is possible to follow the in-situ production of the main long-lived reactive oxygen and nitrogen species like hydrogen peroxide, nitrite, hypochlorite, and chloramine with time. The applicability of this electrochemical sensor for the in-situ assessment of reactive species in redox-based therapies is demonstrated by the precise analysis of hydrogen peroxide dynamics in the presence of blood cancer cells.

Bismarck brown staining is a suitable method for demonstration of mast cells in peripheral tissues. However, apart from the intensive color of the mast cell granules, almost no other structures are visible after this staining, which may compromise the results and discourage the investigator. In the present report, we validate the applicability of the Bismarck brown staining of soft tissue and introduce a modification of the method to improve the quality of the histological preparation. Counterstaining with haematoxylin produces specimens with superb contrast and high analytical value. We consider our method involving counterstaining to be superior to the classical Toluidine blue staining, because of the greater contrast of mast cells, which makes evaluation easier, while the preparations are suitable for automated image analysis as well

Background and Objectives: Early detection and treatment of oral cancer will significantly improve the survival rate and prognosis of the patients. Adjunctive diagnostic aids such as vital staining have been developed to supplement clinical examination and improve the diagnosis. The current study was conducted to evaluate the diagnostic efficacy of acetic acid (2%) (AA), lugol's iodine (3% dilution) (LIS), and toluidine blue (1%) (TBS) in oral leukoplakia (OL) and erosive variant of oral lichen planus (OLP). Materials and Methods: A hospital-based, cross-sectional study was conducted with 30 randomly selected subjects having clinically proven cases of OL and erosive OLP. Every patient underwent AA, LIS, and TBS application to their oral lesions in a sequential manner. Subsequently, clinical and histopathologic diagnosis was compared with staining results of each. Cases which were diagnosed as epithelial hyperplasia were considered as a control group. Data are presented in numbers and percentages. Chi-square test was used to compare between TBS, LIS and AA. Diagnostic efficiency and reliability was calculated by sensitivity and specificity test in terms of its utility in predicting the dysplastic nature of the lesion. Results: Sensitivity of both TBS and LIS staining was calculated as 90.48% whereas the specificity of the former test was 22.22% and latter was 11.11%. AA test showed a sensitivity of 57.14% and specificity of 33.33%. Multiple comparisons of staining with three agents and the histopathologic variants did not show any statistically significant difference. Conclusion: TBS and LIS staining showed high sensitivity in diagnosing OL and erosive OLP compared to AA. The specificity of all three modalities used was low.

Context: Breast cancer constitutes nearly one third of cancers among women. Immune responses caused by neoplastic cells lead to the accumulation of inflammatory cells like mast cells (MCs), macrophages, lymphocytes, and plasma cells around the tumor tissue forming the tumor microenvironment. Aim: The study aims at quantifying the role of MCs in different grades of invasive carcinoma of breast with respect to estrogen receptor (ER), progesterone receptor (PR), and Human Epidermal growth factor Receptor 2 (HER2/neu). Materials and Methods: This study included 60 cases of invasive carcinoma of breast. Toluidine blue staining was used for quantitative MC analysis and correlated with immunohistochemistry analysis for hormonal markers' positivity—ER, PR and HER2/neu. Results: The mean age was 52 years (range: 25–75 years). The average number of MCs in Grade I, II, and III were 24.05, 18.4, and 7.9, respectively, with a significant P value. ER, PR, and HER2/neu positivity was found in 60%, 55%, and 32% of the cases, respectively. ER positivity with mean MC count of 23.55 was found in 36 cases, and 33 cases were positive for PR with a mean MC count of 24.18 and a significant P value. HER2 positive cases were 28 with a mean MC count of 20.82. Conclusion: The presence of MCs in breast cancer is inversely proportional to the grade of tumor, i.e., a maximum number of MCs were seen in low grade tumors. In addition, there is a positive correlation between ER and PR receptor positivity with the presence of MCs in the stroma of breast cancer.

Highlights • FNAC with ROSE helps in triaging of sample for further studies. • Cytologist should be aware of the cytomorphological features of cysticercosis which is common in India. • FNAC can pauci-cellular with histiocytic debris and non-human nuclei., Cysticercosis is a zoonotic disease, caused by the larval form of pork tapeworm Taenia solium. This disease is a public health problem in a country such as India, but its incidence is likely underestimated. With the advent of fine needle aspiration cytology (FNAC) with rapid on-site evaluation (ROSE), early detection of this disease is possible, especially when the lesion is in anatomically approachable superficial locations. We report a case of cysticercosis confirmed by FNAC in the Department of Pathology where ROSE using toluidine blue was done as a part of routine cytology procedure. FNAC diagnosis of cysticercosis can be easily made provided the reporting cytologist is aware of the morphological criteria.

Objective The study aimed to compare the usefulness of two staining methods for imprint cytology for diagnosis of Helicobacter pylori infection. Gastric biopsy specimens (from dyspeptic patients attending routine upper gastrointestinal endoscopy) were placed on glass slides to obtain imprints. The imprints were stained with Toluidine blue and Giemsa stains separately and observed under ×400 magnification using a light microscope. Imprinted biopsies were sectioned and stained with H & E stain and Giemsa stain for histological analysis. Diagnosis of H. pylori infection in both imprint and histological sections were confirmed by a consultant pathologist. The sensitivity, specificity, positive predictive value and negative predictive value of each stain were calculated and benchmarked against histological diagnosis. Results Of the 55 dyspeptic patients enrolled in the study, 5 were positive for H. pylori by Toluidine blue stain and 4 by Giemsa stain. The sensitivity of Toluidine blue stain (57.1%) was higher than Giemsa stain (42.9%) while the specificity of both stains was equal (97.9%). Giemsa stain gave a better discrimination for identification of H. pylori bacteria among the mucosal background. Imprint cytology is a rapid, simple and cost effective diagnosis method that can supplement histological diagnosis. Electronic supplementary material The online version of this article (10.1186/s13104-018-3592-2) contains supplementary material, which is available to authorized users.

Background There are some spaces between abutment and implant body which can be a reservoir of toxic substance, and they can penetrate into subgingival space from microgap at the implant–abutment interface. This penetration may cause periimplantitis which is known to be one of the most important factors associated with late failure. In the present study, three kinds of abutment connection system, external parallel connection (EP), internal parallel connection (IP), and internal conical connection (CC), were studied from the viewpoint of microleakage from the gap between the implant and the abutment and in connection with the loosening of abutment screw. Methods We observed dye leakage from abutment screw hole to outside through microgap under the excessive compressive and tensile load and evaluated the anti-leakage characteristics of these connection systems. Results During the experiment, one abutment screw for EP and two screws for IP, out of seven samples in each group, were fractured. After the 2000 cycles of compressive tensile loadings, removal torque value (RTV) of abutment screw represented no statistical differences among three groups. Standard deviation was largest in the RTV of EP and smallest in that of CC. The results of microleakage of toluidine blue from implant–abutment connection indicated that microleakage generally increased as loading procedure progressed. The amount of microleakage was almost plateau at 2000 cycles in CC, but still increasing in other two groups. The value of microleakage greatly scattered in EP, but the deviation of that in CC is significantly smaller. At 500 cycles of loading, there were no significant differences in the amount of microleakage among the groups, but at 1000, 1500, and 2000 cycles of loading, the amount of microleakage in CC was significantly smaller than that in IP. Throughout the experiment, the amount of microleakage in EP was largest, but no statistical difference was indicated due to the high standard deviation. Conclusions Within the limitation of the present study, CC was stable even after the loading in the RTV of abutment screw and it prevented microleakage from the microgap between the implant body and the abutment, among the three tested connections.

Objectives: One percent toluidine blue being the most effective adjunct is often used to detect dysplasia. Not much attention has been given to the effect of toluidine blue in enhancement of cytological smears. The present study assessed the smears before and after application of toluidine blue in smokers against non-smokers using three different stains [Papanicolaou (PAP), Hematoxylin and Eosin (H and E), and Giemsa]. Study Design: Twenty-five individuals from each group participated in the study. The oral cytosmears were obtained before and after application of toluidine blue and assessed for clumping of squamous cells, cellular and nuclear pleomorphism, micronuclei, binucleation, bacterial colony units, and keratin flakes. Results: In smokers, the maximum enhancement in cytological smears post-toluidine blue application was shown by Giemsa stain than PAP and H and E stains. Among the individual parameters, nuclear pleomorphism exhibited greatest significant difference between smokers and non-smokers. Conclusion: Toluidine blue enhanced the staining characteristics both in terms of sensitivity and specificity and thereby was found to be synergistic in assessment of cytosmears. The cellular alterations noticed in the smears of smokers with clinically normal buccal mucosa can be used as a means of education tool in counselling for smoking cessation.

In the present study, toluidine blue for metachromasia and berberine sulfate for heparin fluorescence were used to determine the localisation and distribution of mast cells as well as the percent of hepa-rin-positive cells (MCH) in normal lumbar spinal ganglia (dorsal root ganglia) of domestic swine. Mast cells density was established after estimating the number of these cells per ganglion. Mast cells with metachromasia (MCTB) were observed predominantly in the ganglion capsule near the blood vessels. The number of mast cells inside the ganglia (2.10±1.45 in males and 2.20±1.03 in females) was significantly lower than in the capsule (13.30±1.95 in males and 13.60±2.42 in females) in both genders (P

International audience; This work describes the modification of a glassy carbon electrode with poly(Toluidine Blue O) (GC/poly-TBO) and single-walled carbon nanotubes (SWCNT) for the electrocatalytic oxidation of nitrite. GC/poly-TBO was prepared by electropolymerization and used as such or after immobilizing SWCNT on the polymeric film to give a composite GC/poly-TBO-SWCNT electrode. The electrochemical and catalytic behavior of both electrodes was studied comparatively. It was observed that the presence of SWCNT contributed to enhance the electrocatalytic response for nitrite oxidation, as measured by amperometry at +0.92 V vs. Ag/AgCl/KClsat and pH 7. The response was linear with respect to the nitrite concentration in the 0.001-4 mM range, with a detection limit of 0.37 mu M (based on signal to noise ratio of 3) for GC/poly-TBO-SWCNT. The proposed method was also applied to the determination of nitrite in a wastewater sample and compared to the spectrophotometric method.

Male factor infertility has been diagnosed as the cause of infertility in about 20% of infertile couples. Sperm analysis is the most common method for diagnosing infertility in a laboratory. However, approximately 15% of infertile men have a normal sperm analysis. Therefore, the result of a routine sperm analysis often cannot be a definitive diagnosis for male factor infertility. Also, approximately 8% of infertile men with normal sperm parameters have high levels of abnormal sperm DNA. This indicates the role of the integrity of sperm DNA in male infertility. Here, we review the current tests available to evaluate the sperm DNA integrity along with their benefits and limitations.

Made available in DSpace on 2018-12-11T17:23:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-01 The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8–2 years), adult (3.5–7 years), and senile (8–14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%–16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy. Department of Medicine Surgery and Animal Reproduction Univ Estadual Paulista (UNESP) Department of Animal Reproduction and Obstetrics University of Rio Preto (UNIRP) Department of Support Production and Animal Health Univ Estadual Paulista (UNESP) Laboratory of Histology Federal University of Uberlândia (UFU) Department of Preventive Veterinarian Medicine and Animal Reproduction Univ Estadual Paulista (UNESP) Department of Medicine Surgery and Animal Reproduction Univ Estadual Paulista (UNESP) Department of Support Production and Animal Health Univ Estadual Paulista (UNESP) Department of Preventive Veterinarian Medicine and Animal Reproduction Univ Estadual Paulista (UNESP)

Aim: Our study aimed to evaluate the infiltration of tissue eosinophils and mast cells in oral squamous cell carcinoma (OSCC) by using special stains. Study Design: Comparative study. Place of Study: Sharad Pawar Dental College, Sawangi (Meghe), Wardha, Maharashtra. Methodology: The study was carried out with the sample size of 30 histopathologically diagnosed cases of oral squamous cell carcinoma and comparison of infiltration of these (tissue eosinophil and mast cells) inflammatory cells with control (normal) group of patients, was done by using special stains. Special stains are wonderful because they allow us to see which we cannot see with routine Haematoxylin and eosin stain. Special stains were used to demonstrate tissue eosinophils and mast cells. Carbol Chromotrope and congo red were used for tissue eosinophil and for mast cells staining toluidine blue and thionin were used. Results: The comparison of infiltration of tissue eosinophil and mast cell in OSCC with control group (normal) of patients shows significantly increased infiltration of these immunological cells in OSCC group of patients (P

Heparin–Toluidine Blue (C.I. Basic Blue 17) interaction was studied using UV–vis spectroscopic analysis of the titration of the functional groups present on the polyanion chain. The study took into consideration both the pH and relative amount of the dye as a function of the Heparin repeat unit number; formation of a dye/polysaccharide aggregate was studied under the same conditions and structural models of the two types of aggregates were proposed. As a consequence of such an investigation a new Heparin determination methodology was developed.

As propriedades eletroquímicas dos corantes catiônicos, azul de meldola e azul de toluidina, imobilizados na superfície do acetato de celulose modificado com dióxido de titânio foram investigadas por voltametria cíclica. Os materiais sintetizados foram empregados como eletrodos de pasta de carbono. As propriedades mediadoras redox dos eletrodos modificados quimicamente com azul de meldola e azul de toluidina foram otimizadas utilizando um planejamento fatorial consistindo de dois níveis e quatro fatores com dois pseudo-pontos centrais (n=20 experimentos). A análise fatorial foi realizada para investigar as melhores condições de reversibilidade do processo redox tais como uma diminuição na separação entre os picos de potenciais anódico e catódico e razão de corrente próxima da unidade. Os fatores que apresentaram efeitos significantes na otimização global do sistema e que permitiram alcançar as melhores condições de reversibilidade de transferência de elétrons foram os fatores principais velocidade de varredura e tipo de eletrodo (azul de meldola ou azul de toluidina) além dos fatores de interação concentração do KCl × tipo de eletrodo (B×D) e a interação pH × [KCl] × velocidade de varredura (A×B×C). As melhores condições de reversibilidade eletroquímica obtidas foram: o eletrodo CA-TiO2-MB, KCl 1,0 mol L-1 como eletrólito suporte e velocidade de varredura de 10,0 mV s-1. Adicionalmente, o eletrodo modificado CA-TiO2-MB foi testado como sensor amperométrico para a determinação de NADH com um limite de detecção de 0,1 μmol L-1. The electrochemical properties of meldola blue and toluidine blue cationic dyes immobilized on cellulose acetate surface modified with titanium dioxide were investigated by cyclic voltammetry. The materials synthesized were employed as carbon paste electrodes. The redox mediator properties of the meldola blue and toluidine blue chemically modified electrodes were optimized using a factorial design, consisting of two levels and four factors with two pseudo-central points (n= 20 experiments). The factorial analysis was carried out by searching for better reversibility of the redox process, such as the lowest separation between anodic and cathodic potential peaks and a current ratio near unity. The factors that presented significant effects on the overall optimization of the system to achieve the best conditions of the reversibility of electron transfer were the main factors scan rate and type of electrode (meldola blue or toluidine blue), besides the interaction factors KCl concentration × type of electrode (B×D) and the pH × [KCl] concentration × scan rate (A×B×C) interaction. The best electrochemical reversibility conditions obtained were: using the CA-TiO2-MB electrode, 1.0 mol L-1 KCl as supporting electrolyte, at scan rate of 10.0 mV s-1. Afterwards, the CA-TiO2-MB modified electrode was tested as an amperometric sensor for the determination of NADH, with a detection limit of 0.1 μmol L-1.

The toluidine blue (TB) aggregation phenomenon in water solutions was investigated by UV-Vis absorption spectroscopy in a wide range of concentrations, from 10−9 to 10−2 M. The experimental data were fitted by assuming that the spectrum was due to the overlapping of the bands of six different aggregation species simultaneously present in solution. The model and the peak centres of the different species were hypothesized on the basis of exciton theory. All the spectra obtained were simultaneously processed thus allowing the derivation of the K constant, which was assumed to be the same for all the equilibria and which was found equal to 10 500. The e spectra of the different aggregation species were also obtained. The overall TB spectrum may be mainly attributed to the H-type aggregation, although some of the species also show the J type bands. The structural features of TB and its dimeric aggregate were also investigated by means of molecular mechanics semi-empirical potential energy calculations. The results obtained for the dimer confirm the H type aggregation and show that the lowest energy model of the four possible is the one obtained by a mirror plane of symmetry containing the aromatic rings, in agreement with the x-ray diffraction data which will be presented in a forthcoming paper.

Introduction and Objectives: Angiogenesis is a complex event facilitated by angiogenic factors released from neoplastic and host immune cells. Among host immune cells, mast cells (MCs) may have greater significance in tumor progression through angiogenesis. The objectives of the study were to evaluate and correlate mast cell density (MCD) and microvessel density (MVD) in normal gingival tissue, leukoplakia with and without dysplasia and oral squamous cell carcinoma (OSCC) cases. Materials and Methods: Among eighty selected cases, twenty were of normal gingiva, twenty each of leukoplakia without and with dysplasia and twenty of OSCC. The slides were stained with CD34 and counterstained with 0.1% toluidine blue, followed by quantification of MCD and MVD per high-power field (×40) using Image-Pro Express software. Statistical Analysis: Chi-square test and correlation coefficient were used for statistical analysis. Observation and Results: A statistically significant difference in the values of MVD and MCD between normal gingival tissue, leukoplakia with and without dysplasia and OSCC (P = 0.000) was observed. MVD and MCD showed a positive correlation between the study groups. Conclusion: MVD and MCD increased significantly in cases of OSCC as compared to leukoplakia with and without dysplasia and normal gingival tissue. It was concluded that MCs may play a significant role in angiogenesis by releasing pro-angiogenic and angiogenic factors which may in turn favor the progression of premalignant lesion to a malignant one.

Collagen is one of the most common fibers in the extracellular matrix, where sulfated mucopolysaccharides are also located. In addition, sulfated mucopolysaccharides are present in some globet cells and secretory glands. The objective of this article is to develop a new staining method that detects these two macromolecules simultaneously in the same sample. The method described stains tissues in five fundamental colors: collagen in red; sulfated mucopolysaccharides in violet; red blood cells in yellow; muscle in orange; and nuclei in green. As a conclusion, it will be interesting in the future to evaluate whether this method could be used as a basic histological method, as a histology teaching tool, or even in histopathological and cytopathological studies.

In order for medical practitioners to adequately explain to the court the findings of their clinical examinations of victims of sexual violence, they must have access to research data which will place their findings in to context. Unfortunately, existing research has reported a very wide range of injury prevalence data. This papers aims to provide an explanation for this wide variation in results and, furthermore, this paper aims to establish if it is possible to carry out a meta-analysis of existing research data, pertaining to the prevalence of injury after sexual assault. It is suggested that pooling of individual study results may allow statistically robust determination of the true prevalence of injury in victims of sexual violence. It is concluded that heterogeneity in research methodology, between existing research studies, is responsible for the broad range of reported prevalence rates. Finally, this heterogeneity is seen to preclude robust meta-analysis.

Con el propósito de determinar el efecto de la integridad de la cromatina sobre las dimensiones morfométricas (largo, ancho, área, perímetro, elipticidad) de la cabeza espermática, se evaluaron mediante un sistema computarizado de análisis de morfometría espermática, 2273 cabezas de espermatozoides de toros Brahman, las cuales fueron teñidas con azul de toluidina para determinar el estatus de la cromatina, resultando 1599 con cromatina normal y 674 con cromatina dañada. La integridad de la cromatina afectó significativamente tres de los parámetros evaluados. Los espermatozoides con cromatina dañada presentaron mayor ancho (4,32 ± 0,009µm, P

Background: Benign vascular lesions such as vascular malformation and hemangioma at times pose difficulty in diagnosis both for clinicians and pathologists. Vascular malformations are difficult to treat while hemangiomas resolve spontaneously in most instances. There are instances when vascular malformations, especially arteriovenous malformations (AVMs) have been misdiagnosed as hemangiomas and vice-versa. Clinical and radiological correlation with histopathological confirmation of these anomalies is important for the management of these lesionsAim: The aim was to study the histological characteristics of hemangiomas and vascular malformations and to study the utility of histochemical stains in their diagnosis. Materials and Methods: We retrospectively studied fifty cases retrieved from the records of Department of Pathology which were diagnosed as hemangioma (n=32) and vascular malformation (n=18) on Hematoxylin and Eosin (H and E) stain over a period of 18 months. The cases were analyzed based on findings of histochemical stains such as Verhoeff-van Gieson (VVG), Masson's trichrome (MT), and toluidine blue. Results: After reviewing all the cases with the use of histochemical stains, two of the three cases originally diagnosed as hemangioma turned out to be AVM and one to be venous malformation. An increased number of intra-lesional nerves were found in 16 of 19 cases of AVM and in both cases of venous and lymphatic malformation. Hemangiomas did not show increase in nerve bundles. Mast cells were found to be increased in proliferating hemangiomas and pyogenic granulomas as compared to AVMs. Conclusion: Hemangiomas and vascular malformations should be clearly differentiated to reduce the risk of treatment failure and recurrence. With the use of histochemical stains such as VVG, MT and toluidine blue, the diagnostic difficulty can be reduced and definitive diagnosis is possible.

Objectives: Dental caries and periodontal diseases are caused by infection of teeth and supporting tissues due to complex aggregate of bacteria known as biofilm, firstly colonized by streptococci. The main purpose of this in vitro study was to evaluate the antimicrobial effects of toluidine blue O (TBO) and Radachlorin® in combination with a diode laser on the viability of Streptococcus mutans. Materials and Methods: Bacterial suspensions of Streptococcus mutans were exposed to either 0.1% TBO associated with (20 mW, 633 nm diode laser, continuous mode, 150 s) or 0.1% Radachlorin® and laser irradiation (100 mW, 662 nm diode laser, continuous mode, 120 s). Those in control groups were subjected to laser irradiation alone or TBO/Radachlorin® alone or received neither TBO/Radachlorin® nor laser exposure. The suspensions were then spread over specific agar plates and incubated aerobically at 37°C. Finally, the bactericidal effects were evaluated based on colony formation. Results: Potential bacterial cell killing was only observed following photosensitization with TBO and 3 j/cm2 laser exposure (p

The objective of this study was to investigate the utility of toluidine blue and brush cytology in patients with clinically detected oral mucosal lesions. Clinical examination of 35 patients was completed before toluidine blue application, oral brush cytology and scalpel biopsy. Lesions were photographed before and after stain application; followed by brush cytology. All findings were compared with histopathologic results. Severe dysplasia and carcinoma-in-situ were determined as 'positive'; no dysplasia and mild to moderate dysplasia were defined as 'negative'. The sensitivity, specificity, positive and negative predictive values of clinical examination and toluidine blue were the same: 0.923, 0.433, 0.414, and 0.929, respectively. Those of brush cytology were 0.923, 0.517, 0.462, and 0.938. The concordance of all methods was 30% for benign and 61% for malignant lesions. Adjuncts identified 92% of carcinoma-in-situ and squamous cell carcinoma as confirmed by histopathology, in contrast to clinical findings alone in which 62% of these lesions were identified (p = 0.046). In conclusion, adjunct diagnostic methods decreased the level of uncertainty for the diagnosis of oral malignancies and lichenoid dysplasias when applied as adjuncts to clinical examination. © 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. 2005-DIS-014 The study was funded by Ege University Scientific Research Projects Fund (2005-DIS-014).

AIM: To test the hypothesis that photo-activated disinfection (PAD) has a bactericidal effect on pathogens inoculated in root canals, with emphasis on biofilm formation/destruction. METHODOLOGY: Root canals of extracted teeth (n = 38) were prepared (size 30, 0.10 taper), autoclaved, divided into three groups and two negative controls inoculated (Streptococcus anginosus, Enterococcus faecalis or Fusobacterium nucleatum) and treated (PAD, laser, dye or positive control) according to a cross-sectional design. Resultant colony-forming unit counts were associated with observations of cell structural changes using environmental scanning electron microscopy (ESEM) on inoculated dentinal surfaces (n = 22, two controls) before (1, 2 and 6 days of incubation) and after treatment with PAD. RESULTS: Treatment of root canals with PAD (15 J) caused a significant reduction of the bacterial load, resulting in a 93.8% kill of S. anginosus (P < 0.0001), a 88.4% kill of E. faecalis (P < 0.05) and a 98.5% kill of F. nucleatum (P < 0.0001), but no sterilization. Laser alone had no significant effect on the load nor did the dye without laser. The ESEM experiment showed that individual cells or monolayers were easily eliminated with PAD. But when biofilms were present (2 and 6 days for E. faecalis, 6 days for S. anginosus), bacterial eradication was substantially reduced in deep layers. CONCLUSIONS: Photo-activated disinfection is not an alternative but a possible supplement to the existing protocols for root canal disinfection as the interaction between light (diode laser) and associated dye (TBO) provides a broad-spectrum effect. Some endodontic pathogens that grow as single-species biofilms, however, are difficult to eradicate. ispartof: International endodontic journal vol:41 issue:3 pages:227-239 ispartof: location:England status: published

A terapia fotodinâmica apresenta-se como uma possível alternativa terapêutica para o tratamento de infecções localizadas, porém, para sua aplicação clínica, as variáveis relacionadas à eficiência fotodinâmica devem ser analisadas para a obtenção de resultados satisfatórios. Os objetivos deste trabalho foram investigar a dinâmica de agregação dos fotossensibilizadores azul de metileno e azul de orto-toluidina em diferentes solventes, caracterizar os solventes em relação ao oxigênio molecular disponível em cada solução e seu respectivo pH, observar o efeito da temperatura, sangue e saliva na agregação dos fotossensibilizadores, monitorar a dinâmica de fotodegradação em função da exposição radiante e irradiância, avaliar a eficiência fotodinâmica microbiologicamente e através da produção de 1O2. Os solventes utilizados foram água deionizada, água destilada, água estéril, solução salina 0,9% e solução salina 0,45%. Em cada uma das soluções, foi realizado teste de pH e medição de oxigênio dissolvido por sonda apropriada. Os efeitos de fluidos, temperatura e concentração na agregação dos fotossensibilizadores foram analisados através de espectroscopia de absorção óptica. A dinâmica de fotodegradação foi testada com diferentes exposições radiantes e irradiâncias, nos comprimentos de onda adequados para cada composto. As irradiações foram realizadas com emissão de banda larga e banda estreita. A eficiência fotodinâmica foi avaliada através da medição direta da luminescência do oxigênio singleto em ?=1270nm, em dois solventes. Testes microbiológicos foram realizados com culturas de Escherichia coli. Os resultados obtidos demonstram que a dinâmica de agregação está diretamente relacionada com solvente e concentração: a solução salina apresentou diferença em relação aos demais solventes em todos os quesitos analisados. Dos solventes testados, a água deionizada apresentou os melhores resultados. A dinâmica de fotodegradação está relacionada, em sua magnitude, com a exposição radiante e, em sua dinâmica, com a irradiância. O uso de banda larga para irradiação foi mais efetivo para na fotossensibilização letal. Portanto, para a aplicação clínica da terapia, o substrato deve ser considerado. Apropriadas condições de irradiação, solvente e concentração de fotossensibilizador aumentam a eficiência do processo. Photodynamic therapy may be a viable alternative to treat localized infections, although to obtain a resourceful clinical application, parameters linked with photodynamic efficiency must be analyzed in order to achieve quantifiable results. The aims of this study were to investigate the aggregation dynamic of the photosensitizers methylene blue and orto-toluidine blue in different solvents; to characterize the solvents regarding to available molecular oxygen and pH; to observe the effect caused by temperature, blood and saliva on photosensitizer aggregation, to monitor the photobleaching dynamic as a function of fluence and irradiance, and to evaluate the photodynamic efficiency in such conditions. The tested solvents were deionized water, distillated water, sterile water, and saline solution 0.9%. In each solution, the pH and molecular oxygen availability were measured with the appropriated device. The effects of organic fluids, temperature and concentration on aggregation were analyzed through optical absorption spectroscopy. The photobleaching dynamic was tested regarding radiant exposure and irradiance with the appropriated wavelength for each compound. The irradiations were performed with broad band and narrow band light sources. The photodynamic efficiency was evaluated through singlet oxygen luminescence measurement in ?=1270nm and microbiological tests were performed with cultures of Escherichia coli. The results demonstrated that the aggregation dynamic is directly linked with solvent and concentration: the saline solution presented different results in all tested conditions. Deionized water presented the best results. The photobleaching dynamic is correlated in its magnitude with the radiant exposure and its dynamic is associated with irradiance. The use of broad band to perform the irradiation was considered more effective for lethal photosensitization. Therefore, for the clinical application of this therapy, the substrate must be taking into account, and appropriated parameters of irradiation and photosensitizer presentation increase the efficiency of the process.

Background: Periapical lesions occur in response to chronic irritation in periapical tissue, generally resulting from an infected root canal. Specific etiological agents of induction, participating cell population and growth factors associated with maintenance and resolution of periapical lesions are incompletely understood. Among the cells found in periapical lesions, mast cells have been implicated in the inflammatory mechanism. Aim: Quantifications and the possible role played by mast cells in the periapical granuloma and radicular cyst. Hence, this study is to emphasize the presence (localization) and quantification of mast cells in periapical granuloma and radicular cyst. Materials and Methods: A total of 30 cases and out of which 15 of periapical granuloma and 15 radicular cyst, each along with the case details from the previously diagnosed cases in the department of oral pathology were selected for the study. The gender distribution showed male 8 (53.3%) and females 7 (46.7%) in periapical granuloma cases and male 10 (66.7%) and females 5 (33.3%) in radicular cyst cases. The statistical analysis used was unpaired t‑test.Results: Mean mast cell count in periapical granuloma subepithelial and deeper connective tissue, was 12.40 (0.99%) and 7.13 (0.83%), respectively. The mean mast cell counts in subepithelial and deeper connective tissue of radicular cyst were 17.64 (1.59%) and 12.06 (1.33%) respectively, which was statistically significant. No statistical significant difference was noted among males and females.Conclusion: Mast cells were more in number in radicular cyst. Based on the concept that mast cells play a critical role in the induction of inflammation, it is logical to use therapeutic agents to alter mast cell function and secretion, to thwart inflammation at its earliest phases. These findings may suggest the possible role of mast cells in the pathogenesis of periapical lesions. Keywords: Mast cells, Periapical granuloma, Radicular cyst, Toluidine blue

Made available in DSpace on 2014-10-09T12:46:02Z (GMT). No. of bitstreams: 0 Made available in DSpace on 2014-10-09T14:04:58Z (GMT). No. of bitstreams: 1 07496.pdf: 4298184 bytes, checksum: 0ea1a09cef64fb7f7cb7c36f765c879b (MD5) Dissertacao (Mestrado Profissionalizante Lasers em Odontologia) IPEN/D-MPLO Instituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo

In the adult rat brain mast cells reside selectively in the thalamus. We investigated thalamic mast cells stained by acidic toluidine blue or pinacyanol, and with histamine immunocytochemistry, focusing on their state of activity revealed by degranulation. Mast cells exhibited perivascular prevalence and high quantitative variability, between cases and in different sections, with no asymmetry or topographical selectivity in thalamic nuclei. Pinacyanol, alone or with erythrosine, stained mast cells with higher sensitivity than toluidine blue. However, toluidine blue was highly predictive of pinacyanol staining and provided the best resolution of mast cell cytoplasmic features. Histamine immunocytochemistry labeled 61% of pinacyanol-stained mast cells. Intensely toluidine blue-stained granulated cells, as well as cells exhibiting different degrees of degranulation that paralleled lighter staining, were observed. The response of thalamic mast cells to intracerebroventricular administration of nerve growth factor (NGF) and control cytochrome-c injections was evaluated after 2, 24, and 72 hours. No obvious changes in mast cell number or distribution were found after treatment, but massive degranulation was frequently observed after NGF administration. Significant decrease of staining intensity of mast cells, supporting enhanced degranulation, was documented in NGF-treated animals by quantitative image analysis. Ultrastructural features of mast cell degranulation, with granule coalescence and matrix dissolution, were detected in untreated and NGF-treated cases. The findings point out that mast cells are active in the thalamus in basal conditions and that NGF has the potential to elicit long-lasting degranulation of thalamic mast cells in vivo, exerting a direct effect and/or priming these cells to react to endogenous stimuli.

The distribution of toluidine blue stained mast cells was evaluated in 38 biliary ducts (Ductus choledochus), collected from patients with secondary chronic cholangitis with different inflammatory activities. The mast cells counts in chronic exacerbated and chronic sclerotic cholangitis were significantly higher as compared to that in controls (45.75 cells/mm2 and 19.5 cells/mm2 vs. 5.9 cells /mm2, p

Context: Carcinoma in an early stage of development is hard to detect clinically because the lesion may not be palpable and color of the lesional tissue is not necessarily different from the color of the surrounding mucosa. In order to improve the efficacy of the diagnosis, techniques are being developed to complement clinical examination and to facilitate the identification of initial carcinomas. Aims: To find out the efficacy of chemiluminescent illumination (ViziLite TM ) for the diagnosis in precancer and cancer patients and compare this result to toluidine blue staining and oral exfoliative cytology. Materials and Methods: This study was done in 3 groups. Each group consists of 10 cases. Group I consists of normal appearing mucosa. Group II and III consist of clinically diagnosed pre-cancer and clinically suggestive of cancer respectively. Chemiluminescent illumination, toluidine blue supravital staining, oral exfoliative cytology and biopsy were performed in all cases. Statistical analysis used: SPSS version 10.05 was used to calculate positive and negative predictive values. Results: In Group I, all 10 patients showed negative result to ViziLite TM . 8 patients showed positivity and 2 patients showed negativity to ViziLite TM test in Group II. 9 patients were positive and one patient was negative for ViziLite TM . Conclusions: Chemiluminescent illumination test was sensitive for precancerous and cancerous lesions, which presented as keratotic lesions and red-white lesions. It showed negative result to erosive lesions. Toluidine blue staining test was reliable in precancerous and cancerous lesions, which present as erosive and red-white lesions. It showed negative result to keratotic lesions. Oral exfoliative cytology has diagnostic value in cancer patients than in precancer patients. These Results indicate that chemiluminescent illumination test is relatively reliable and accurate than toluidine blue staining test and useful chair side diagnostic test.

Background : Enterococcus faecalis has been widely used as a valuable microbiological pathogen for in vitro studies due to its ability to successfully colonize the root canal in a biofi lm-like style, invade dentinal tubules, and resist endodontic treatment procedures.The aim of this study was to compare the bactericidal effi cacy of photodynamic therapy (PDT), 2% chlorhexidine, 2.5% NaOCl, and combination of PDT and 2.5% NaOCl against E. faecalis . Materials and Methods: Sixty single-rooted teeth had their canals contaminated with E. faecalis in brain heart infusion broth and were incubated for 48 hours.The canals were then subjected to 2% chlorhexidine, 2.5% NaOCl, PDT (red light emitting diode 625 nm+ Toludine Blue) and PDT + 2.5% NaOCl. Controls consisted of no treatment (positive control) and without inoculation of bacterium (negative control). Following treatment, the canal contents were sampled with sterile paper points. The samples were dispersed in transport medium, serially diluted, and cultured on blood agar to determine the number of colony forming units (CFU). Data were analyzed by Mann-Whitney U test at 5% signifi cance level. The signifi cance level for all analyses was set at P

Photodynamic Therapy (PDT) is a minimally invasive approach that has shown promising results in management of oral, head and neck lesions. PDT can be used alone or in combination with other conventional treatments (surgery, chemotherapy, radiotherapy). Oral Lichen Planus (OLP) is a mucosal and cutaneous chronic disease characterized by an autoimmune insult of basal keratinocytes. We aim to evaluate the feasibility of topical toluidine blue-mediated PDT for the treatment of oral cavity multifocal homogeneous white lesions by oral lichen planus without dysplastic features.