Classification of Multiple H&E Images via an Ensemble Computational Scheme.

In this work, a computational scheme is proposed to identify the main combinations of handcrafted descriptors and deep-learned features capable of classifying histological images stained with hematoxylin and eosin. The handcrafted descriptors were those representatives of multiscale and multidimensional fractal techniques (fractal dimension, lacunarity and percolation) applied to quantify the histological images with the corresponding representations via explainable artificial intelligence (xAI) approaches. The deep-learned features were obtained from different convolutional neural networks (DenseNet-121, EfficientNet-b2, Inception-V3, ResNet-50 and VGG-19). The descriptors were investigated through different associations. The most relevant combinations, defined through a ranking algorithm, were analyzed via a heterogeneous ensemble of classifiers with the support vector machine, naive Bayes, random forest and K-nearest neighbors algorithms. The proposed scheme was applied to histological samples representative of breast cancer, colorectal cancer, oral dysplasia and liver tissue. The best results were accuracy rates of 94.83% to 100%, with the identification of pattern ensembles for classifying multiple histological images. The computational scheme indicated solutions exploring a reduced number of features (a maximum of 25 descriptors) and with better performance values than those observed in the literature. The presented information in this study is useful to complement and improve the development of computer-aided diagnosis focused on histological images.

Correlation of H3K9ac and H4K12ac With Cell Proliferation Marker Ki-67 in Oral Leukoplakia: An Immunohistochemical Study.

This study aimed to analyze the immunohistochemical expression of H3K9ac and H4K12ac in oral leukoplakia (OL) and its association with cell proliferation marker Ki-67 and clinicopathologic data. Paraffin-embedded, formalin-fixed tissue samples from 50 OLs and 15 fragments of the normal oral mucosa (NOM) were submitted to immunohistochemical assay using the streptavidin-biotin-peroxidase method. Quantitative analysis of the antigen-antibody reaction was performed by obtaining integrated optical density (IOD) and the percentage of positive nuclei (PPN) with ImageJ software. OL samples presented higher PPN ( P =0.02) and lower IOD values ( P =0.007) for H4K12ac in comparison to NOM. The area under the receiver operating characteristic curve for PPN and IOD values of H4K12ac immunostaining were 0.70 ( P =0.02) and 0.73 ( P =0.007), respectively. No differences were found between OL and NOM for H3K9ac. Cell proliferation marker Ki-67 had a positive correlation with PPN ( P <0.0001) and IOD ( P =0.0007) for H3K9ac expression and with IOD values ( P =0.002) for H4K12ac expression. The present findings suggest that alterations in the acetylation pattern of H4K12 occur in the early stages of oral carcinogenesis and that both H3K9ac and H4K12ac might have a role in the regulation of epithelial cell proliferation of OL.

Prognostic value of histone H3 serine 10 phosphorylation and histone H4 lysine 12 acetylation in oral squamous cell carcinoma.

AIMS: Studies on epigenetics in oral squamous cell carcinoma (OSCC) are rare. Histone modifications comprise epigenetic mechanisms that perform a key role in gene transcription and may regulate tumour development. Thus, the aim of this study was to determine whether two post-translational histone modifications, i.e. phosphorylation of serine 10 in histone H3 and acetylation of lysine 12 in histone H4, have prognostic value for OSCC patients. METHODS AND RESULTS: Paraffin-embedded tissue samples of 90 patients diagnosed with OSCC were obtained and subjected to immunohistochemical staining with antibodies against histone H3 with phosphorylation of serine 10 (H3S10ph) and histone H4 with acetylation of lysine 12 (H4K12ac). The associations of H3S10ph and H4K12ac expression levels with clinicopathological factors were determined. Five-year survival analysis and univariate and multivariate analyses were also performed. Both H3S10ph and H4K12ac were expressed in the nuclei of tumour cells. A low median of H3S10ph expression was significantly associated with cervical lymph node metastasis. Tumours with high H4K12ac expression were significantly associated with gender, alcohol consumption, and cervical lymph node metastasis. H4K12ac was also shown to have independent prognostic value in the multivariate analysis. Tumours with high H3S10ph expression, size >40 mm, an advanced stage and the presence of cervical lymph node metastases were associated with a better 5-year survival rate. Tumours with low H4K12ac expression, size >40 mm, an advanced stage and cervical lymph node metastasis were associated with a better 5-year survival rate. CONCLUSIONS: These findings suggest that H3S10ph, and mainly H4K12ac, may play a role in OSCC progression and the occurrence of cervical lymph node metastasis. Also, the expression level of H4K12ac could be an independent prognostic factor for OSCC patients.

Ameloblastic carcinoma: a Brazilian collaborative study of 17 cases.

AIMS: Ameloblastic carcinoma (AMECA) is an odontogenic malignancy that combines the histological features of ameloblastoma and cytological atypia. Because of its rarity, it poses difficulties in diagnosis. The aim of this study was to investigate the socio-demographic data, histopathology, immunohistochemical features, treatment and outcomes of 17 cases. METHODS AND RESULTS: Descriptive statistical analyses were used to portray the clinicopathological data collected, retrospectively. Log-rank tests were performed to determine new prognostic factors. Lesions were immunostained for Ki67, p16, p53, and cytokeratins (CKs), and compared with solid/multicystic ameloblastomas (n = 15). AMECA was mostly diagnosed at a late stage, affecting the posterior mandible of male patients in their fifth decade of life. Recurrence was diagnosed in nearly 90% of treated patients, and metastasis occurred in four patients. The mean number of Ki67-positive cells was 86.4 ± 66 per field. Tumours were focally positive for CK7, CK8, CK14, and CK18, and diffusely positive for CK19, p53, and p16. AMECA showed increased immunoexpression of CK18, CK19, p16, p53 and Ki67 as compared with benign cases. CONCLUSIONS: Our study has contributed to the improved characterization of the epidemiology, prognostic markers, treatment options and outcomes of AMECA. Current criteria must be reviewed to simplify the diagnostic process for these neoplasms.

Metallothionein gene expression is altered in oral cancer and may predict metastasis and patient outcomes.

AIMS: Metallothioneins (MTs) are proteins associated with the carcinogenesis and prognosis of various tumours. Previous studies have shown their potential as biomarkers in oral squamous cell carcinoma (OSCC). Aiming to understand more clearly the function of MTs in OSCC we evaluated, for the first time, the gene expression profile of MTs in this neoplasm. MATERIALS AND RESULTS: Tissue samples from 35 cases of tongue and/or floor of mouth OSCC, paired with their corresponding non-neoplastic oral mucosa (NNOM), were retrieved (2007-09). All tissues were analysed for the following genes using TaqMan(®) reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 and MT4. The expression of MT1B and MT1H was seldom detected in both OSCC and NNOM. A significant loss of MT1A, MT1X, MT3 and MT4 expression and gain of MT1F expression was observed in OSCC, compared to NNOM. Cases with MT1G down-regulation exhibited the worst prognoses. The up-regulation of MT1X was restricted to non-metastatic cases, whereas up-regulation of MT3 was related to cases with lymph node metastasis. CONCLUSIONS: Metallothionein mRNA expression is altered significantly in oral squamous cell carcinomas. The expression of MT1G, MT1X and MT3 may aid in the prognostic discrimination of OSCC cases.

Loss of heterozygosity (LOH) in tumour suppressor genes in benign and malignant mixed odontogenic tumours.

Although molecular alterations are reported in different types of odontogenic tumours, their pathogenesis remains to be established. Loss of heterozygosity (LOH) studies allow the identification of minimal regions of deletions of known or putative tumour suppressor genes, the losses of which may promote neoplastic growth. The purpose of this study was to investigate LOH in a set of odontogenic mixed tumours. Tumour suppressor gene loci on 3p, 9p, 11p, 11q and 17p chromosomes were analysed in five samples of ameloblastic fibroma (AF), three samples of ameloblastic fibro-odontoma (AFO) and three samples of ameloblastic fibrosarcoma (AFS). The most frequently lost genetic loci were p53 (17p13, 62%) and CHRNB1 (17p13, 55%). LOH at the chromosome regions 3p24.3, 9p22 and 9p22-p21 was identified only in AFS. No sample showed LOH at the chromosomal loci 3p21.2 and 11q13.4. For the region 9p22-p13, LOH occurred in one sample of AFO. The fractional allelic loss (FAL) was calculated for each sample. The mean FAL of the benign lesions (i.e. AF and AFO) was 22%, whereas the mean FAL of the malignant lesions (i.e. AFS) was 74.6%. In conclusion, our results show a higher FAL in AFS compared to its benign counterparts and reveal a different pattern of LOH of tumour suppressor genes in AFS, which may regulate changes in tumour behaviour.

Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig.

This study aimed to evaluate by the intra-osseous implant technique the most commonly used materials for pulp therapy in pediatric dentistry: calcium hydroxide (CH), Guedes Pinto paste and CTZ paste, according to FDI (1980) and ANSI/ADA (1982) recommendations. Thirty guinea pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks received one implant on each side of the lower jaw symphysis. The external lateral tube wall served as control for the technique. At the end of the observation periods, the animals were euthanized and specimens were prepared for routine histological examination. It was observed that CH and CTZ paste induced severe inflammation, a large amount of necrotic tissue, lymphocytes, foreign body cells and bone resorption, while Guedes Pinto Paste induced little or no inflammation in the 4-week observation period. After 12 weeks, the reactions to CH and Guedes Pinto paste were also absent/mild, presenting a general pattern of replacement by recently formed bone tissue while a moderate to severe inflammatory response was observed with CTZ paste. Guedes Pinto paste presented acceptable biocompatibility levels in both analyzed periods; CH only showed acceptable biocompatibility in the 12-week period while CTZ paste showed no biocompatibility in both periods. Among the tested materials, only Guedes Pinto paste presented an acceptable biocompatibility.

Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig

This study aimed to evaluate by the intra-osseous implant technique the most commonly used materials for pulp therapy in pediatric dentistry: calcium hydroxide (CH), Guedes Pinto paste and CTZ paste, according to FDI (1980) and ANSI/ADA (1982) recommendations. Thirty guinea pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks received one implant on each side of the lower jaw symphysis. The external lateral tube wall served as control for the technique. At the end of the observation periods, the animals were euthanized and specimens were prepared for routine histological examination. It was observed that CH and CTZ paste induced severe inflammation, a large amount of necrotic tissue, lymphocytes, foreign body cells and bone resorption, while Guedes Pinto Paste induced little or no inflammation in the 4-week observation period. After 12 weeks, the reactions to CH and Guedes Pinto paste were also absent/mild, presenting a general pattern of replacement by recently formed bone tissue while a moderate to severe inflammatory response was observed with CTZ paste. Guedes Pinto paste presented acceptable biocompatibility levels in both analyzed periods; CH only showed acceptable biocompatibility in the 12-week period while CTZ paste showed no biocompatibility in both periods. Among the tested materials, only Guedes Pinto paste presented an acceptable biocompatibility.

A pesquisa teve como objetivo avaliar a biocompatibilidade através da técnica de implantes intra-ósseos dos materiais utilizados em odontopediatria para tratamento pulpar: hidróxido de cálcio, pastas Guedes Pinto e CTZ, de acordo com as recomendações da FDI (1980) e ANSI/ADA(1982). Trinta guinea pigs, dez para cada material, divididos em períodos experimentais de 4 e 12 semanas receberam um implante em cada lado da sínfise mandibular. A parede lateral externa do copo serviu como controle para a técnica. No final dos períodos experimentais, os animais foram sacrificados e os espécimes preparados para o exame histológico de rotina. Observou-se que o hidróxido de cálcio e a pasta CTZ mostraram reação inflamatória severa, grande quantidade de tecido necrosado, linfócitos, células de corpo estranho e reabsorção óssea; enquanto a pasta Guedes Pinto induziu pouca ou nenhuma inflamação no período de 4 semanas. Após 12 semanas as reações para o hidróxido de cálcio e pasta Guedes Pinto foram ausentes/suaves apresentando um padrão geral de substituição por tecido ósseo neoformado, enquanto uma resposta inflamatória de moderada a severa foi observada para a pasta CTZ. A pasta Guedes Pinto apresentou níveis aceitáveis de biocompatibilidade nos dois períodos analisados; hidróxido de cálcio apresentou biocompatibilidade aceitável somente no período de 12 semanas e a pasta CTZ não mostrou biocompatibilidade em ambos os períodos. Entre estes, apenas a pasta Guedes Pinto apresentou níveis de biocompatibilidade nos dois períodos analisados.

Central odontogenic fibroma: a case report with long-term follow-up.

An osteolytic tumour of the mandible with prominent expansive growth on the alveolar ridge and displacement of the involved teeth is described in a 28-year-old man. The lesion was diagnosed as a central odontogenic fibroma, an uncommon benign neoplasm derived from dental apparatus, and was removed by curettage. The patient remains asymptomatic after thirteen years of follow-up, which supports the claimed indolent behavior of this poorly documented disease and the adequacy of a conservative surgical treatment.

Assessment of angiogenesis by CD105 antigen in epithelial salivary gland neoplasms with diverse metastatic behavior.

BACKGROUND: Information on the biology of metastasis development in salivary gland tumors is scarce. Since angiogenesis seems associated with this phenomenon in other tumors, we sought to compare salivary gland tumors with diverse metastatic behavior in order to improve the knowledge and management of these lesions. METHODS: Samples from the most important salivary gland tumors were segregated according to its metastatic behavior and submitted to routine immunohistochemistry to identify vessels positive for CD105 expression. Frequency of positive cases and intratumoral microvessel density (IMD) was compared among the group of lesions. RESULTS: CD105 positive vessels were absent in normal salivary gland tissue, were rare in pleomorphic adenomas and adenoid cystic carcinomas (ACC), more common in polymorphous low-grade adenocarcinomas and highest in mucoepidermoid carcinomas. Only ACC with such feature were metastatic. IMD was higher in malignant rather than benign tumors. CONCLUSION: Immunostaining of CD105 in salivary gland tumors implies participation of angiogenesis in the development of malignant lesions, as well as some role for myoepithelial cells in the control of new vessel formation. In addition, suggest that ACC with positive CD105 vessels are at higher risk for metastasis.

Keratoacanthoma of the inferior lip: review and report of case with spontaneous regression

Keratoacanthoma (KA) is a self-limited benign epithelial proliferative lesion that eventually presents with very similar clinical features to squamous cell carcinoma. Many KA appear in the vermilion border of the lips and therefore dental professionals must be familiar of the disease. This article reports the case of a 40-year-old female patient presenting with an exophytic ulcerative tumor in her lower lip that resolved after incisional biopsy. Photographic documentation of the case is presented and topics that are relevant to the clinical management of the disease are addressed.

Keratoacanthoma of the inferior lip: review and report of case with spontaneous regression.

Keratoacanthoma (KA) is a self-limited benign epithelial proliferative lesion that eventually presents with very similar clinical features to squamous cell carcinoma. Many KA appear in the vermilion border of the lips and therefore dental professionals must be familiar of the disease. This article reports the case of a 40-year-old female patient presenting with an exophytic ulcerative tumor in her lower lip that resolved after incisional biopsy. Photographic documentation of the case is presented and topics that are relevant to the clinical management of the disease are addressed.

Lack of galectin-3 alters the balance of innate immune cytokines and confers resistance to Rhodococcus equi infection.

Galectin-3 is a beta-galactoside-binding lectin implicated in the fine-tuning of innate immunity. Rhodococcus equi, a facultative intracellular bacterium of macrophages, causes severe granulomatous bronchopneumonia in young horses and immunocompromised humans. The aim of this study is to investigate the role of galectin-3 in the innate resistance mechanism against R. equi infection. The bacterial challenge of galectin-3-deficient mice (gal3-/-) and their wild-type counterpart (gal3+/+) revealed that the LD50 for the gal3(-/-) mice was about seven times higher than that for the gal3+/+ mice. When challenged with a sublethal dose, gal3(-/-) mice showed lower bacteria counts and higher production of IL-12 and IFN-gamma production, besides exhibiting a delayed although increased inflammatory reaction. Gal3(-/-) macrophages exhibited a decreased frequency of bacterial replication and survival, and higher transcript levels of IL-1beta, IL-6, IL-10, TLR2 and MyD88. R. equi-infected gal3+/+ macrophages showed decreased expression of TLR2, whereas R. equi-infected gal3(-/-) macrophages showed enhanced expression of this receptor. Furthermore, galectin-3 deficiency in macrophages may be responsible for the higher IL-1beta serum levels detected in infected gal3(-/-) mice. Therefore galectin-3 may exert a regulatory role in innate immunity by diminishing IL-1beta production and thus affecting resistance to R. equi infection.

Metallothionein immunolocalization in actinic skin nonmelanoma carcinomas.

Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent skin cancer. Its pathogeny is linked to genotoxic effects of actinic radiation exposure, especially to ultraviolet wavelength. Metallothionein (MT) is a low-molecular weight protein with high affinity for heavy metal. Its intracellular function has been related to heavy metals and free-radical detoxification, although many studies linked MT to protective action against actinic mutagenesis. In other way, overexpression in malignant tumors has been related to worse prognosis. We aimed to evaluate MT immunohistochemical expression in skin cancer associated to actinic radiation. Twenty-six BCC cases, 20 SCC, and 6 normal skin fragments were investigated. Immunohistochemical assay were performed by streptavidin-biotin-peroxidase technique with standard monoclonal antibody (E9). In normal skin, immunostaining was observed in basal layer of the epithelium. In the epithelium adjacent to tumors, suprabasal layer was also intensely labeled. Mean MT immunostaining indices were 18.5+21.2% for BCC and 69.1+14.4% for SCC. This difference was statistically significant. Higher MT expression in SCC as compared with BCC suggests association with tumoral aggressiveness.

Histological and serological evidence of experimental paracoccidioidomycosis in Calomys callosus (Rodentia: Cricetidae).

The responses of animal experimental models related to the infectivity, virulence and pathogenicity of Paracoccidioides brasiliensis is constantly used to develop new perspectives of investigation. The rodent Calomys callosus, Rengger 1830 (Rodentia: Cricetidae) is an indigenous inhabitant of the savannah environment found in the central regions of Brazil. The aim of the present work was to evaluate the histopathological and serological features of C. callosus after inoculation with the Pb18 strain of P. brasiliensis. Furthermore, A/Sn and B10.A mice strains were also tested to compare the results obtained in C. callosus to these well-established experimental models of resistance and susceptibility respectively. In every instance, survival analysis was performed, and histopathological study of the lungs, liver and spleen was employed to investigate tissue involvement, degree of inflammation and fungal presence. Levels of antibodies to P. brasiliensis were measured by using an enzyme-linked immunosorbent assay after 4 weeks and at the advanced stage of infection. The mortality rate was proportional to inoculation dose in all groups, but overall it was much superior in C. callosus than in the B10.A-susceptible mice. Macroscopical and microscopical pathological alterations were also more extensive and remarkable for C. callosus, once again proportional to inoculation dose, but more noticeable differences among the studied groups were found with 0.6x10(5) inoculum. In addition, the serological profile of C. callosus was similar to that found for B10.A-susceptible mice. Infection of C. callosus with 0.6x10(8) Pb18 inoculum resulted in more serious illness, and it decreased in severity in proportion to the inoculum dose. This difference was more pronounced in C. callosus, and the clinical, serological and pathological findings in this animal were more intense and precocious compared with the B10.A-susceptible mice. The present results suggest that C. callosus is a potentially alternative experimental animal model for paracoccidioidomycosis infection.

Comparison of the intraosseous biocompatibility of AH Plus, EndoREZ, and Epiphany root canal sealers.

To evaluate the intraosseous biocompatibility of AH Plus, EndoREZ, and Epiphany root canal sealers as recommended by the Technical Report #9 of the Fédération Dentaire Internationale (FDI). Thirty guinea pigs, 10 for each material, divided into experimental periods of 4 and 12 weeks, received one implant on each side of the lower jaw symphysis. At the end of the observation periods, the animals were killed and the specimens prepared for routine histological examination. After analyzing both periods, the inflammatory tissue reaction to EndoREZ was considered severe. In the AH Plus group, the reaction changed from severe to moderate, while it was observed biological compatibility to Epiphany with bone formation and none to slight inflammatory reaction. It was concluded that Epiphany root canal sealer was the only material that presented intraosseous biocompatibility within the two analyzed periods.

Immunolocalization of HLA-DR and metallothionein on amalgam tattoos.

Despite studies concerning toxic reactions related to amalgam components in the literature, few studies have been devoted to evaluate local noxious effects of amalgam tattoos (AT) on biological tissues. In addition, little is known about activation of inflammatory cells by mucosa-implanted amalgam debris. Tissue reaction to AT depends on the particle size. Human leukocyte antigen DR (HLA-DR) is an activation marker of inflammatory cells associated with antigen presentation. Metallothioneins (MT) are proteins involved with metal detoxication, including mercury and silver. The purpose of the present study was to investigate the immunolocalization of HLA-DR and MT in AT with large or powdered particles. Paraffin-embedded AT tissue blocks were sectioned and subjected to immunohistochemistry for HLA-DR and MT localization. The results demonstrated a dense mononuclear inflammatory infiltrate associated with large and powdered debris and positivity for HLA-DR and MT in inflammatory cells. While blood vessel walls and connective fibers impregnated with powdered particles were negative for HLA-DR, they were positive for MT. In addition, wherever epithelial basement membrane impregnation by powdered amalgam particles was observed, a strong positivity for MT was detected. These findings demonstrate that residual elements of AT still have noxious local effects over tissues.

Immunolocalization of HLA-DR and metallothionein on amalgam tattoos

Poucos estudos têm investigado a toxicidade tecidual local das tatuagens por amálgama (TA), embora diversos trabalhos demonstrem efeitos nocivos desse material restaurador. Pouco se sabe sobre a ativação de células inflamatórias nesse tipo de lesão. A reação tecidual contra os restos de amálgama varia com o tamanho das partículas implantadas. O antígeno leucocitário humano DR (HLA-DR) está associado com a ativação de células inflamatórias, sendo relacionado à apresentação de antígeno. Metalotioneínas (MT) são proteínas envolvidas com neutralização de metais pesados, tais como mercúrio e prata. O objetivo do presente trabalho foi investigar a imunolocalização de HLA-DR e MT em TA compostas por depósitos teciduais de diferentes tamanhos. Cortes histológicos de lesões fixadas em formol e embebidas em parafina foram submetidos a técnica imunoistoquímica para a detecção dos antígenos mencionados. Os resultados demonstraram denso infiltrado inflamatório associado com partículas grandes ou pulverizadas, observando-se presença de células HLA-DR e MT positivas. Paredes de vasos sangüíneos e fibras de tecido conjuntivo impregnadas por restos de amálgama foram negativas para HLA-DR, mas positivas para MT. Impregnação da membrana basal por partículas de amálgama correspondia a forte positividade para MT no epitélio. Esses resultados demonstram a existência de efeitos nocivos locais das TA sobre os tecidos.

Prognostic impact of metallothionein on oral squamous cell carcinoma.

Metallothionein (MT), a low-molecular-weight protein with high cysteine content, seems to be related to neoplastic resistance to oncologic treatment and therefore has been studied as a prognostic factor for a variety of human malignant tumors. MT overexpression in neoplasms of ectodermal origin is usually associated with a poor prognosis. MT expression was evaluated in 60 samples of oral squamous cell carcinoma by immunohistochemistry to study its prognostic influence on oral cancer. Possible associations of MT immunoexpression were also investigated with respect to clinical stage (TNM), histological grading, and proliferation index (Ki-67) of the lesions. No significant statistical correlation was observed among these variables. The impact on overall survival was assessed by uni and multivariate statistical tests. Mean MT labeling index was 60%. High MT labeling indexes (over 76%) predicted shorter survival in univariate statistical analysis. In multivariate analysis, MT labeling index and clinical stage were independent prognostic factors. MT overexpression in oral squamous cell carcinoma seems to be related to a worse prognosis for patients.