Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.

In vitro evaluation of the effect of dietary acids and toothbrushing on human root dentin permeability

OBJECTIVES: The purpose of this in vitro study was to quantify the alterations in human root dentin permeability after exposure to dietary acids and to evaluate the effect of toothbrushing after acid application. METHOD AND MATERIALS: Extracted human third molars had their crowns sectioned above the CEJ, pulp tissue removed, and cervical root dentin exposed using a high-speed bur (approximately 1 mm in depth of substance loss). From each root fragment, one specimen was prepared. A total of 25 specimens were used and distributed randomly into five groups. The specimens were attached to a hydraulic pressure apparatus to evaluate the alterations of root dentin permeability after exposure to different acids. Dentin permeability was measured after the following sequential steps: (1) treatment with EDTA for 3 minutes to obtain the maximum permeability; (2) root planing to create a smear layer; (3) exposure to different acidic substances for 5 minutes (vinegar, cola drink, lemon juice, white wine, and orange juice); and (4) brushing for 3 minutes. RESULTS: All acidic substances increased dentin permeability after root planing. Lemon juice produced higher values for permeability when compared to the other substances (P = .009); moreover, orange juice showed similar results (P < .02) except when compared to vinegar (P = .12). Brushing right after acid exposure significantly reduced dentin permeability except in the vinegar group (P = .07). CONCLUSION: Under the experimental conditions, dietary acids increased root dentin permeability, and immediate brushing reduced permeability levels.

In vitro analysis with human bone marrow stem cells on Ti-15Mo alloy for dental and orthopedic implants application

Aim: Nowadays, research on orthopedic and dental implants is focused on titanium alloys for their mechanical properties and corrosion resistance in the human body environment. Another important aspect to be investigated is their surface topography, which is very important to osseointegration. With laser beam irradiation for roughening the implants surface an easier control of the microtopography is achieved, and surface contamination is avoided. The aim of this study was to assess human bone marrow stem cells response to a newly developed titanium alloy, Ti-15Mo, with surface topography modified by laser beam irradiation. Materials and methods: A total of 10 Ti machined disks (control), 10 Ti-15Mo machined disks and 10 Ti-15Mo disks treated by laser beam-irradiation were prepared. To study how Ti-15Mo surface topografy can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction. Results: In Test 1 (comparison between Ti-15Mo machined disks and Ti-machined disks) quantitative real-time RT-PCR showed a significant induction of ALPL, FOSL1 and SPP1, which increase 20% or more. In Test 2 (comparison between Ti-15Mo laser treated disks and Ti-machined disks) all investigated genes were up-regulated. By comparing Test 1 and Test 2 it was detected that COL1A1, COL3A1, FOSL1 and ENG sensibly increased their expression whereas RUNX2, ALPL and SPP1 expression remained substantially unchanged. Conclusion: The present study demonstrated that laser treated Ti-15Mo alloys are promising materials for implants application.

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P < 0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P < 0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P > 0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P > 0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P > 0.05) in the presence of heparin and PHE (P < 0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development. © 2013 The Society for In Vitro Biology.

Tutela penal do embrião humano in vitro: uma releitura à luz da proteção jurídica das dimensões da vida

Pós-graduação em Direito - FCHS

Cytotoxicity and genotoxicity of low doses of mercury chloride and methylmercury chloride on human lymphocytes in vitro

Mercury is a xenobiotic metal that is a highly deleterious environmental pollutant. The biotransformation of mercury chloride (HgCl2) into methylmercury chloride (CH3HgCl) in aquatic environments is well-known and humans are exposed by consumption of contaminated fish, shellfish and algae. The objective of the present study was to determine the changes induced in vitro by two mercury compounds (HgCl2 and CH3HgCl) in cultured human lymphocytes. Short-term human leukocyte cultures from 10 healthy donors (5 females and 5 males) were set-up by adding drops of whole blood in complete medium. Cultures were separately and simultaneously treated with low doses (0.1 to 1000 µg/l) of HgCl2 and CH3HgCl and incubated at 37ºC for 48 h. Genotoxicity was assessed by chromosome aberrations and polyploid cells. Mitotic index was used as a measure of cytotoxicity. A significant increase (P < 0.05) in the relative frequency of chromosome aberrations was observed for all concentrations of CH3HgCl when compared to control, whether alone or in an evident sinergistic combination with HgCl2. The frequency of polyploid cells was also significantly increased (P < 0.05) when compared to control after exposure to all concentrations of CH3HgCl alone or in combination with HgCl2. CH3HgCl significantly decreased (P < 0.05) the mitotic index at 100 and 1000 µg/l alone, and at 1, 10, 100, and 1000 µg/l when combined with HgCl2, showing a synergistic cytotoxic effect. Our data showed that low concentrations of CH3HgCl might be cytotoxic/genotoxic. Such effects may indicate early cellular changes with possible biological consequences and should be considered in the preliminary evaluation of the risks of populations exposed in vivo to low doses of mercury.

Maturação in vitro de oócitos caninos: influência da suplementação hormonal seriada sobre a ultraestrutura oocitária

Pós-graduação em Medicina Veterinária - FCAV

In Vitro Regulation of CCL3 and CXCL12 by Bacterial By-products Is Dependent on Site of Origin of Human Oral Fibroblasts

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of human whole saliva on the in vitro adhesion of Candida albicans to a denture base acrylic resin: a focus on collection and preparation of saliva samples

Objective In studies on Candida albicans adhesion to surfaces, diverse protocols have been used for collection and preparation of saliva samples. Thus, this study investigated whether variations in the centrifugation parameters and number of donors of saliva would influence the adhesion of C. albicans to a denture base resin. Methods Resin acrylic samples (n = 72) were made and then divided into four groups: (a) control – specimens were left without preconditioning in saliva; (b) three experimental groups, in which the specimens were preconditioned with saliva collected from 15 volunteers and centrifuged at 12 000 g for 5 min (G1); from 15 volunteers and centrifuged at 18 000 g for 30 min (G2); and from one volunteer and centrifuged at 12 000 g for 5 min (G3). Candida adhesion was evaluated by both the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction method and crystal violet staining. Data were analyzed by one-way analyses of variance (P = 0.05). Results For XTT reduction assay, groups G2, G3, and control were not significantly different, whereas group G1 showed significantly higher absorbance value than control. For crystal violet staining there were no significant differences among all groups. Conclusion Variations in the centrifugation parameters and number of donors of saliva may influence C. albicans adhesion to denture base resins.

Human follicle stimulating hormone (hFSH) and thyroxine (T4) in survival maintenance and in vitro growth promotion of caprine preantral follicles

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of Er:YAG laser on CaF2 formation and its anticariogenic action on human enamel: An in vitro study

Objective: The objective of this study was to evaluate the effect of Er: YAG laser on the formation of CaF2, after the application of acidulated phosphate fluoride (APF), and its influence on the anti-cariogenic action in human dental enamel. Background Data: Er:YAG laser was designed to promote ablation of the enamel. However, the possibility of using this energy to increase the enamel's resistance to caries has hardly been explored, and neither has its interaction with the use of fluorides. Materials and Methods: One hundred and twenty blocks of enamel were allocated to four groups of 30 blocks each: (1) C, control group; (2) Er:YAG, laser; (3) APF; and (4) Er:YAG+APF. Of these, 80 blocks were submitted to pH cycling for 14 days. In the other 40 blocks, fluoride (CaF2) was measured before cycling. After pH cycling, surface microhardness (SMH), microhardness in cross-section (converted to mineral contents % vol. min.), and fluoride after cycling (40 blocks) were also determined. Results: SMH decreased in all groups. The control group showed the highest decrease, and Er:YAG+APF showed the lowest decrease (p < 0.05). Groups APF and Er:YAG showed the same results (p > 0.05). Mineral content at depths 10, 20, and 40 μm was lower in the control and Er:YAG groups, and higher in groups APF and Er:YAG+APF. CaF2 (μgF/cm2) deposited before pH cycling was higher in the APF group when compared to the Er:YAG+APF group. Control and Er:YAG groups showed the lowest values (p > 0.05). Conclusion: It was concluded that Er:YAG laser influenced the deposition of CaF2 on the enamel and showed a superficial anti-cariogenic action, but not in depth.

Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

Study of quercetin-loaded liposomes as potential drug carriers: in vitro evaluation of human complement activation

Liposomes have been employed as potential drug carriers. However, after their in vivo administration, they can be destabilized by proteins of complement system, contributing to the clearance of vesicles from blood circulation. Antioxidant flavonoids such as quercetin have been reported to be beneficial to human health, but their low water solubility and bioavailability limit their enteric administration. Therefore, the development of appropriate flavonoid-carriers could be of great importance to drug therapy. The aim of the present study was to evaluate the activation of human complement system proteins by liposomes composed of soya phosphatidylcholine (SPC) and cholesterol (CHOL) or cholesteryl ethyl ether (CHOL-OET) loaded with quercetin or not. The consumption of complement, via classical (CP) and alternative (AP) pathways, by different vesicles was evaluated using a hemolytic assay and quantitative determination of iC3b and natural antibodies deposited on empty liposomal surfaces by ELISA. The main results showed that empty liposomes composed of large amounts of CHOL consumed more complement components than the others for both CP and AP. Furthermore, replacement of CHOL with CHOL-OET reduced complement consumption via both CP and AP. Incorporation of quercetin did not change CP and AP consumption. Deposition of iC3b, IgG and IgM in vesicles composed of SPC: CHOL-OET at a molar ratio of 1.5:1 was lower compared to the others. Taken together, these observations suggest that liposomes composed of SPC: CHOL-OET at a molar ratio of 1.5:1 are the most appropriate among the vesicles studied herein to be used as a drug carrier system in further investigations.