IMMUNOHISTOCHEMICAL DETECTION OF L CELLS IN GASTROINTESTINAL TRACT MUCOSA OF PATIENTS AFTER SURGICAL TREATMENT FOR CONTROL OF TYPE 2 DIABETES MELLITUS.

OBJECTIVE: Type 2 diabetes mellitus (T2DM) is a disease of global impact that has led to an increase in comorbidities and mortality in several countries. Immunoexpression of the incretin hormones such as glucagon-like peptide-1 (GLP-1) and peptide YY (3-36) (PYY3-36) can be used as a scorer in the gastrointestinal tract to analyze L-cell activity in response to T2DM treatment. This study aimed to investigate the presence, location, and secretion of L cells in the small intestine of patients undergoing the form of bariatric surgery denominated adaptive gastroenteromentectomy with partial bipartition. METHODS: Immunohistochemical assays, quantitative real-time polymerase chain reaction (qPCR), and Western blot analysis were performed on samples of intestinal mucosa from patients with T2DM in both the preoperative and postoperative periods. RESULTS: All results were consistent and indicated basal expression and secretion of GLP-1 and PYY3-36 incretins by L cells. A greater density of cells was demonstrated in the most distal portions of the small intestine. No significant difference was found between GLP-1 and PYY3-36 expression levels in the preoperative and postoperative periods because of prolonged fasting during which the samples were collected. CONCLUSION: The greater number of L cells in activity implies better peptide signaling, response, and functioning of the neuroendocrine system.

High reduction of staphylococcal biofilm by aqueous extract from marine sponge-isolated Enterobacter sp.

Staphylococcus aureus and Staphylococcus epidermidis are among the most important bacterial species responsible for biofilm formation on indwelling medical devices, including orthopaedic implants. The increasing resistance to antimicrobials, partly attributed to the ability to form biofilms, is a challenge for the development of new antimicrobial agents. In this study, the cell-free supernatant obtained from sponge-associated Enterobacter strain 84.3 culture inhibited biofilm formation (>65%) and dissociated mature biofilm (>85%) formed by S. aureus and S. epidermidis strains. The culture supernatant was subjected to solvent partitioning and the aqueous extract presented a concentration-dependent antibiofilm activity for each strain with a minimum biofilm eradication concentration (MBEC) ranging from 16 to 256 µg/mL. The effect of the aqueous extract on mature S. aureus biofilm was analyzed by confocal scanning laser microscopy, showing a significant reduction of the biofilm layer as well as diminished interactions among the cells. This extract is not toxic for mammalian cells (L929 cell line). Studies targeting substances with antibiofilm activity gained significant attention in recent years due to difficult-to-treat biofilm infections. Here, sponge-associated Enterobacter 84.3 proved to be a source of substances capable of eradicating staphylococcal biofilm, with potential medical use in the future.

Synergistic effects of ketamine and azole derivatives on Candida spp. resistance to fluconazole.

The emergence of Candida spp. with resistance to antifungal molecules, mainly the azole class, is an increasing complication in hospitals around the globe. Aim: In the present research, we evaluated the synergistic effects of ketamine with two azole derivatives, itraconazole and fluconazole, on strains of Candida spp. to fluconazole. Materials & methods: The drug synergy was evaluated by quantifying the fractional inhibitory concentration index and by fluorescence microscopy and flow cytometry techniques. Results: Our achievements showed a synergistic effect between ketamine in addition to the two antifungal agents (fluconazole and itraconazole) against planktonic cells and biofilms of Candida spp. Conclusion: This combination promoted alteration of membrane integrity, generation of reactive oxygen species, damage to and DNA and externalization of phosphatidylserine.

Modulation of cancer cells' radiation response in the presence of folate conjugated Au@Fe2O3 nanocomplex as a targeted radiosensitizer.

OBJECTIVES: To investigate the effects of Au@Fe2O3 core-shell nanoparticle (NP), with and without conjugation to folic acid (FA) as a targeting ligand, on radiosensitization of both cancer and healthy cells. METHODS: Au@Fe2O3 NPs were first synthesized, then modified with FA, and finally characterized. Radiation dose enhancement studies were performed on KB cancer cells and L929 healthy cells. NPs at the concentration of 20 µg/ml were first incubated with both cell lines and then different doses of 6 MV X-ray radiation were examined. The end effects were evaluated via MTT assay and flow cytometry using AnnexinV/PI kit. RESULTS: It was indicated that viability of KB cells has a much lower rate than L929 cells when the cells were treated by {(FA-Au@Fe2O3) + (X-ray)} regimen. Cell viability was even decreased significantly when X-ray dose increased. Moreover, flow cytometry studies revealed that FA-targeted NPs induced higher level of apoptosis for KB cancer cells than L929 healthy cells. CONCLUSION: Our findings provide a new perspective on high ability of the synthesized FA-targeted Au@Fe2O3 NPs which may be considered as an efficient radiosensitizer in the process of targeted radiation therapy of cancer.

Evaluation of recombinant human parathyroid hormone by CZE method and its correlation with in vitro bioassay and LC methods.

A stability-indicating capillary zone electrophoresis (CZE) method was validated to assess the content/potency of the recombinant human parathyroid hormone (rhPTH 1-34), using ranitidine as internal standard (IS). A fused-silica capillary, (i.d. of 50µm; effective length of 40cm) was used at 25°C; the applied voltage was 20kV. The background electrolyte solution consisted of 50mmolL-1 sodium dihydrogen phosphate solution at pH 3.0. Injections were performed using a pressure mode at 50 mbar for 45s, with detection by photodiode array (PDA) detector set at 200nm. Separation was obtained with a migration time of 5.3min, and was linear over the concentration range of 0.25-250µgmL-1 (r2 =0.9992). Specificity and stability-indicating capability were established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.28% with bias lower than 0.85%. Analyses of the same batches showed mean differences of the estimated content/potencies of 0.61%, 1.31% higher and 0.86% lower as compared to the validated reversed-phase and size exclusion liquid chromatography methods, and to the UMR-106 cell culture bioassay, respectively, with non-significant differences (p>0.05). Degraded forms were also subjected to the in vitro cytotoxicity test. The results obtained showed the capabilities of each one of the methods, and constitute an alternative strategy to monitor stability, improve the quality control and ensure the batch-to-batch consistency of bulk and finished biotechnology-derived medicine.

Berberine Antifungal Activity in Fluconazole-Resistant Pathogenic Yeasts: Action Mechanism Evaluated by Flow Cytometry and Biofilm Growth Inhibition in Candida spp.

The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, and Hydrastis canadensis, and of Phellodendron amurense Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistant Candida and Cryptococcus neoformans strains, as well as against the biofilm form of Candida spp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 µg/ml and 16 µg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P < 0.001).

Dual properties of hispidulin: antiproliferative effects on HepG2 cancer cells and selective inhibition of ABCG2 transport activity.

Hepatocellular carcinoma is the third most common cause of cancer-related deaths worldwide. Furthermore, the existing pharmacological-based treatments are insufficiently effective and generate many side effects. Hispidulin (6-methoxy-5,7,4'-trihydroxyflavone) is a flavonoid found in various medicinal herbs that present antineoplastic properties. Here we evaluated how modulation of reactive oxygen species (ROS) and alterations of antioxidant defenses could be associated to the antiproliferative effects of hispidulin in HepG2 cells. In addition, we studied the inhibitory activity of hispidulin on the efflux of drugs mediated by ABC transporters involved in multidrug resistance. In order to understand the increase of intracellular ROS promoted by hispidulin, we investigated the mRNA expression levels and activities of antioxidant enzymes, and the GSH/GSSG ratio. We showed that hispidulin significantly down-regulated the transcription levels of catalase, leading to reduction of enzyme activity and decrease of the GSH content. We also observed that, in the presence of N-acetylcysteine or exogenous catalase, the proliferation was lowered back to the control levels. These data clearly indicate a strong involvement of intracellular ROS levels for triggering the antiproliferative effects. We also demonstrated that the inhibition produced by hispidulin on drug efflux was specific for ABCG2, since no effects were observed with ABCB1 and ABCC1. Furthermore, HepG2 cells were more sensitive to hispidulin-mediated cell death than immortalized L929 fibroblasts, suggesting a differential toxicity of this compound between tumor and non-tumor cell lines. Our results suggest that hispidulin constitutes a promising candidate to sensitize chemoresistant cancer cells overexpressing ABCG2.

Phototoxic effect of curcumin on methicillin-resistant Staphylococcus aureus and L929 fibroblasts.

Photodynamic therapy has been investigated as an alternative method of killing pathogens in response to the multiantibiotic resistance problem. This study evaluated the photodynamic effect of curcumin on methicillin-resistant Staphylococcus aureus (MRSA) compared to susceptible S. aureus (MSSA) and L929 fibroblasts. Suspensions of MSSA and MRSA were treated with different concentrations of curcumin and exposed to light-emitting diode (LED). Serial dilutions were obtained from each sample, and colony counts were quantified. For fibroblasts, the cell viability subsequent to the curcumin-mediated photodynamic therapy was evaluated using the MTT assay and morphological changes were assessed by SEM analysis. Curcumin concentrations ranging from 5.0 to 20.0 µM in combination with any tested LED fluences resulted in photokilling of MSSA. However, only the 20.0 µM concentration in combination with highest fluence resulted in photokilling of MRSA. This combination also promoted an 80% reduction in fibroblast cell metabolism and morphological changes were present, indicating that cell membrane was the main target of this phototherapy. The combination of curcumin with LED light caused photokilling of both S. aureus strains and may represent an alternative treatment for eradicating MRSA, responsible for significantly higher morbidity and mortality and increased healthcare costs in institutions and hospitals.

Antimicrobial properties and cytotoxicity of an antimicrobial monomer for application in prosthodontics.

PURPOSE: This study aimed to investigate the antimicrobial properties and cytotoxicity of the monomer methacryloyloxyundecylpyridinium bromide (MUPB), an antiseptic agent capable of copolymerizing with denture base acrylic resins. MATERIALS AND METHODS: The antimicrobial activity of MUPB was tested against the species Candida albicans, Candida dubliniensis, Candida glabrata, Lactobacillus casei, Staphylococcus aureus, and Streptococcus mutans. The minimum inhibitory and fungicidal/bactericidal concentrations (MIC, MFC/MBC) of MUPB were determined by serial dilutions in comparison with cetylpyridinium chloride (CPC). The cytotoxic effects of MUPB at concentrations ranging from 0.01 to 1 g/L were assessed by MTT test on L929 cells and compared with methyl methacrylate (MMA). The antimicrobial activity of copolymerized MUPB was tested by means of acrylic resin specimens containing three concentrations of the monomer (0, 0.3, 0.6% w/w). Activity was quantified by means of a disc diffusion test and a quantification of adhered planktonic cells. Statistical analysis employed the Mann-Whitney test for MIC and MFC/MBC, and ANOVA for the microbial adherence test (α = 0.05). RESULTS: MUBP presented lower MIC values when compared with CPC, although differences were significant for C. dubliniensis and S. mutans only (p= 0.046 and 0.043, respectively). MFC/MBC values were similar for all species except C. albicans; in that case, MUPB presented significantly higher values (p = 0.046). MUPB presented higher cytotoxicity than MMA for all tested concentrations (p < 0.001) except at 0.01 g/L. Irrespective of the concentration incorporated and species, there was no inhibition halo around the specimens. The incorporation of MUPB influenced the adhesion of C. albicans only (p = 0.003), with lower CFU counts for the 0.6% group. CONCLUSIONS: It was concluded that non-polymerized MUPB has an antimicrobial capacity close to that of CPC and high cytotoxicity when compared with MMA. The antimicrobial activity of MUPB after incorporation within a denture base acrylic resin did not depend on its elution, but was shown to be restricted to C. albicans.

Evaluation of cytotoxicity and degree of conversion of glass ionomer cements reinforced with resin.

The objective of the present study was to evaluate the cytotoxicity and degree of monomer conversion of resin-reinforced glass ionomer cements (RGIC) over different time periods. Four RGICs: Fuji Ortho LC (FOLC), Fuji Ortho Band (FOB), Orthoglass (OGL), and Multicure Glass Ionomer (MCI) were evaluated for cytotoxicity in fibroblastic L929 cells and for their degree of monomer conversion over different time periods. Three control groups were also analysed: positive control (C+), consisting of Tween 80 cell detergent; negative control (C-), consisting of phosphate-buffered saline; and cell control (CC), consisting of cells exposed to any material. To evaluate the cytotoxicity, the dye-uptake technique was used and the degree of conversion was evaluated using infrared spectroscopy. The data obtained were analysed by analysis of variance and the Tukey's test. The results showed cytotoxicity of the RGICs at 1 and 24 hours; the viability values of these materials were statistically different from the C- and CC groups (P < 0.05). After 48 hours, the FOLC group was statistically similar to the CC and C- groups but different from the others. At 1 hour, there was no difference in the degree of conversion between the FOLC and OGL groups (P > 0.05) or between the FOB and MCI (P < 0.05) groups. However, at 48 hours, the FOLC group had greater conversion values than the other groups (P < 0.05). There is a direct relationship between the degree of conversion and RGIC cytotoxicity. Following initial polymerization, cytotoxicity decreases and, consequently, the degree of conversion of the material increases.

Annexin VI is a mannose-6-phosphate-independent endocytic receptor for bovine ß-glucuronidase.

Endocytosis and transport of bovine liver ß-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine ß-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine ß-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine ß-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine ß-glucuronidase.

Antiviral activity of Bignoniaceae species occurring in the State of Minas Gerais (Brazil): part 1.

AIMS: To evaluate the antiviral activity of Bignoniaceae species occurring in the state of Minas Gerais, Brazil. METHODS AND RESULTS: Ethanol extracts of different anatomical parts of bignoniaceous plant species have been evaluated in vitro against human herpesvirus type 1 (HSV-1), vaccinia virus (VACV) and murine encephalomyocarditis virus (EMCV) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A total of 34 extracts from 18 plant species selected according to ethnopharmacological and taxonomic criteria were screened. Fifteen of the 34 extracts (44.1%) have disclosed antiviral activity against one or more of the viruses assayed with EC(50) values in the range of 23.2 ± 2.5-422.7 ± 10.9 µg ml(-1). CONCLUSIONS: Twelve of the 34 extracts (35.3%) might be considered promising sources of antiviral natural products, as they have shown EC50 ≤ 100 µg ml(-1). The present screening discloses the high potential of the Bignoniaceae family as source of antiviral agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Active extracts were identified and deserve bioguided studies for the isolation of antiviral compounds and studies on mechanism of action.

Cytotoxicity of monomers, plasticizer and degradation by-products released from dental hard chairside reline resins.

OBJECTIVES: The aim of this study was to evaluate the cytotoxic effect of the monomers isobutyl methacrylate (IBMA) and 1,6-hexanediol dimethacrylate (1,6-HDMA), the plasticizer di-n-butyl phthalate (DBP), and the degradation by-products methacrylic acid (MA) and benzoic acid (BA) on L929 cells. Based on previous investigations on the release of these compounds from hard chairside reline resins, a range of concentrations (micromol/L) were selected for the cytotoxicity tests (IBMA, 5.49-1406.57; 1,6-HDMA, 1.22-39.32; DBP, 1.12-143.8; MA, 9.07-581; BA, 3.19-409). METHODS: Cytotoxic effects were assessed using MTT and (3)H-thymidine assays after the cells had been exposed to the test compounds at the given concentrations for 24 h. Cytotoxicity was rated based on cell viability relative to controls (cells exposed to medium without test substances). RESULTS: DNA synthesis activity was inhibited by all compounds. Mitochondrial dehydrogenase activity decreased in cells treated with monomers, plasticizer and MA by-product, whereas no cytotoxic effect was observed on contact with BA at the majority of concentrations tested. The ranges of suppression for (3)H-thymidine assay were: IBMA, 25-95%; 1,6-HDMA, 95-98%; DBP, 40-98%; MA, 97-99%; BA, 54-71%. For MTT assay, the ranges of suppression were: IBMA, 0-96%; 1,6-HDMA, 26-89%; DBP, 17-80%; MA, 52-66%; BA, 0-27%. The (3)H-thymidine assay was more sensitive than the MTT assay. SIGNIFICANCE: This study evaluated the cytotoxicity of a wide range of concentrations of monomers (IBMA and 1,6-HDMA), plasticizer (DBP) and degradation by-products (MA and BA), including those expected to be released from hard chairside reline resins. The differences observed in the cytotoxicity of these compounds, along with other properties, may assist the dental practitioners in the selection of reline materials with improved service life performance and low risk of adverse reactions in patients who wear relined dentures.

The effects of low-level diode laser treatment and dental pulp-capping materials on the proliferation of L-929 fibroblasts.

Low-level laser therapy (LLLT) has been reported to improve tissue healing and might therefore be useful in dental pulp capping after trauma. We evaluated the effects of a low-level diode laser (lambda = 680 nm) and dental pulp-capping substances on cell proliferation. Calcium hydroxide and adhesive resin were applied as conditioned media to cultures. Half of the samples received irradiation with the diode laser at a fluence of 4 J/cm(2) for 60 s. Using a hemocytometer, cells were counted at 1, 3, 5, and 7 days, and the data were analyzed by ANOVA. All cultures exhibited continuous growth, except those treated with adhesive resin. As compared to the other two groups, cell proliferation was significantly lower in cultures treated with adhesive resin; it was also significantly lower in cultures treated with calcium hydroxide, as compared to the control group. When combined with dental pulp-capping materials, LLLT had no effect on L-929 cell proliferation.

Cytotoxicity of endodontic irrigants containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from mouse L929 cell line.

The aim of this study was to evaluate the cytotoxicity of root canal irrigating solutions containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from L929 cell line. Saturated calcium hydroxide aqueous solution (CH), sodium lauryl sulphate (SLS) and SLS associated with calcium hydroxide (HCT20) were diluted with sterile distilled water at 50%, 20%, 10% and 5% concentrations. Minimum essential medium (MEM) served as the control group. The cytotoxicity of the solutions was evaluated on L929 mouse fibroblast cell line, at 4 and 24 h of contact time by the 51Cr radiotracer method. Data were compared and statistical inferences were made with the chi-square test. In all analysis, significance level was set at 5%. CH and HCT20 showed toxicity at 50% concentration, while at concentrations lower than 50% these solutions showed cell tolerance. SLS was cytotoxic at all concentrations. In conclusion, the association of calcium hydroxide and SLS (HCT20) combines the beneficial properties of these solutions and was not harmful to the fibroblast cell line, seeming to be a suitable endodontic irrigating solution.

Actividad citotóxica de physalis peruviana (aguaymanto) en cultivos celulares de adenocarcinoma colorectal, próstata y leucemia mieloide crónica / Cytotoxic effect of physalis peruviana in cell culture of colorectal and prostate cancer and chronic myeloid leukemia

INTRODUCCIÓN: Las plantas han sido empleadas como drogas por siglos. Sin embargo, se ha investigado poco sobre su gran potencial como fuentes de nuevos agentes terapéuticos. El objetivo del presente trabajo fue evaluar la actividad citotóxica de los extractos etanólicos de tallos y hojas de Physalis peruviana, sobre las líneas celulares HT-29, PC-3, K-562 y VERO. MATERIALES Y MÉTODOS: Las líneas celulares HT-29, PC-3, K-562 y VERO, fueron expuestas a cuatro concentraciones de extractos etanólicos de tallos y hojas de Physalis peruviana. Asimismo, a diferentes concentraciones de cisplatino y 5-Fluorouracilo (5-FU), que se usaron como controles positivos. Se hallaron los porcentajes de crecimiento en 48 horas, luego se determinó la concentración inhibitoria 50 (IC50) mediante análisis de regresión lineal y el índice de selectividad de cada muestra. RESULTADOS: Los extractos etanólicos de tallos y hojas de Physalis peruviana mostraron actividad citotóxica: Los CI50 en μg/mL en hojas y tallos fueron, 0.35 (r=-0.95 p<0.025) y 0.37 (r=-0,90 p<0.05) para HT-29; 0.87 (r=-0.98 p<0.01) y 1.01 (r=-0.95 p<0.025) para PC-3; 0.02 (r=-0.98 p<0.01) y 0.03 (r=-0.98 p<0.01) para K-562; 4.9 (r=-0.95 p<0.025) y 6.2 (r=-0.98 p<0.01) para VERO. Los CI50 para los antineoplásicos fueron: para el cisplatino: 4.2 (r=-0.96 p<0.025), 10.3 (r=-0.97 p<0.025), 0.15 (r=-0.98 p=0.01), y 1.1 (r=-0.98 p=0.01). Parael 5-FU: 2.3 (r=-0.97 p<0.025), 17.9 (r=-0.95 p<0.025), 0.15 (r=-0.98 p=0.01), y 1.1 (r=-0.94 p=0.05) para HT-29, PC-3, K562 y VERO, respectivamente. Los índices de selectividad de los extractos de tallos y hojas, estuvieron entre 5.6 y 245 para las líneas celulares tumorales evaluadas; por el contrario, el cisplatino y el 5-FU, solo alcanzaron valores entre 0.11 y 7.3...

INTRODUCTION: The plants have been used as drugs for centuries. However, limited research has been done on its great potential as sources of new therapeutic agents. The purpose of this study was to evaluate Physalis peruviana cytotoxic activity on cell lines HT-29, PC-3, K-562 and VERO. Materials and Methods: The HT-29 cell lines, PC-3, K-562 and VERO, were exposed to four concentrations of P. peruviana ethanolic leave and stem extracts, also at different concentrations of cisplatin and 5-fluorouracil (5-FU), which were used as positive controls. We found rates of growth within 48 hours, then we determined the inhibitory concentration 50 (IC50) using linear regression analysis and the index of selectivity of each sample. RESULTS: The P. peruviana ethanolic leave and stem extracts showed cytotoxic activity. The IC50 in μg/mL in leaves and stems were, 0.35 (r =-0.95 p <0.025) and 0.37 (r =- 0.90 p <0.05 ) for HT-29; 0.87 (r =-0.98 p <0.01) and 1.01 (r =-0.95 p <0.025) for PC-3; 0.02 (r =-0.98p <0.01) and 0.03 (r =-0.98 p <0.01) for K-562; 4.9 (r =-0.95 p <0.025) and 6.2 (r =-0.98 p <0.01) for VERO. The IC50 for antineoplastic were: for cisplatin: 4.2 (r =-0.96 p <0.025),10.3 (r =-0.97 p <0.025), 0.15 (r =-0.98 p = 0.01) and 1.1 (r =- 0.98 p = 0.01); for 5-FU: 2.3 (r =-0.97 p <0.025), 17.9 (r =-0.95 p <0.025), 0.15 (r =-0.98 p = 0.01) and 1.1 (r =-0.94 p = 0.05) for HT-29, PC-3, K562 and VERO respectively. The leaves and stems extracts selectivity index were between 5.6 and 245 for tumor cell lines evaluated, by contrast, cisplatin and 5-FU, only showed values between 0.11 and 7.3. CONCLUSIONS: The P. peruviana leaves and steams ethanolic extracts were more cytotoxic than cisplatin and 5 FU, on the lines HT-29, PC-3 and K562. Furthermore the P. peruviana cytotoxic effects were less than cisplatin and 5-FU for VERO control cells lines.

L929 cell response to root perforation repair cements: an in vitro cytotoxicity assay.

This study compared the cytotoxicity of an experimental epoxy-resin and calcium hydroxide-based cement (MBPc), gray mineral trioxide aggregate (MTA) and white mineral trioxide aggregate (WMTA) using the agar overlay method with neutral red dye. L929 cells were seeded into 6-well culture plates where 48-h set test materials were placed on the agar overlay, in triplicate. Teflon and natural rubber served as negative and positive controls. After an incubation period of 24 h at 37 degrees C in a humidified atmosphere of 5% CO2 in air, a discolored area around the samples and the positive controls could be observed and measured per quadrant. The mean values were compared and converted into grades to classify the results according to the table of cytotoxicity grades according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation, Brazil. The nonviable cell areas and the morphological changes in the cells were observed with an inverted microscope. The results showed grade 1 (slight) for the two types of MTA (p>0.05) and grade 2 (mild) for the MBPc (p<0.001). All samples met the requirements of the test as none of the cultures showed reactivity higher than grade 2.

Alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in Venezuelan equine encephalitis virus infection.

There are no widely available vaccines or antiviral drugs capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), although an adenovirus vector expressing VEEV structural proteins protects mice from challenge with VEEV and is potentially a vaccine suitable for human use. This work examines whether alpha interferon (IFN-alpha) could act as an adjuvant for the adenovirus-based vaccine. IFN-alpha was either expressed by a plasmid linked to the adenovirus vaccine or encoded by a separate adenovirus vector administered as a mixture with the vaccine. In contrast to previous reports with other vaccines, the presence of IFN-alpha reduced the antibody response to VEEV. When IFN-alpha was encoded by adenovirus, the lack of a VEEV-specific response was accompanied by an increase in the immune response to the adenovirus vector. IFN-alpha also plays a direct role in defence against virus infection, inducing the expression of a large number of antiviral proteins. Adenovirus-delivered IFN-alpha protected mice from VEEV disease when administered 24 h prior to challenge, but not when administered 6 h post-challenge, suggesting that up to 24 h is required for the development of the IFN-mediated antiviral response.

L929 cell response to root perforation repair cements: an in vitro cytotoxicity assay

This study compared the cytotoxicity of an experimental epoxy-resin and calcium hydroxide-based cement (MBPc), gray mineral trioxide aggregate (MTA) and white mineral trioxide aggregate (WMTA) using the agar overlay method with neutral red dye. L929 cells were seeded into 6-well culture plates where 48-h set test materials were placed on the agar overlay, in triplicate. Teflon and natural rubber served as negative and positive controls. After an incubation period of 24 h at 37ºC in a humidified atmosphere of 5 percent CO2 in air, a discolored area around the samples and the positive controls could be observed and measured per quadrant. The mean values were compared and converted into grades to classify the results according to the table of cytotoxicity grades according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation, Brazil. The nonviable cell areas and the morphological changes in the cells were observed with an inverted microscope. The results showed grade 1 (slight) for the two types of MTA (p>0.05) and grade 2 (mild) for the MBPc (p<0.001). All samples met the requirements of the test as none of the cultures showed reactivity higher than grade 2.

O objetivo deste estudo foi comparar a citotoxicidade de um cimento experimental à base de resina epóxica e hidróxido de cálcio (MBPc), do agregado trióxido mineral (MTA) cinza e do MTA branco, utilizando o ensaio de difusão em agar com o corante vermelho neutro. Células L929 foram semeadas em placas de 6 poços e sobre elas a camada de agar, onde foram colocados os materiais endurecidos por 48 h, em triplicata, além de teflon como controle negativo e látex como controle positivo. Após 24 h em estufa umidificada a 37ºC com 5 por cento CO2, um halo claro se formou ao redor das amostras e dos controles positivos. As medidas foram tomadas, por quadrante, e as médias foram comparadas e convertidas em graus para qualificar os resultados, de acordo com a tabela de grau de citotoxicidade do POP/FIOCRUZ. As zonas de inibição e as alterações da morfologia celular foram avaliadas sob microscópio invertido. Os resultados revelaram grau 1 (leve) para os dois tipos de MTA (p>0,05) e grau 2 (branda) para o MBPc (p<0,001). Todas as amostras foram consideradas satisfatórias, pois nenhuma cultura exposta aos cimentos revelou toxicidade superior ao grau 2.

Cytotoxicity of endodontic irrigants containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from mouse L929 cell line

The aim of this study was to evaluate the cytotoxicity of root canal irrigating solutions containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from L929 cell line. Saturated calcium hydroxide aqueous solution (CH), sodium lauryl sulphate (SLS) and SLS associated with calcium hydroxide (HCT20) were diluted with sterile distilled water at 50 percent, 20 percent, 10 percent and 5 percent concentrations. Minimum essential medium (MEM) served as the control group. The cytotoxicity of the solutions was evaluated on L929 mouse fibroblast cell line, at 4 and 24 h of contact time by the 51Cr radiotracer method. Data were compared and statistical inferences were made with the chi-square test. In all analysis, significance level was set at 5 percent. CH and HCT20 showed toxicity at 50 percent concentration, while at concentrations lower than 50 percent these solutions showed cell tolerance. SLS was cytotoxic at all concentrations. In conclusion, the association of calcium hydroxide and SLS (HCT20) combines the beneficial properties of these solutions and was not harmful to the fibroblast cell line, seeming to be a suitable endodontic irrigating solution.

O objetivo desta pesquisa foi avaliar a citotoxicidade de soluções irrigadoras de canais radiculares contendo hidróxido de cálcio e lauril sulfato de sódio em linhagem de fibroblastos L929. Solução aquosa saturada de hidróxido de cálcio, lauril sulfato de sódio e HCT20 (lauril sulfato de sódio e hidróxido de cálcio) foram diluídos em água destilada em concentrações de 50 por cento, 20 por cento, 10 por cento e 5 por cento. O grupo controle foi representado por meio de cultura de células (MEM - minimum essential medium). A citotoxicidade das soluções sobre os fibroblastos foi avaliada em 4 e 24 h de contato, pelo método do cromo radioativo. Os resultados foram analisados estatisticamente pelo teste do qui-quadrado. Em todas as análises, o intervalo de confiança referente às médias entre os grupos foi estabelecido em 95 por cento. As soluções saturadas de hidróxido de cálcio e o HCT20 apresentaram toxicidade nas concentrações de 50 por cento. O lauril sulfato de sódio foi tóxico em todas as concentrações. As soluções de hidróxido de cálcio em concentrações menores que 50 por cento apresentaram tolerância celular, assim como combinadas ao lauril sulfato de sódio. Tal comportamento não foi observado na solução pura de lauril sulfato de sódio em todas as concentrações.