Differential response of hepatocellular carcinoma glycolytic metabolism and oxidative stress markers after exposure to human amniotic membrane proteins.

BACKGROUND: The human Amniotic Membrane (hAM) has been studied as a potential therapeutic option in cancer, namely in hepatocellular carcinoma. Previously, our research group evaluated the effect of human Amniotic Membrane Protein Extracts (hAMPE) in cancer therapy, demonstrating that hAMPE inhibit the metabolic activity of human hepatocellular carcinoma cell lines: Hep3B2.1-7, HepG2 and Huh7. Therefore, and considering the close relationship between metabolic activity and oxidative stress, the aim of this study was to evaluate the effect of hAMPE treatment in glucose metabolism and its role in oxidative stress of hepatocellular carcinoma. METHODS AND RESULTS: Glucose uptake and lactate production was assessed by 1 H-NMR, and the expression of several mediators of the glycolytic pathway was evaluated by Western blot or fluorescence. Total antioxidant capacity (TAC) and biomarkers of oxidative stress effects in proteins were detected. Our results showed that hAMPE treatment increased glucose consumption on Hep3B2.1-7, HepG2, and Huh7 through the increase of GLUT1 in Hep3B2.1-7 and Huh7, and GLUT3 in HepG2 cells. It was observed an increased expression of 6-phosphofrutokinase (PFK-1L) in all cell lines though glucose was not converted to lactate on HepG2 and Huh7 cells, suggesting that hAMPE treatment may counteract the Warburg effect observed in carcinogenesis. In Hep3B2.1-7, hAMPE treatment induced an increase in expression of lactate dehydrogenase (LDH) and monocarboxylate transporter isoform 4 (MCT4). We further detected that hAMPE enhances the TAC of culture media after 2 and 8 h. This was followed by a degree of protection against proteins nitration and carbonylation. CONCLUSIONS: Overall, this work highlights the potential usefulness of hAMPE as anticancer therapy through the modulation of the glycolytic and oxidative profile in human hepatocellular carcinoma.

GLP-1 improves adipose tissue glyoxalase activity and capillarization improving insulin sensitivity in type 2 diabetes.

Methylglyoxal was shown to impair adipose tissue capillarization and insulin sensitivity in obese models. We hypothesized that glyoxalase-1 (GLO-1) activity could be diminished in the adipose tissue of type 2 diabetic obese patients. Moreover, we assessed whether such activity could be increased by GLP-1-based therapies in order to improve adipose tissue capillarization and insulin sensitivity. GLO-1 activity was assessed in visceral adipose tissue of a cohort of obese patients. The role of GLP-1 in modulating GLO-1 was assessed in type 2 diabetic GK rats submitted to sleeve gastrectomy or Liraglutide treatment, in the adipose tissue angiogenesis assay and in the HUVEC cell line. Glyoxalase-1 activity was decreased in visceral adipose tissue of pre-diabetic and diabetic obese patients, together with other markers of adipose tissue dysfunction and correlated with increased HbA1c levels. Decreased adipose tissue GLO-1 levels in GK rats were increased by sleeve gastrectomy and Liraglutide, being associated with overexpression of angiogenic and vasoactive factors, as well as insulin receptor phosphorylation (Tyr1161). Moreover, GLP-1 increased adipose tissue capillarization and HUVEC proliferation in a glyoxalase-dependent manner. Lower adipose tissue GLO-1 activity was observed in dysmetabolic patients, being a target for GLP-1 in improving adipose tissue capillarization and insulin sensitivity.

Endometrial Cancer Spheres Show Cancer Stem Cells Phenotype and Preference for Oxidative Metabolism.

This study aimed to characterize endometrial cancer regarding cancer stem cells (CSC) markers, regulatory and differentiation pathways, tumorigenicity and glucose metabolism. Endometrial cancer cell line ECC1 was submitted to sphere forming protocols. The first spheres generation (ES1) was cultured in adherent conditions (G1). This procedure was repeated and was obtained generations of spheres (ES1, ES2 and ES3) and spheres-derived cells in adherent conditions (G1, G2 and G3). Populations were characterized regarding CD133, CD24, CD44, aldehyde dehydrogenase (ALDH), hormonal receptors, HER2, P53 and ß-catenin, fluorine-18 fluorodeoxyglucose ([18F]FDG) uptake and metabolism by NMR spectroscopy. An heterotopic model evaluated differential tumor growth. The spheres self-renewal was higher in ES3. The putative CSC markers CD133, CD44 and ALDH expression were higher in spheres. The expression of estrogen receptor (ER)α and P53 decreased in spheres, ERß and progesterone receptor had no significant changes and ß-catenin showed a tendency to increase. There was a higher 18F-FDG uptake in spheres, which also showed a lower lactate production and an oxidative cytosol status. The tumorigenesis in vivo showed an earlier growth of tumours derived from ES3. Endometrial spheres presented self-renewal and differentiation capacity, expressed CSC markers and an undifferentiated phenotype, showing preference for oxidative metabolism.

Dental caries and bacterial load in saliva and dental biofilm of type 1 diabetics on continuous subcutaneous insulin infusion.

OBJECTIVES: Since most of the studies evaluates diabetics on multiple daily injections therapy and continuous subcutaneous insulin infusion may help gain better metabolic control and prevent complications, the objective of this study was to evaluate the prevalence of dental caries, the unstimulated salivary flow rate and the total bacteria load, Streptococcus spp. levels and Lactobacillus spp. levels in saliva and supragingival dental biofilm of type 1 diabetics on insulin pump. MATERIAL AND METHODS: Sixty patients with type 1 diabetes on insulin pump and 60 nondiabetic individuals were included. The dental caries evaluation was performed using ICDAS and the oral hygiene was assessed according to Greene and Vermillion Simplified Oral Hygiene Index. Unstimulated saliva and supragingival dental biofilm were collected. Total bacteria, Streptococcus spp. and Lactobacillus spp. was quantified by qPCR. RESULTS: Patients with type 1 diabetes had a higher prevalence of dental caries and filled and missing teeth when compared with the control group. These patients were associated with more risk factors for the development of dental caries, namely a lower unstimulated salivary flow rate and a higher bacterial load in saliva and dental biofilm. CONCLUSION: Some risk factors related to dental caries were associated with type 1 diabetics. An early diagnosis combined with the evaluation of the risk profile of the diabetic patient is imperative, allowing the dental caries to be analyzed through a perspective of prevention and the patient to be integrated into an individualized oral health program.

Dental caries and bacterial load in saliva and dental biofilm of type 1 diabetics on continuous subcutaneous insulin infusion

Abstract Objectives Since most of the studies evaluates diabetics on multiple daily injections therapy and continuous subcutaneous insulin infusion may help gain better metabolic control and prevent complications, the objective of this study was to evaluate the prevalence of dental caries, the unstimulated salivary flow rate and the total bacteria load, Streptococcus spp. levels and Lactobacillus spp. levels in saliva and supragingival dental biofilm of type 1 diabetics on insulin pump. Material and Methods Sixty patients with type 1 diabetes on insulin pump and 60 nondiabetic individuals were included. The dental caries evaluation was performed using ICDAS and the oral hygiene was assessed according to Greene and Vermillion Simplified Oral Hygiene Index. Unstimulated saliva and supragingival dental biofilm were collected. Total bacteria, Streptococcus spp. and Lactobacillus spp. was quantified by qPCR. Results Patients with type 1 diabetes had a higher prevalence of dental caries and filled and missing teeth when compared with the control group. These patients were associated with more risk factors for the development of dental caries, namely a lower unstimulated salivary flow rate and a higher bacterial load in saliva and dental biofilm. Conclusion Some risk factors related to dental caries were associated with type 1 diabetics. An early diagnosis combined with the evaluation of the risk profile of the diabetic patient is imperative, allowing the dental caries to be analyzed through a perspective of prevention and the patient to be integrated into an individualized oral health program.

Autotransplantation combined with orthodontic treatment: a case involving the maxillary central incisors with root resorption after traumatic injury.

Traumatic dental injury can result in avulsion of anterior teeth. In young patients, it is a challenge to the dental professional because after replantation, late complications such as ankylosis require tooth extraction. Although prosthetic and orthodontic treatment, and implant placement have been described as the options for intervention, autogenous tooth transplantation could be an effective procedure in growing patients if there is a suitable donor tooth available. This case presents the treatment of a patient who suffered a traumatic injury at 9 years old with avulsion of tooth 21, which had been replanted, and intrusion of tooth 11. Both teeth ankylosed; thus they were removed and autotransplantation of premolars was carried out. After transplantation, the tooth underwent root canal treatment because of pulpal necrosis. Orthodontic treatment began 3 months after transplantation and during 7 years' follow-up the aesthetics and function were maintained without signs of resorption.

Hematogenic Dissemination of Triple-negative Versus Hormonal Receptor-positive Breast Cancer Cells.

The aim of this study was to characterize the hematogeneous spread, in vivo, of breast cancer (BC) cell lines that express hormonal receptors (HR) comparing with triple-negative (TN) BC, particularly considering the lung and liver. Female Balb/c nu nu mice (n=30) were injected with two breast cancer cell lines (MCF7 and HCC1806). Nuclear medicine imaging with Technetium ((99m)Tc)-hydroxymethylene diphosphonate ((99m)Tc-HMDP) and (99m)Tc-Hexakis 2-methoxy-2-methylpropylisonitrile (MIBI) were performed between the 7th and 8th weeks after injection. The histological metastatic foci were analyzed by morphometric and immunohistochemistry studies regarding estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (ERBB2) and cytokeratin (CK)-5/6. The mean area of lung metastasis in MCF7 cases was significantly higher (p=0.023), although the number of liver foci was higher in the HCC1806 group (p=0.006). Logistic regression revealed a potentiating model for liver metastasis with HCC1806 cells (odds ratio=16; p=0.03). The number and area of lung-metastatic foci were not predictive of liver dissemination. Lung metastasis study showed ER positivity in 57.1% of the MCF7 group, compared to 80% of the HCC1806 group. PR was positive in 42.9% of MCF7 cases and negative in 60% of HCC1806 cases. HR-positive cells developed massive lung metastization. TN cells seem to potentiate liver metastasis. ER, PR, ERBB2 and basal-like CK expression in metastases was not uniformly correlated with that of primary tumor cells.

2-Bromo-5-hydroxyphenylporphyrins for photodynamic therapy: photosensitization efficiency, subcellular localization and in vivo studies.

BACKGROUND: Photodynamic therapy (PDT) is a therapeutic modality capable of inducing cell death by oxidative stress through activation of a sensitizer by light. Aryl-porphyrin with hydroxyl groups are good photosensitizers and presence of bromine atoms can enhance the photodynamic activity through heavy atom effect. These facts and our previous work made pertinent to compare the photodynamic capacity of tetraaryl brominated porphyrin (TBr4) with the corresponding diaryl (BBr2) derivative. METHODS: Cell cultures were incubated with the sensitizers, ranging from 50nM to 10µM and irradiated until 10J. Cell proliferation was analysed by MTT assay. Flow cytometry studies evaluated cell death pathways, mitochondrial membrane potential and ROS. For in vivo studies Balb/c nu/nu mice were injected with 4×10(6)cells. After PDT, monitoring was carried out for 12 days to establish Kaplan-Meier survival curves. Tumours were excised and histological analysis was performed. RESULTS: Both sensitizers seem to accumulate in the mitochondria. The molecules have no intrinsic cytotoxicity or in non-tumour cells at therapeutic concentrations. Both sensitizers induced a significant decrease of cell proliferation and growth of xenografts of melanoma and colorectal adenocarcinoma. Diaryl BBr2 is more efficient than tetraaryl TBr4, concerning intracellular ROS production, mitochondrial disruption and induction of cell death. The main cell death pathway is necrosis. CONCLUSIONS: TBr2 and BBr4 are promising sensitizers with good photodynamic properties and have the ability to induce cell death in human melanoma and colorectal adenocarcinoma in vitro and in vivo. We consider that BBr2 is a molecule that should be the subject of extensive studies towards clinical use.

Effect of calcium hydroxide as intracanal medication on the apical sealing ability of mineral trioxide aggregate (MTA): an in vitro apexification model / Efeito da medicação intracanal de hidróxido de cálcio na capacidade seladora do mineral trióxido agregado (MTA): um modelo de apicificação in vitro

Objective - To evaluate the effect of calcium hydroxide premedication on the apical seal of White MTA, placed as an apical barrier in permanent teeth with simulated immature apices. Furthermore, we intended to compare potential changes, under the influence of calcium hydroxide, in the apical seal of MTA over time. Methods - Thirty-four single-rooted extracted teeth were prepared in order to simulate a divergent open apex. Two experimental groups of 10 teeth were created: group 1 (G1) and group 2 (G2) with and without calcium hydroxide intracanal medication previous to the placement of MTA apical plug. Two control groups, positive and negative, each with 7 teeth were created. On the 7th and on the 28th day after placement of the MTA apical plug, the apices of the teeth were submersed in a solution of sodium pertechnetate (99mTcO4Na) for 3 hours. The radioactivity was measured using a gamma camera. Results - Results revealed statistically significant differences between the 2 control groups and the 2 experimental groups with respect to the microleakage. Within the experimental groups no statistically significant differences were found; nor between the two observed periods. Conclusion - Based on the results obtained in this study, it was concluded that intracanal medication with calcium hydroxide did not affect the sealing ability of WMTA, placed as an apical plug, neither on the 7th, nor on the 28th day.

Objetivo - Avaliar o efeito do hidróxido de cálcio como medicação intracanal, no selamento apical do MTA branco, colocado como uma barreira apical em dentes permanentes com ápices imaturos simulados. Métodos - Trinta e quatro dentes unirradiculares extraídos foram preparados para simular um ápice aberto divergente. Foram separados em dois grupos experimentais (n=10): G1 e G2 com e sem medicação intracanal de hidróxido de cálcio antes da colocação do tampão apical com o MTA e dois grupos controle, positivos e negativos (n=7 em cada). Em 7 e 28 dias após a colocação do tampão apical de MTA, os ápices dos espécimes foram submersos em uma solução de pertecnetato de sódio (99mTcO4Na) por 3 horas. A radioatividade foi medida usando uma câmara gama. Resultados - Os resultados revelaram diferenças estatisticamente significativas entre os grupos controle e os grupos experimentais com relação a microinfiltração. Não houve diferença entre os grupos experimentais nos dois períodos. Conclusão - Com base nos resultados obtidos neste estudo, concluiu-se que a medicação intracanal com hidróxido de cálcio não influenciou na capacidade seladora do MTA.

Estrogen receptor ligands for targeting breast tumours: a brief outlook on radioiodination strategies.

The design and development of radiolabelled estradiol derivatives has been an important area of research due to their recognized value in breast cancer management. The estrogen receptor (ER) is a relevant biomarker in the diagnosis, prognosis and prediction of the therapeutic response in estrogen receptor positive breast tumours. Hence, many radioligands based on estradiol derivatives have been proposed for targeted functional ER imaging. The main focus of this review is to survey the current knowledge on estradiol-based radioiodinated receptor ligands synthesis for breast tumour functional imaging. The main preclinical and clinical achievements in the field will also be briefly presented to make the manuscript more comprehensive.

Dynamics of CD86 expression on allergic inflammation--new insights.

CD86 is a well-known costimulatory molecule in its interaction with CD28 and/or CTLA present on T cells, and is essential for full activation of naïve T-cell and subsequent differentiation. Usually the B7 molecules are expressed mainly on APCs and B cells and in specific conditions on other activated cells. These costimulatory molecules are involved in the development of allergic inflammation and airways hyperreactivity (AHR) in allergen-challenged mice. Activated T cells, CD4(+)CD25(+), express CD86 in the first 60 minutes after the specific inhalatory exposure. These T cells can be relevant in IgE mediated allergic reaction possibly by an autocrine costimulation via CD28/CTLA activation pathway. The blockage of the expression of CD86 could be a potential therapeutical target to reduce the magnitude or the progression of the allergic reaction. The review article also discussed relevant patents.

Radioiodinated ligands for the estrogen receptor: effect of different 7-cyanoalkyl chains on the binding affinity of novel iodovinyl-6-dehydroestradiols.

Three novel 17 alpha-ethynyl-Delta(6,7)-estra-3,17beta-diols and their 17 alpha-[(125)I]-iodovinyl derivatives, containing different C7-cyanoalkyl chains, were studied as potential radioligands for the estrogen receptor. The influence of the chain length on the biological behaviour of the compounds was assessed through in vitro ER binding assays of the ethynyl derivatives and breast cancer cell uptake studies of the 17 alpha-[(125)I]-iodovinyl-Delta(6,7)-estra-3,17beta-diols. A difference in alkyl chain induced a decrease in ER binding affinities of substances, however, the receptor-binding affinities (RBA) of all compounds were lower than that of estradiol itself. In addition, a non-specific cell binding was observed which is in accordance with the encountered ethynyl RBA values suggesting that the uptake is not ER mediated.

Imunoterapia específica - avaliação dinâmica e cinética in vivo do extrato terapêutico em doentes alérgicos / Specific immunotherapy - in vivo dinamic and kinetic evaluation of a therapeutic extract in allergic patients

Introdução: A imunoterapia específica (IT) é uma forma comum de tratamento da doença alérgica. O mecanismo preciso desta terapêutica não é conhecido, embora a eficácia clínica esteja plenamente documentada. O objetivo deste estudo foi avaliar a cinética da IT , aplicando uma técnica de Medicina Nuclear, marcação de leucócitos com 99mTc-HMPAO, em doentes em fase de manutenção e com excelente eficácia clínica à terapêutica.Pacientes e métodos: Foram estudados 14 doentes alérgicos agrupados de acordo com o tipo de extrato e via de administração: extratos aquosos subcutâneos (látex= 4 doentes; veneno de abelha= 2 doentes), extrato depot subcutâneo (ácaros= 2 doentes; gramíneas= 2 doentes); extrato modificado subcutâneo (gramíneas= 1 doente; Parietária= 1 doente) extrato sublingual (ácaros= 2 doentes). O grupo controle foi constituído por dois doentes alérgicos a ácaros, submetidos respectivamente a: injecção subcutânea de soluto salino e injecção de extrato bacteriano por via subcutânea (controle positivo).Simultaneamente à administração do extrato alergênico terapêutico, procedeu-se à re-injecção de leucócitos marcados com 99mTc-HMPAO, em veia periférica contralateral. A aquisição dinâmica decorreu durante 60 minutos, com matriz de 64x64, 2 imagens/ minuto em projeção torácica anterior. As aquisições estáticas, com matrizes 256x256, durante 5 minutos cada foram adquiridas aos 60, 90, 120, 180, 240, 300 e 360 minutos, em projeção torácica (anterior e posterior) e abdominal (anterior).Resultados: A atividade inflamatória no ocal de administração da IT para os extratos subcutâneos aquosos e depot iniciou-se na primeira hora e manteve um aumento ao longo do tempo de estudo. Para os extratos sublinguais a atividade inflamatória foi observada logo nos primeiros minutos. Todos os estratos subcutâneos condicionaram dreanagem linfática ascendente para áreas axilares homolaterais nos primeiros minutos após a adminstração do extrato e, posteriormente, para tecido linfóide do mediastino superior e anterior, e áreas cervicais. As focalizações torácicas estiveram presentes em todos os doentes estudados, o mesmo não acontecendo para a atividade de intestinal. A via sublingual não induziu focalizações axilares ou instetinais, mesmo tendo havido deglutição do alérgeno. Foram calculados coeficientes corrigidos de captação em áreas individualizadas (ROIs- region of interest) em relação aos coeficientes de captação das áreas de background.