In Vitro Susceptibility of Environmental Isolates of Exophiala dermatitidis to Five Antifungal Drugs

Several dematiaceous fungi frequently isolated from nature are involved in cases of superficial lesions to lethal cerebral infections. Antifungal susceptibility data on environmental and clinical isolates are still sparse despite the advances in testing methods. The objective of this study was to examine the activities of 5-flucytosine, amphotericin B, itraconazole, voriconazole and terbinafine against environmental isolates of Exophiala strains by minimum inhibition concentration (MIC) determination. The strains were obtained from hydrocarbon-contaminated soil, ant cuticle and fungal pellets from the infrabuccal pocket of attine gynes. Broth microdilution assay using M38-A2 reference methodology for the five antifungal drugs and DNA sequencing for fungal identification were applied. Terbinafine was the most active drug against the tested strains. It was observed that amphotericin B was less effective, notably against Exophiala spinifera, also studied. High MICs of 5-flucytosine against Exophiala dermatitidis occurred. This finding highlights the relevance of studies on the antifungal resistance of these potential opportunistic species. Our results also contribute to a future improvement of the standard methods to access the drug efficacy currently applied to black fungi. © 2012 Springer Science+Business Media Dordrecht.

In vitro analysis of shear bond strength and adhesive remnant index comparing light curing and self-curing composites

OBJETIVO: Avaliar in vitro a resistência ao cisalhamento de compósitos autopolimerizáveis (Concise e Alpha Plast) e fotopolimerizáveis (Transbond XT e Natural Ortho) utilizados na colagem de braquetes metálicos da marca Morelli, analisando o índice de adesivo remanescente (ARI) e da integridade da superfície do esmalte por meio de microscopia eletrônica de varredura (MEV). MÉTODOS: foram utilizados 40 pré-molares humanos extraídos. As raízes dos dentes foram incluídas em gesso-pedra especial, no interior de tubos de PVC usados para a confecção dos corpos de prova. Esses corpos de prova foram divididos em quatro grupos: grupo G1, braquetes associados ao compósito Concise; grupo G2, braquetes associados ao compósito Alpha Plast; grupo G3, braquetes associados ao compósito Transbond XT; e grupo G4, braquetes associados ao compósito Natural Ortho. Os grupos foram submetidos ao teste de cisalhamento em máquina universal de ensaios, a uma velocidade de 0,5mm por minuto. RESULTADOS: houve diferença estatística entre os grupos G3 e G4, sendo os valores de G4 superiores; no entanto, não foram encontradas diferenças estatisticamente significativas entre os grupos G1, G2 e G3 e G1, G2 e G4. Na análise do ARI não foram encontradas diferenças estatísticas entre os grupos, predominando escores baixos. De acordo com a análise da MEV, constatou-se o rompimento dos compósitos e a integridade do esmalte entre os grupos. CONCLUSÃO: a resistência ao cisalhamento foi satisfatória e semelhante entre os compósitos utilizados, sendo que a resina Natural Ortho apresentou-se superior à Transbond XT.

In vitro comparison of the force degradation of orthodontic intraoral elastics from different compositions

The synthetic intermaxillary elastic emerged as an alternative for clinical use in patients with latex sensitivity. However, there are disagreements about this elastic protocol use according to the force degradation. The aim of this study was to evaluate, in vitro, the forces generated by latex and synthetic elastics over time. Material and methods: Sample size of 840 elastics were used (420 latex and 420 synthetic), delivering medium strength (Dental Morelli®) with internal diameter of 1/8", 3/16", 1/4" and 5/16". The elastics were randomly divided into 7 groups according to the time of the force measuring and immersed into distilled water at 37°C. To measure the force in each group, the elastics were stretched in six progressive increases of 100% of its internal diameter with the aid of a testing machine Emic and measured up to 72 hours. Data were analyzed with SPSS 16.0, using one-way analysis of variance (ANOVA). Results: Immediate force level of synthetic elastics was statistically higher than latex elastics in all strains, for the same size. However, the latex elastics mean forceslightly decreased over time, while the synthetic elastics presented an abrupt decrease. Conclusion: In view of these findings, Sudanese homemade alcoholic beverages cause oral epithelial atypical changes, which lead to oral precancerous and cancerous lesions. OEFC is a useful procedure for detection and assessment of oral ET.

In vitro comparison of the force degradation of orthodontic intramural elastic from different compositions

Introduction and Objective: The synthetic intermaxillary elastic emerged as an alternative for clinical use in patients with latex sensitivity. However, there are disagreements about this elastic protocol use according to the force degradation. The aim of this study was to evaluate, in vitro, the forces generated by latex and synthetic elastics over time. Material and methods: Sample size of 840 elastics were used (420 latex and 420 synthetic), delivering medium strength (Dental Morelli®) with internal diameter of 1/8”, 3/16”, 1/4” and 5/16”. The elastics were randomly divided into 7 groups according to the time of the force measuring and immersed into distilled water at 37°C. To measure the force in each group, the elastics were stretched in six progressive increases of 100% of its internal diameter with the aid of a testing machine Emic and measured up to 72 hours. Data were analyzed with SPSS 16.0, using one-way analysis of variance (ANOVA). Results: Immediate force level of synthetic elastics was statistically higher than latex elastics in all strains, for the same size. However, the latex elastics mean force slightly decreased over time, while the synthetic elastics presented an abrupt decrease. Conclusion: The synthetic elastic presented severe force degradation, jeopardizing the cost-benefit ratio, which indicates a higher replacement frequency. The latex elastic showed better mechanical performance in comparison to synthetic ones.

Biomechanical in vitro evaluation of three stable internal fixation techniques used in sagittal osteotomy of the mandibular ramus: a study in sheep mandibles

Among the osteotomies performed in orthognathic surgery, the sagittal osteotomy of the mandibular ramus (SOMR) is the most common, allowing a great range of movements and stable internal fixation (SIF), therefore eliminating the need of maxillomandibular block in the postoperative period. Objectives: The purpose of this study was to evaluate the biomechanical resistance of three national systems used for SIF in SOMR in sheep mandibles. Material and methods: The study was performed in 30 sheep hemi-mandibles randomly divided into 3 experimental groups, each containing 10 hemi-mandibles. The samples were measured to avoid discrepancies and then subjected to SOMR with 5-mm advancement. In group I, 2.0x12 mm screws were used for fixation, inserted in an inverted "L" pattern (inverted "L" group). In group II, fixation was performed with two 2.0x12 mm screws, positioned in a linear pattern and a 4-hole straight miniplate and four 2.0x6.0 mm monocortical screws (hybrid group). In group III, fixation was performed with two-hole straight miniplates and eight 2.0x6.0 mm monocortical screws (mini plate group). All materials used for SIF were supplied by Osteosin - SIN. The hemimandibles were subjected to vertical linear load test by Kratos K2000MP mechanical testing unit for loading registration and displacement. Results: All groups showed similar resistance during mechanical test for loading and displacement, with no statistically significant differences between groups according to analysis of variance. Conclusion: These results indicate that the three techniques of fixation are equally effective for clinical fixation of SOMR.

Plasma corticosterone levels in mouse models of pain

Background: Pain markedly activates the hypothalamic-pituitary-adrenal (HPA) axis and increases plasma corticosterone release interfering significantly with nociceptive behaviour as well as the mechanism of action of analgesic drugs. Aims/Methods: In the present study, we monitored the time course of circulating corticosterone in two mouse strains (C57Bl/6 and Balb/C) under different pain models. In addition, the stress response was investigated following animal handling, intrathecal (i.t.) manipulation and habituation to environmental conditions commonly used in nociceptive experimental assays. We also examined the influence of within-cage order of testing on plasma corticosterone. Results: Subcutaneous injection of capsaicin precipitated a prompt stress response whereas carrageenan and complete Freund's adjuvant induced an increased corticosterone release around the third hour post-injection. However, carrageenan induced a longer increased corticosterone in C57Bl/6 mice. In partial sciatic nerve ligation, neuropathic pain model corticosterone increased only in the first days whereas mechanical hypersensitivity remained much longer. Animal handling also represents an important stressor whereas the i.t. injection per se does not exacerbate the handling-induced stress response. Moreover, the order of testing animals from the same cage does not interfere with plasma corticosterone levels in the intrathecal procedure. Animal habituation to the testing apparatus also does not reduce the immediate corticosterone increase as compared with non-habituated mice. Conclusion: Our data indicate that HPA axis activation in acute and chronic pain models is time dependent and may be dissociated from evoked hyperalgesia. Therefore, HPA-axis activation represents an important variable to be considered when designing experimental assays of persistent pain as well as for interpretation of data.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

Introduction: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. Methods: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36 degrees C with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. Results: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075 mu M and an LLC MK2 toxicity of 2.003 mu M. Pyrimethamine was effective at an IC50 of 0.482 mu M and a toxicity of 11.178 mu M. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. Conclusions: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

In Vitro Characterization of Chitosan Gels for Buccal Delivery of Celecoxib: Influence of a Penetration Enhancer

Celecoxib (Cx) shows high efficacy in the treatment of osteoarthritis and rheumatoid arthritis as a result of its high specificity for COX-2, without gastrolesivity or interference with platelet function at therapeutic concentrations. Besides of anti-inflammatory effects, Cx also has a potential role for oral cancer chemoprevention. For these conditions, oral administration in long-term treatment is a concern due to its systemic side effects. However, local application at the site of injury (e.g., buccal inflammation conditions or chemoprevention of oral cancer) is a promising way to reduce its toxicity. In this study, the in vitro characterization of mucoadhesive chitosan (CHT) gels associated to AzoneA (R) was assessed to explore the potential buccal mucosal administration of Cx in this tissue. Rheological properties of gels were analyzed by a rheometer with cone-plate geometry. In vitro Cx release and permeability studies used artificial membranes and pig cheek mucosa, respectively. Mucoadhesion were measured with a universal test machine. CHT gels (3.0%) containing 2.0% or 3.0% Az showed more appropriate characteristics compared to the others: pH values, rheology, higher amount of Cx retained in the mucosa, and minimal permeation through mucosa, besides the highest mucoadhesion values, ideal for buccal application. Moreover, the flux (J) and amounts of drug released decreased with increased CHT and Az concentrations. CHT gels (3.0%) associated with 2.0% or 3.0% Az may be considered potential delivery systems for buccal administration of Cx.

Influence of caffeine on the survival of leaf-cutting ants Atta sexdens rubropilosa and in vitro growth of their mutualistic fungus

BACKGROUND: Leaf-cutting ants collect plant fresh material for the cultivation of their mutualistic fungus. Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) cause great economic losses through their foraging activity, mainly in agriculture. The main control method is the application of granulated toxic baits incorporated with an active ingredient (AI). The present goal is to evaluate the effect of caffeine on in vitro growth of the mutualistic fungus and on the survival of the leaf-cutting ants, aiming to verify the potential toxicity of this secondary metabolite over these organisms. RESULTS: Three distinct patterns of fungal growth correlated with caffeine concentration were observed: (1) no effect (0.01% caffeine); (2) intermediate growth reduction (0.05% caffeine); (3) drastic growth reduction (0.10 and 0.50% caffeine). The highest caffeine concentration causes fungus death in the first week. As for insect survival, caffeine does not seem to exert any effect. The treatments with diet containing caffeine showed similar values of M50, irrespective of caffeine concentration. CONCLUSION: As caffeine was shown to reduce growth of the mutualistic fungus of Atta sexdens rubropilosa, but with no conclusive effect on insect survival, a hypothetical explanation for the selection of different Coffea species by this leaf-cutting ant species might be associated with caffeine toxicity to the fungus. Copyright (C) 2011 Society of Chemical Industry

In vitro activity of extracts and isolated polyphenols from West African medicinal plants against Plasmodium falciparum

The aim of the study was to screen 11 selected traditional medicinal plants from West Africa for their in vitro antiplasmodial activity in order to determine the activity of single and of combination of plant extracts and to examine the activity of isolated pure compounds. Ethanolic and aqueous extracts of the 11 selected plants and pure compounds from Phyllanthus muellerianus and Anogeissus leiocarpus were tested in vitro against Plasmodium falciparum 3D7. Proliferation inhibitory effects were monitored after 48 h. Among the plants and pure compounds investigated in this study, geraniin from P. muellerianus, ellagic, gentisic, and gallic acids from A. leiocarpus, and extracts from A. leiocarpus, P. muellerianus and combination of A. leiocarpus with P. muellerianus affected the proliferation of P. falciparum most potently. Significant inhibitory activity was observed in combination of A. leiocarpus with P. muellerianus (IC50 = 10.8 mu g/ml), in combination of A. leiocarpus with Khaya senegalensis (IC50 = 12.5 mu g/ml), ellagic acid (IC50 = 2.88 mu M), and geraniin (IC50 = 11.74 mu M). In general growth inhibition was concentration-dependent revealing IC50 values ranging between 10.8 and -40.1 mu g/ml and 2.88 and 11.74 mu M for plant extracts and pure substances respectively. Comparison with literature sources of in vivo and in vitro toxicity data revealed that thresholds are up to two times higher than the determined IC50 values. Thus, the present study suggests that geraniin from P. muellerianus; ellagic acid, gallic acid, and gentisic acid from A. leiocarpus; and combination of extracts from A. leiocarpus with either P. muellerianus or K. senegalensis could be a potential option for malaria treatment.

In vitro cytotoxicity of white MTA, MTA Fillapex® and Portland cement on human periodontal ligament fibroblasts

The aim of this study was to compare the in vitro cytotoxicity of white mineral trioxide aggregate (MTA), MTA Fillapex® and Portland cement (PC) on human cultured periodontal ligament fibroblasts. Periodontal ligament fibroblast culture was established and the cells were used for cytotoxic tests after the fourth passage. Cell density was set at 1.25 X10 4 cells/well in 96-well plates. Endodontic material extracts were prepared by placing sealer/cement specimens (5X3mm) in 1mL of culture medium for 72 h. The extracts were then serially two-fold diluted and inserted into the cell-seeded wells for 24, 48 and 72 h. MTT assay was employed for analysis of cell viability. Cell supernatants were tested for nitric oxide using the Griess reagent system. MTA presented cytotoxic effect in undiluted extracts at 24 and 72 h. MTA Fillapex® presented the highest cytotoxic levels with important cell viability reduction for pure extracts and at ½ and ¼ dilutions. In this study, PC did not induce alterations in fibroblast viability. Nitric oxide was detected in extract-treated cell supernatants and also in the extracts only, suggesting presence of nitrite in the soluble content of the tested materials. In the present study, MTA Fillapex displayed the highest cytotoxic effect on periodontal ligament fibroblasts followed by white MTA and PC.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.

In vitro efficacy of Coriandrum sativum, Lippia sidoides and Copaifera reticulata against Leishmania chagasi

The increased incidence of visceral leishmaniasis (VL) in Brazil is due to a lack of effective disease control measures. In addition to that, no effective treatment exists for canine VL in response to synthetic drugs. Thus, the objective of this study was to evaluate the effect of the essential oils of Coriandrum sativum and Lippia sidoides, and oleoresin from Copaifera reticulata, on Leishmania chagasi promastigotes and amastigotes. We also examined the toxicity of these treatments on the murine monocyte cell line RAW 264.7. To determine the IC50 a MTT test (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was performed on promastigotes, and an in situ ELISA assay was conducted on amastigotes. Here, we demonstrate that oleoresin from C. reticulata was effective against both promastigotes (IC50 of 7.88 µg.mL-1) and amastigotes (IC50 of 0.52 µg.mL-1), and neither of the two treatments differed significantly (p > 0.05) from pentamidine (IC50 of 2.149 µg.mL-1) and amphotericin B (IC50 of 9.754 µg.mL-1). Of the three plant oils tested, only oleoresin showed no toxicity toward monocyte, with 78.45% viability after treatment. Inhibition of promastigote and amastigote growth and the lack of cytotoxicity by C. reticulata demonstrate that oleoresin may be a viable option for analyzing the in vivo therapeutic effects of leishmanicidal plants

Neuroprotective activity of guanosine in an in vitro model of Alzheimer's disease

The β-Amyloid (βA) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer’s Disease (AD). It is well recognized that Aβ exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common βA-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against Aβ peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 μM) in presence of monomeric βA25-35 (neurotoxic core of Aβ), oligomeric and fibrillar βA1-42 peptides showed a strong dose-dependent inhibitory effects on βA-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 μM of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 μM for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against βA-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.

In vivo and in vitro investigation of the tissue-specific activity of the human CYP3A4 and CYP3A5 promoters

The human cytochrome P450 3A4 (CYP3A4), the predominant but variably expressed cytochrome P450 in adult liver and small intestine is involved in the metabolism of over 50% of currently used drugs. Its paralog CYP3A5 plays a crucial role in the disposition of several drugs with low therapeutic index, including tacrolimus. Limited information is available for the CYP3A5 transcriptional regulation and its induction by xenobiotics remains controversial. In the first part of this study, we analysed the CYP3A5 transcriptional regulation and its induction by xenobiotics in vivo using transgenic mice. To this end, two transgenic strains were established by pronuclear injection of a plasmid, expressing firefly luciferase driven by a 6.2 kb of the human CYP3A5 promoter. A detailed analysis of both strains shows a tissue distribution largely reflecting that of CYP3A5 transcripts in humans. Thus, the highest luciferase activity was detected in the small intestine, followed by oesophagus, testis, lung, adrenal gland, ovary, prostate and kidney. However, no activity was observed in the liver. CYP3A5-luc transgenic mice were similarly induced in both sexes with either PCN or TCPOBOP in small intestine in a dose-dependent manner. Thus, the 6.2 kb upstream promoter of CYP3A5 mediates the broad tissue activity in transgenic mice. CYP3A5 promoter is inducible in the small intestine in vivo, which may contribute to the variable expression of CYP3A in this organ. rnThe hepato-intestinal level of the detoxifying oxidases CYP3A4 and CYP3A5 is adjusted to the xenobiotic exposure mainly via the xenosensor and transcriptional factor PXR. CYP3A5 is additionally expressed in several other organs lacking PXR, including kidney. In the second part of this study, we investigated the mechanism of the differential expression of CYP3A5 and CYP3A4 and its evolutionary origin using renal and intestinal cells, and comparative genomics. For this examination, we established a two-cell line models reflecting the expression relationships of CYP3A4 and CYP3A5 in the kidney and small intestine in vivo. Our data demonstrate that the CYP3A5 expression in renal cells was enabled by the loss of a suppressing Yin Yang 1 (YY1)-binding site from the CYP3A5 promoter. This allowed for a renal CYP3A5 expression in a PXR-independent manner. The YY1 element is retained in the CYP3A4 gene, leading to its suppression, perhaps via interference with the NF1 activity in renal cells. In intestinal cells, the inhibition of CYP3A4 expression by YY1 is abrogated by a combined activating effect of PXR and NF1 acting on their respective response elements located adjacent to the YY1-binding site on CYP3A4 proximal promoter. CYP3A4 expression is further facilitated by a point mutation attenuating the suppressing effect of YY1 binding site. The differential expression of CYP3A4 and CYP3A5 in these organs results from the loss of the YY1 binding element from the CYP3A5 promoter, acting in concert with the differential organ expression of PXR, and with the higher accumulation of PXR response elements in CYP3A4. rn