Cytotoxicity of Etch-and-Rinse, Self-Etch, and Universal Dental Adhesive Systems in Fibroblast Cell Line 3T3.

The aim of this study was to evaluate in fibroblast cultures the direct cytotoxic effects of etch-and-rinse, self-etch, and universal adhesive systems. The sterile glass cover slips (n = 3) were then immersed in culture medium to obtain the eluates for the experimental groups: (1) Adper™ Single Bond 2; (2) Ambar; (3) Adper™ Scotchbond™ Multi-Purpose; (4) Scotchbond™ Universal; (5) Ambar Universal; and (6) OptiBond All-In-One. As a negative control, sterile glass cover slips were immersed in culture medium only. After 24 h, the eluate obtained was applied on fibroblast culture. Cell viability and cell morphology were evaluated by MTT assay and SEM, respectively. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α = 0.05). All adhesive systems except universal reduced cell viability in 3T3 cells to between 26.04% and 56.57%, and Scotchbond Universal and Ambar Universal reduced cell viability to 2.13% and 3.57%, respectively, when compared to the negative control. Cytoplasmic membrane shrinkage and cell-free areas with residual membrane fragments from dead cells were observed. In conclusion, improvements in universal adhesive system formulations and their mechanisms of action are not accompanied by increased toxicity compared with those in other systems, warranting commitment to the use of these dentin-pulp complexes.

Imipramine alters the sterol profile in Leishmania amazonensis and increases its sensitivity to miconazole.

BACKGROUND: Imipramine, a tricyclic antidepressant widely used clinically, has other pharmacological effects, such as antileishmanial activity. Tricyclic antidepressants interact with lipid bilayers, and some studies have shown that imipramine inhibits methyltransferases. Leishmania spp. produces compounds with an ergostane skeleton instead of a cholesterol skeleton, and the inhibition of enzymes of the sterol biosynthesis pathway is an interesting therapeutic target. Among these enzymes, C-24 methyltransferase has been suggested to play an essential role, as its inhibition kills the parasites. In this context, we investigated whether imipramine alters the biosynthesis of sterols in L. amazonensis and evaluated the efficacy of imipramine alone and in combination with miconazole, a classical inhibitor of another step in this pathway. METHODS: To analyze the interference of imipramine with sterol metabolism, promastigotes of L. amazonensis were cultured with medium alone, 15 or 30 µM imipramine or 4 µM miconazole, and their lipids were extracted with methanol/chloroform/water (1:0.5:0.4 v/v) and analyzed by GC/MS. To assess the antileishmanial activity of the treatments, promastigotes of L. amazonensis were incubated with various concentrations of imipramine up to 100 µM and up to 24 µM miconazole. Promastigotes were also treated with the combination of imipramine and miconazole at concentrations up to 12.5 µM of imipramine and 24 µM of miconazole. Parasite growth was evaluated by the MTT assay. The fractional inhibitory concentration index (FICI) was calculated to determine whether there were synergistic effects. Peritoneal macrophages with and without L. amazonensis infection were treated with miconazole (0 - 16 µM) or imipramine (0 to 50 µM) for 72 hours. For assays of the combined treatment in amastigotes, the concentration of imipramine was fixed at 12.5 µM and various concentrations of miconazole were used up to 16 µM. The infection rate was determined by counting the infected macrophages under a light microscope. FINDINGS: Promastigotes treated with imipramine accumulated cholesta-5,7,22-trien-3ß-ol and cholesta-7-24-dien- 3ß-ol, sterols that normally increase after treatment with classical inhibitors of C-24 methyltransferase. The IC50 of miconazole in promastigotes decreased when it was used in combination with imipramine, resulting in an additive effect, with a FICI value of 0.83. Imipramine also showed activity against intracellular amastigotes and enhanced the activity of miconazole, without apparent toxicity to the host cells. CONCLUSIONS: Imipramine was confirmed to have antileishmanial activity in both forms of the parasite, affecting the sterol biosynthesis of the organisms. Using imipramine in combination with azoles may be advantageous for the treatment of leishmaniasis.

Growing Candida albicans Biofilms on Paper Support and Dynamic Conditions.

A stainless steel paper-embedded biofilm reactor (PEBR) was developed for Candida spp. growth, permitting confluent distribution of nutrients by capillary diffusion through ordinary laboratory filter paper. Antibiogram disks were distributed along the filter paper rim, and the PEBR received 0.1 or 0.01 % crystal violet (CV) at 200 µL min(-1) and at 37 °C, for 48 h. CV was recovered from the disks and measured at 540 nm. Candida albicans SC5314 cells were applied onto antibiogram disks. The bioreactor was assembled, and YEPD broth was admitted (200 µL min(-1)) at 37 °C, for 72 h. Biofilm growth was estimated via the MTT reduction test. Controls were disks that received the same treatments, except for the fungus. The PEBR was considered high-throughput table, low-cost, and feasible to grow C. albicans biofilms.

Comparison of tetrazolium salt assays for evaluation of drug activity against Leishmania spp.

In French Guiana, leishmaniasis is an essentially cutaneous infection. It constitutes a major public health problem, with a real incidence of 0.2 to 0.3%. Leishmania guyanensis is the causal species most frequently encountered in French Guiana. The treatment of leishmaniasis is essentially drug based, but the therapeutic compounds available have major side effects (e.g., liver damage and diabetes) and must be administered parenterally or are costly. The efficacy of some of these agents has declined due to the emergence of resistance in certain strains of Leishmania. There is currently no vaccine against leishmaniasis, and it is therefore both necessary and urgent to identify new compounds effective against Leishmania. The search for new drugs requires effective tests for evaluations of the leishmanicidal activity of a particular molecule or extract. Microculture tetrazolium assays (MTAs) are colorimetric tests based on the use of tetrazolium salts. We compared the efficacies of three tetrazolium salts-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8)-for quantification of the promastigotes of various species of Leishmania. We found that the capacity of Leishmania to metabolize a tetrazolium salt depended on the salt used and the species of Leishmania. WST-8 was the tetrazolium salt best metabolized by L. guyanensis and gave the best sensitivity.

Psoralen derivatives and longwave ultraviolet irradiation are active in vitro against human melanoma cell line.

Cutaneous malignant melanoma is a very serious form of skin cancer that arises from melanocytes. Currently there is no effective treatment for metastatic melanoma so intense clinical trials are evaluating new drugs for this human malignancy. Psoralens are a group of compounds that bind to DNA in rapidly dividing cells and with ultraviolet light in the A band (UVA) cause DNA crosslinking, thereby preventing cellular division. They are used in the treatment of psoriasis and cutaneous T-cell lymphoma among other skin and blood diseases. We have investigated the cytotoxic potential of three psoralen derivatives plus UVA exposure (PUVA) on a established cell line of human melanoma. Cells were treated with different concentrations of 8-methoxypsoralen (8-MOP), 4,5',8-trimethylpsoralen (TMP) and 7-methylpyridopsoralen (MPP), for 1 h and after exposure to UVA light (0.3 J/cm(2)) were allowed to recover over a 24-72 h period. Viability was assessed by the microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cisplatin, one of the most important drugs in the chemotherapy of melanoma, was included for comparative studies. All the psoralen derivatives tested were markedly cytotoxic in a dose and post-exposure-time dependent manner. The IC(50) values for 72 h of post-exposure time were as follows: MPP=0.05+/-0.01, TMP=0.13+/-0.003 and 8-MOP=10.79+/-1.85 micromol/L. Regardless of the limitations of the in vitro model, our results suggested that the lower IC(50) values of TMP and MPP might be of clinical importance.

Frequency of 2, 3, 5-triphenyltetrazolium chloride (TTC) non-reducing bacteria in pasteurized milk

2, 3, 5-triphenyltetrazolium chloride (TTC) is a dye largely used for enumeration of microbial colonies in solid culture media, being a key component of the dry rehydratable film system used for microbiological analysis of food. This dye is colorless in the oxidized form and red when reduced by microorganisms, due to formation of formazan. In this study, TTC was added to Plate Count Agar (PCA) for enumeration of microorganisms in thirty four pasteurized milk samples, with the aim to verify the frequency of microorganisms that are unable to reduce TTC. Milk samples were decimally diluted in saline and pour-plated in PCA plus 0.015(per cent) TTC. Colonies were counted after 24h and 48 h of incubation at 35(degree)C. From a total of 50,574 colonies 19,665(38.88 per cent) did not reduce TTC in 48h. It was observed that 571(6.36 per cent) colonies that were colorless in 24h became red in 48h. From those that didn't reduce TTC in 48h, 233 were purified and Gram stained. 229(98.71 per cent) of them were Gram positive cocci and bacilli. The results show that there is a high percentage of microorganisms unable to reduce TTC in pasteurized milk, which cannot be detected by laboratory procedures based on the formation of red colonies.