In vitro diagnosis of T cell-mediated drug allergy

Diagnosis of drug hypersensitivity relies on history, skin tests, in vitro tests and provocation tests. In vitro tests are of great interest, due to possible reduction of drug provocation tests. In this review we focus on best investigated in vitro techniques for the diagnosis of T cell-mediated drug hypersensitivity reactions. As drug hypersensitivity relies on different pathomechanisms and as a single diagnostic test usually does not cover all possible reactions, it is advisable to combine different tests to increase the overall sensitivity. Recently, proliferation-based assays have been supplemented by a panel of novel in vitro tests including analysis of cytotoxic potential of effector cells (granzyme B, granulysin, CD107a), evaluation of cytokine secretion (IL-2, IL-5, IL-13, and IFN-γ) and up-regulation of cell surface activation markers (CD69). We discuss the latest findings and readout systems to identify causative drugs by detecting functional and phenotypic markers of drug-reacting cells, and their ability to enable a more conclusive diagnosis of drug allergy.

Application of an in vitro drug screening assay based on the release of phosphoglucose isomerase to determine the structure-activity relationship of thiazolides against Echinococcus multilocularis metacestodes

OBJECTIVES: The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame. METHODS: Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy. RESULTS: We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study. CONCLUSIONS: The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

In vitro bioactivity of MOEP grafted ePTFE membranes for craniofacial applications

The bioactivity of three methacryloyloxyethyl phosphate (MOEP) grafted expanded polytetrafluoroethylene (ePTFE) membranes with varying surface coverage as well as unmodified ePTFE was investigated through a series of in vitro tests: calcium phosphate (CaP) growth in simulated body fluid (SBF), serum protein adsorption, and a morphology and attachment study of human osteoblast-like SaOS-2 cells. The graft copolymers were prepared by means of gamma irradiation induced grafting and displayed various surface morphologies and wettabilities depending on the grafting conditions used. Unmodified ePTFE did not induce nucleation of Cal? minerals, whereas all the grafted membranes revealed the growth of Cal? minerals after 7 days immersion in SBF. The sample with lowest surface grafting yield (24% coverage), a smooth graft morphology and relatively high hydrophobicity (theta(adv) = 120 degrees, theta(rec) = 80 degrees) showed carbonated hydroxyapatite growth covering the surface. On the other hand, the samples with high surface grafting yield (76% and 100%), a globular graft morphology and hydrophilic surfaces (theta(adv) = 60 degrees and 80 degrees, theta(rec) = 25 degrees and 15 degrees, respectively) exhibited irregular growth of non-apatitic Cap minerals. Irreversibly adsorbed protein measured after a 1 h immersion in serum solution was quantified by the amount of nitrogen on the surface using XPS, as well as by weight increase. All grafted membranes adsorbed 3-6 times more protein than the unmodified membrane. The sample with the highest surface coverage adsorbed the most protein. Osteoblast-like SaOS-2 cells cultured for 3 h revealed significantly higher levels of cell attachment on all grafted membranes compared to unmodified ePTFE. Although the morphology of the cells was heterogeneous, in general, the higher grafted surfaces showed a much better cell morphology than both the low surface-grafted and the control unmodified sample. The suite of in vitro tests confirms that a judicious choice of grafted monomer such as the phosphate-containing methacrylate monomer (MOEP) significantly improves the bioactivity of ePTFE in vitro. (c) 2005 Elsevier Ltd. All rights reserved.

Degradable poly(ethylene glycol)-based hydrogels:synthesis, physico-chemical properties and in vitro characterization

Designing degradable hydrogels is complicated by the structural and temporal complexities of the gel and evolving tissue. A major challenge is to create scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. Poly(ethylene glycol) was used in this work, to form biodegradable poly(ethylene glycol)-based hydrogels with hydrolyzable poly-l-lactide segments in the backbone. Non-degradable poly(ethylene glycol) was also introduced in the formulation to obtain control of the degradation profile that encompasses cell growth and new tissue formation. The dependence on polymer composition was observed by higher degradation profiles and decreased mechanical properties as the content of degradable segments was increased in the formulation. Based on in vitro tests, no toxicity of extracts or biomaterial in direct contact with human adipose tissue stem cells was observed, and the ultraviolet light treatment did not affect the proliferation capacity of the cells.

In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of IgE-mediated reactions in RAO remains unclear. The aim of the study was to investigate with a serological IgE ELISA test (Allercept), an in vitro sulfidoleukotriene (sLT) release assay (CAST) and with intradermal testing (IDT) whether serum IgE and IgE-mediated reactions against various mould, mite and pollen extracts are associated with RAO. IDT reactions were evaluated at different times in order to detect IgE-mediated immediate type reactions (type I hypersensitivity reactions, 0.5-1 h), immune complex-mediated late type reactions (type III reactions, 4-10 h) and cell-mediated delayed type reactions (type IV hypersensitivity reactions 24-48 h). In the serological test, overall the control horses displayed more positive reactions than the RAO-affected horses but the difference was not significant. Comparison of the measured IgE levels showed that the RAO-affected horses had slightly higher IgE levels against Aspergillus fumigatus than controls (35 and 16 AU, respectively, p<0.05), but all values were below the cut off (150 AU) of the test. In the sLT release assay, seven positive reactions were observed in the RAO-affected horses and four in the controls but this difference was not significant. A significantly higher proportion of late type IDT reactions was observed in RAO-affected horses compared to controls (25 of 238 possible reactions versus 12 of 238 possible reactions, respectively, p<0.05). Interestingly, four RAO-affected but none of the control horses reacted with the recombinant mould allergen A. fumigatus 8 (rAsp f 8, p<0.05), but only late phase and delayed type reactions were observed. In all three tests the majority of the positive reactions was observed with the mite extracts (64%, 74% and 88% of all positive reactions, respectively) but none of the tests showed a significant difference between RAO-affected and control animals. Our findings do not support that IgE-mediated reactions are important in the pathogenesis of RAO. Further studies are needed to investigate whether sensitisation to mite allergens is of clinical relevance in the horse and to understand the role of immune reactions against rAsp f 8.

The development of functional in vitro toxicity tests

In vitro toxicity tests which detect evidence of the formation of reactive metabolites have previously relied upon cell death as a toxicity end point. Therefore these tests determine cytotoxicity in terms of quantitative changes in specified cell functions. In the studies involving the CaC0-2 cell model, there was no significant change in the transport of [3H] L-proline by the cell after eo-incubation with either dapsone or cyclophosphamide (50µM) and rat liver microsomal metabolite generating system. The pre incubation of the cells with N-ethylmalemide to inhibit Phase II sulphotransferase activity, prior to the microsomal incubations, resulted in cytotoxcity in all incubation groups. Studies involving the L6 cell model showed that there was no significant effect in the cell signalling pathway producing the second messenger cAMP, after incubation with dapsone or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. There was also no significant affect on the vasopressin stimulated production of the second messenger IP3, after incubation with the hydroxylamine metabolite of dapsone, although there were some morphological changes observed with the cells at the highest concentration of dapsone hydroxylamine (100µM). With the test involving the NG115-401 L-C3 cell model, there was no significant changes in DNA synthesis in terms of [3H] thymidine incorporation, after eo-incubation with either phenytoin or cyclophosphamide (50µM) and the rat microsomal metabolite generating system. In the one compartment erythrocyte studies, there were significant decreases in glutathione with cyclophosphamide (50µM) (0.44 ± 0.04 mM), sulphamethoxazole (50µM) (0.43 ± 0.08mM) and carbamazepine (50µM) (0.47 ± 0.034 mM), when eoincubated with the rat microsomal system, compared to the control (0.52 ± 0.07mM). There was no significant depletion in glutathione when the erythrocytes were eoincubated with phenytoin and the rat microsomal system. In the two compartment erythrocyte studies, there was a significant decrease in the erythrocyte glutathione with cyclophosphamide (50µM) (0.953 ± 0110mM) when co-incubated the rat microsomal system, compared to the control (1.124 ± 0.032mM). Differences were considered statistically significant for p<0.05, using the Student's two tailed 't' test with Bonferroni's correction. There was no significant depletion of glutathione with phenytoin, carbamazepine and sulphamethoxazole when co-incubated with the rat microsomalsystem, compared to the control.

In Vitro Evaluation Of Sun Protection Factor And Stability Of Commercial Sunscreens Using Mass Spectrometry.

Sunlight exposure causes several types of injury to humans, especially on the skin; among the most common harmful effects due to ultraviolet (UV) exposure are erythema, pigmentation and lesions in DNA, which may lead to cancer. These long-term effects are minimized with the use of sunscreens, a class of cosmetic products that contains UV filters as the main component in the formulation; such molecules can absorb, reflect or diffuse UV rays, and can be used alone or as a combination to broaden the protection on different wavelengths. Currently, worldwide regulatory agencies define which ingredients and what quantities must be used in each country, and enforce companies to conduct tests that confirm the Sun Protection Factor (SPF) and the UVA (Ultraviolet A) factor. Standard SPF determination tests are currently conducted in vivo, using human subjects. In an industrial mindset, apart from economic and ethical reasons, the introduction of an in vitro method emerges as an interesting alternative by reducing risks associated to UV exposure on tests, as well as providing assertive analytical results. The present work aims to describe a novel methodology for SPF determination directly from sunscreen formulations using the previously described cosmetomics platform and mass spectrometry as the analytical methods of choice.

Estudo in vitro da resistência ao cisalhamento da colagem direta de tubos ortodônticos em molares

INTRODUÇÃO: apesar da colagem direta despender menor tempo clínico, com maior preservação da integridade gengival, ainda hoje se observa uma alta incidência de bandagem dos molares. Portanto, torna-se interessante a idealização de recursos para o aumento da eficiência desse procedimento para dentes submetidos a maiores impactos mastigatórios, como, por exemplo, os molares. OBJETIVO: esse estudo teve o propósito de avaliar se a resistência à adesão com a aplicação de uma camada de resina adicional na região oclusal da interface tubo/dente aumenta a qualidade do procedimento de colagem direta de tubos em molares. MÉTODOS: selecionou-se uma amostra composta por 40 terceiros molares inferiores, que foram aleatoriamente divididos em 2 grupos: Grupo 1 - colagem direta convencional, seguida pela aplicação de uma camada de resina na oclusal da interface tubo/dente; e Grupo 2 - colagem direta convencional. O teste de resistência ao cisalhamento foi realizado 24 horas após a colagem, utilizando-se uma máquina de ensaio universal, operando a uma velocidade de 0,5mm/min. Os resultados foram analisados por meio do teste t independente. RESULTADOS: os valores médios obtidos nos testes de cisalhamento foram: 17,08MPa para o Grupo 1 e 12,60MPa para o Grupo 2. O Grupo 1 apresentou uma resistência ao cisalhamento estatisticamente significativa mais alta do que o Grupo 2. CONCLUSÃO: a aplicação de uma camada adicional de resina na oclusal da interface tubo/dente aumenta a qualidade da adesão do procedimento de colagem direta de tubos ortodônticos em molares.

Effect of ultrasonic instrumentation on the bond strength of crowns cemented with zinc phosphate cement to natural teeth. An in vitro study

Several studies have reported the benefits of sonic and/or ultrasonic instrumentation for root debridement, with most of them focusing on changes in periodontal clinical parameters. The present study investigated possible alterations in the tensile bond strength of crowns cemented with zinc phosphate cement to natural teeth after ultrasonic instrumentation. Forty recently extracted intact human third molars were selected, cleaned and stored in physiologic serum at 4°C. They received standard preparations, at a 16º convergence angle, and AgPd alloy crowns. The crowns were cemented with zinc phosphate cement and then divided into four groups of 10 teeth each. Each group was then subdivided into two subgroups, with one of the subgroups being submitted to 5,000 thermal cycles ranging from 55 ± 2 to 5 ± 2°C, while the other was not. Each group was submitted to ultrasonic instrumentation for different periods of time: group 1 - 0 min (control), group 2 - 5 min, group 3 - 10 min, and group 4 - 15 min. Tensile bond strength tests were performed with an Instron testing machine (model 4310). Statistical analysis was performed using ANOVA and Tukey's test at the 5% level of significance. A significant reduction in the tensile bond strength of crowns cemented with zinc phosphate and submitted to thermal cycles was observed at 15 min (196.75 N versus 0 min = 452.01 N, 5 min = 444.23 N and 10 min = 470.85 N). Thermal cycling and ultrasonic instrumentation for 15 min caused a significant reduction in tensile bond strength (p < .05).

In Vitro Evaluation of Enamel Erosion After Nd:YAG Laser Irradiation and Fluoride Application

Objective: Previous investigations have demonstrated improved enamel demineralization resistance after laser irradiation. Due to the possibility of a synergistic effect between laser and fluoride, this study investigated the effect of fluoridated agents and Nd:YAG irradiation separately and in combination on enamel resistance to erosion. Methods: One hundred bovine enamel blocks were randomly divided into 10 groups: G1, untreated (control); G2, acidic phosphate fluoride (APF) (1.23% F) for 4 min; G3, fluoride varnish for 6 h (NaF, 2.26%); G4, 0.5 W Nd: YAG laser (250 mm pulse width, 10 Hz, 35 J/cm(2), with uniform velocity for 30 sec in each application); G5, 0.75 W Nd:YAG laser (52.5 J/cm(2)); G6, 1.0 W Nd:YAG laser (70 J/cm(2)); G7, APF + 0.75 W Nd:YAG laser; G8, 0.75 W Nd:YAG laser + APF; G9, fluoride varnish + 0.75 W Nd:YAG laser; and G10, 0.75 W Nd:YAG laser + fluoride varnish. During 10 d the erosive cycle was conducted by immersion of the blocks in Sprite light for 1 min, followed by immersion in artificial saliva for 59 min. This procedure was consecutively repeated four times per day. In each day, during the remaining 20 h, the blocks were maintained in artificial saliva. The wear was evaluated by profilometry (days 5 and 10). Data were tested by two-way ANOVA and Bonferroni's tests (p < 0.05). Results: The mean wear at days 5 and 10 was, respectively: G1, 1.83 and 2.67 mu m; G2, 1.04 and 2.60 mu m; G3, 1.03 and 2.48 mu m; G4, 1.13 and 2.47 mu m; G5, 1.07 and 2.44 mu m; G6, 1.0 and 2.35 mu m; G7, 0.75 and 2.27 mu m; G8, 0.80 and 2.12 mu m; G9, 0.76 and 2.47 mu m; and G10, 1.09 and 2.46 mu m. At day 5, all the experimental groups presented significant lesser wear when compared to control group. However, at 10 d, only G7 and G8 were still different from control. Conclusions: The association between APF application and laser irradiation seems to be an alternative preventive measure against dental erosion.

The effect of low-level laser irradiation (In-Ga-Al-AsP-660 nm) on melanoma in vitro and in vivo

Background: It has been speculated that the biostimulatory effect of Low Level Laser Therapy could cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the present study is to analyze the behavior of melanoma cells (B16F10) in vitro and the in vivo development of melanoma in mice after laser irradiation. Methods: We performed a controlled in vitro study on B16F10 melanoma cells to investigate cell viability and cell cycle changes by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation. The in vivo mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and histological characteristics. Laser irradiation was performed three times (once a day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm(2), irradiance 2.5 W/cm(2) and irradiation times of 60s (dose 150 J/cm(2)) and 420s (dose 1050 J/cm(2)) respectively. Results: There were no statistically significant differences between the in vitro groups, except for an increase in the hypodiploid melanoma cells (8.48 +/- 1.40% and 4.26 +/- 0.60%) at 72 h postirradiation. This cancer-protective effect was not reproduced in the in vivo experiment where outcome measures for the 150 J/cm(2) dose group were not significantly different from controls. For the 1050 J/cm(2) dose group, there were significant increases in tumor volume, blood vessels and cell abnormalities compared to the other groups. Conclusion: LLLT Irradiation should be avoided over melanomas as the combination of high irradiance (2.5 W/cm(2)) and high dose (1050 J/cm(2)) significantly increases melanoma tumor growth in vivo.

Knoop Microhardness and FT-Raman Spectroscopic Evaluation of a Resin-Based Dental Material Light-Cured by an Argon Ion Laser and Halogen Lamp: An in Vitro Study

Objective: The purpose of this study was to evaluate in vitro the Knoop microhardness (Knoop hardness number [KHN]) and the degree of conversion using FT-Raman spectroscopy of a light-cured microhybrid resin composite (Z350-3M-ESPE) Vita shade A3 photopolymerized with a halogen lamp or an argon ion laser. Background Data: Optimal polymerization of resin-based dental materials is important for longevity of restorations in dentistry. Materials and Methods: Thirty specimens were prepared and inserted into a disc-shaped polytetrafluoroethylene mold that was 2.0 mm thick and 3 mm in diameter. The specimens were divided into three groups (n = 10 each). Group 1 (G1) was light-cured for 20 sec with an Optilux 501 halogen light with an intensity of 1000 mW/cm(2). Group 2 (G2) was photopolymerized with an argon laser with a power of 150 mW for 10 sec, and group 3 (G3) was photopolymerized with an argon laser at 200 mW of power for 10 sec. All specimens were stored in distilled water for 24 h at 37 degrees C and kept in lightproof containers. For the KHN test five indentations were made and a depth of 100 mu m was maintained in each specimen. One hundred and fifty readings were obtained using a 25-g load for 45 sec. The degree of conversion values were measured by Raman spectroscopy. KHN and degree of conversion values were obtained on opposite sides of the irradiated surface. KHN and degree of conversion data were analyzed by one-way ANOVA and Tukey tests with statistical significance set at p < 0.05. Results: The results of KHN testing were G1 = 37.428 +/- 4.765; G2 = 23.588 +/- 6.269; and G3 = 21.652 +/- 4.393. The calculated degrees of conversion (DC%) were G1 = 48.57 +/- 2.11; G2 = 43.71 +/- 3.93; and G3 = 44.19 +/- 2.71. Conclusions: Polymerization with the halogen lamp ( G1) attained higher microhardness values than polymerization with the argon laser at power levels of 150 and 200 mW; there was no difference in hardness between the two argon laser groups. The results showed no statistically significant different degrees of conversion for the polymerization of composite samples with the two light sources tested.

Bovine pericardium coated with biopolymeric films as an alternative to prevent calcification: In vitro calcification and cytotoxicity results

Bovine pericardium, for cardiac valve fabrication, was coated with either chitosan or silk fibroin film. In vitro calcification tests of coated and non coated bovine pericardium were performed in simulated body fluid solution in order to investigate potential alternatives to minimize calcification on implanted heart valves. Complementary, morphology was assessed by scanning electron microscopy - SEM; X-ray diffraction (XRD) and infrared spectroscopy (FTIR-ATR) were performed for structural characterization of coatings and biocompatibility of chitosan. Silk fibroin films were assayed by in vitro cytotoxicity and endothelial cell growth tests. Bovine pericardium coated with silk fibroin or chitosan did not present calcification during in vitro calcification tests, indicating that these biopolymeric coatings do not induce bovine pericardium calcification. Chitosan and silk fibroin films were characterized as non cytotoxic and silk fibroin films presented high affinity to endothelial cells. The results indicate that bovine pericardium coated with silk fibroin is a potential candidate for cardiac valve fabrication, since the affinity of silk fibroin to endothelial cells can be explored to induce the tissue endothelization and therefore, increase valve durability by increasing their mechanical resistance and protecting them against calcification. (C) 2010 Elsevier B.V. All rights reserved.

In vitro study of the antimicrobial activity of Brazilian propolis against Paenibacillus larvae

The honey bee disease American foulbrood (AFB) is a serious problem since its causative agent (Paenibacillus larvae) has become increasingly resistant to conventional antibiotics. The objective of this study was to investigate the in vitro activity of propolis collected from various states of Brazil against P. larvae. Propolis is derived from plant resins collected by honey bees (Apis mellifera) and is globally known for its antimicrobial properties and particularly valued in tropical regions. Tests on the activity of propolis against P. larvae were conducted both in Brazil and Minnesota, USA using two resistance assay methods that measured zones of growth inhibition due to treatment exposure. The propolis extracts from the various states of Brazil showed significant inhibition of P. larvae. Clear dose responses were found for individual propolis extracts, particularly between the concentrations of 1.7 and 0.12 mg propolis/treatment disk, but the source of the propolis, rather than the concentration, may be more influential in determining overall activity. Two of the three tested antibiotics (tylosin and terramycin) exhibited a greater level of inhibition compared to most of the Brazilian samples, which could be due to the low concentrations of active compounds present in the propolis extracts. Additionally, the majority of the Brazilian propolis samples were more effective than the few collected in MN, USA. Due to the evolution of resistance of P. larvae to conventional antibiotic treatments, this research is an important first step in identifying possible new active compounds to treat AFB in honey bee colonies. (C) 2007 Elsevier Inc. All rights reserved.