The in vitro impact of toothpaste extracts on cell viability.

Toothpastes contain three main components: detergents, abrasives, and fluoride. Detergents, particularly sodium lauryl sulfate, have been proposed as components that enable toothpastes to produce cytotoxic effects in vitro. However, not all toothpastes contain sodium lauryl sulfate, and almost no studies have found an association between detergents and the in vitro cytotoxicity of toothpastes. The present study examined the in vitro cytotoxicity of nine commercially available toothpastes containing four different detergents. Toothpastes were diluted in serum-free medium, centrifuged, and filter sterilized. The half-lethal concentration of the toothpaste-conditioned medium (TCM) was calculated based on the formation of formazan by gingival fibroblasts, oral squamous cell carcinoma HSC-2 cells, and L929 cells. Cell proliferation was analyzed, and live-dead staining was performed, after exposure of cells to conditioned medium prepared with 1% toothpaste (1% TCM). It was found that toothpastes containing sodium lauryl sulfate and amine fluoride strongly inhibited cell viability with the half-lethal concentration being obtained with conditioned medium prepared with approximately 1% toothpaste (1% TCM). Toothpastes containing cocamidopropyl betaine and Steareth-20 showed higher half-lethal concentration values, with the half-lethal concentration being obtained with conditioned medium prepared with 10% (10% TCM) and 70% (70% TCM) toothpaste, respectively. Proliferation and live-dead data were consistent with the cell-viability analyses. These results demonstrate that the type of detergent in toothpastes can be associated with changes in in vitro cell toxicity.

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16 +/- 0.27 (mean +/- S.D.), 1.35 +/- 0.285 and 2.72 +/- 0.47 per 1000 mu m(2) resulted in percent deaths of 3.96 +/- 5.22, 45.91 +/- 10.89, 90.52 +/- 12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death. (c) 2005 Elsevier Ltd. All rights reserved.

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds.

Regulation of beta-cell viability and gene expression by distinct agonist fragments of adiponectin

Obesity is an established risk factor for type 2 diabetes. Activation of the adiponectin receptors has a clear role in improving insulin resistance although conflicting evidence exists for its effects on pancreatic beta-cells. Previous reports have identified both adiponectin receptors (ADR-1 and ADR-2) in the beta-cell. Recent evidence has suggested that two distinct regions of the adiponectin molecule, the globular domain and a small N-terminal region, have agonist properties. This study investigates the effects of two agonist regions of adiponectin on insulin secretion, gene expression, cell viability and cell signalling in the rat beta-cell line BRIN-BD11, as well as investigating the expression levels of adiponectin receptors (ADRs) in these cells. Cells were treated with globular adiponectin and adiponectin (15-36) +/-leptin to investigate cell viability, expression of key beta-cell genes and ERK1/2 activation. Both globular adiponectin and adiponectin (15-36) caused significant ERK1/2 dependent increases in cell viability. Leptin co-incubation attenuated adiponectin (15-36) but not globular adiponectin induced cell viability. Globular adiponectin, but not adiponectin (15-36), caused a significant 450% increase in PDX-1 expression and a 45% decrease in LPL expression. ADR-1 was expressed at a higher level than ADR-2, and ADR mRNA levels were differentially regulated by non-esterified fatty acids and peroxisome-proliferator-activated receptor agonists. These data provide evidence of roles for two distinct adiponectin agonist domains in the beta-cell and confirm the potentially important role of adiponectin receptor agonism in maintaining beta-cell mass.

Resistin down-regulates insulin receptor expression, and modulates cell viability in rodent pancreatic beta-cells

The adipokine resistin is known to induce insulin resistance in rodent tissues. Increases in adipose tissue mass are known to have a negative effect on pancreatic beta-cell function, although the mechanisms are poorly understood. This study investigated the effects of resistin on insulin secretion, insulin receptor expression and cell viability in pancreatic beta-cells. BTC-6 or BRIN-BD11 cells were treated for 24h with resistin, and insulin receptor expression, insulin secretion and cell viability were measured. Incubation with 40ng/ml resistin caused significant decreases in insulin receptor mRNA and protein expression, but did not affect insulin secretion. At low concentrations, resistin caused significant increases in cell viability. These data implicate resistin as a factor that may regulate beta-cell function/viability, and suggests a potential mechanism by which increased adiposity causes beta-cell dysfunction.

Luminescent techniques for studying free radical production and cell viability in relation to cardiovascular disease and HRT

Epidemiological studies have suggested that hormone replacement therapy (HRT) offers protection from atherosclerosis, a precursor of cardiovascular disease (CVD), in postmenopausal women. There is good evidence that oxidation of low-density lipoprotein (LDL) by leucocyte-derived reactive oxygen species plays a key role in development of an atherosclerotic plaque. Therefore we have investigated whether the possible protection against CVD by HRT could be due to immunomodulation, specifically of free radical production. The study involves 2 approaches: I) analysing the production of free radicals by leucocytes from women on HRT, 2) investigating the effect of I7p-oestradiol and progesterone on cultured myeloid cells (HL60 and U937). Free radical production by leucocytes was determined using a recently developed bioluminescent assay. In the assay, Pholasin® emits light in the presence of free radicals produced by the NADPH oxidase system of leucocytes stimulated with PMA or fMLP. Cell viability was also investigated using a bioluminescent assay (Cell Titer-Glo®) in which cytosolic ATP levels were measured by the production of luminescence in the presence of Luciferin/Luciferase reagent. Studies of leucocytes from HRT patients showed considerable variation in free radical production, which appeared to be dependent on HRT regime. Studies on the cultured cells showed that there was no cell proliferation at low hormone concentrations, while high concentrations caused cytotoxicity. The effect of hormones on free radical production in this in vitro model system is currently being investigated. The results show that the effects of the hormones on cells of the immune system are very dose dependent, and that both beneficial and adverse effects may occur. In conclusion, luminescent techniques offer a valuable and sensitive approach to studying inflammatory and oxidative processes both in vivo and in vitro.

Nicotine exposure reduces cell viability but induces anti-inflammatory effects in human cystic fibrosis bronchial epithelial cells

Background: Mouse models of cystic fibrosis (CF) fail to truly represent the respiratory pathology. We have consequently developed human airways cell culture models to address this. The impact of cigarette smoke within the CF population is well documented, with exposure being known to worsen lung function. As nicotine is often perceived to be a less harmful component of tobacco smoke, this research aimed to identify its effects upon viability and inflammatory responses of CF (IB3-1) and CF phenotype corrected (C38) bronchial epithelial cells. Methods: IB3-1 and C38 cell lines were exposed to increasing concentrations of nicotine (0.55-75μM) for 24 hours. Cell viability was assessed via Cell Titre Blue and the inflammatory response with IL-6 and IL-8 ELISA. Results: CF cells were more sensitive; nicotine significantly (P<0.05) reduced cell viability at all concentrations tested, but failed to have a marked effect on C38 viability. Whilst nicotine induced anti-inflammatory effects in CF cells with a significant reduction in IL-6 and IL-8 release, it had no effect on chemokine release by C38 cells. Conclusion: CF cells may be more vulnerable to inhaled toxicants than non-CF cells. As mice lack a number of human nicotinic receptor subunits and fail to mimic the characteristic pathology of CF, these data emphasise the importance of employing relevant human cell lines to study a human-specific disease.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

Rat visceral yolk sac cells: viability and expression of cell markers during maternal diabetes

The function of the visceral yolk sac (VYS) is critical for embryo organogenesis until final fetal development in rats, and can be affected by conditions such as diabetes. In view of the importance of diabetes during pregnancy for maternal and neonatal health, the objective of this study was to assess fetal weight, VYS cell markers, and viability in female Wistar rats (200-250 g) with induced diabetes (alloxan, 37 mg/kg) on the 8th gestational day (gd 8). At gd 15, rats from control (n=5) and diabetic (n=5) groups were anesthetized and laparotomized to remove the uterine horns for weighing of fetuses and collecting the VYS. Flow cytometry was used for characterizing VYS cells, and for determining mitochondrial activity, cell proliferation, DNA ploidy, cell cycle phases, and caspase-3 activity. Fetal weight was reduced in the diabetic group. Expression of the cell markers CD34, VEGFR1, CD115, CD117, CD14, CCR2, CD90, CD44, STRO-1, OCT3/4, and Nanog was detected in VYS cells in both groups. In the diabetic group, significantly decreased expression of CD34 (P<0.05), CCR2 (P<0.001), and OCT3/4 (P<0.01), and significantly increased expression of CD90 (P<0.05), CD117 (P<0.01), and CD14 (P<0.05) were observed. VYS cells with inactive mitochondria, activated caspase-3, and low proliferation were present in the rats with diabetes. Severe hyperglycemia caused by maternal diabetes had negative effects on pregnancy, VYS cell viability, and the expression of cell markers.

Cell population, viability, and some key immunomodulatory molecules in different milk somatic cell samples in dairy cows

Immune cells in the milk are most important in combating pathogens that invade the mammary gland. This study investigated the immune competence and viability of somatic milk cells that are already resident in milk and udders free of infection. Cells were studied in freshly removed milk to simulate conditions in the udder. Effects of incubation, cell preparation, and immunological stimulation with 0.5 mug/ml lipopolysaccharide (LPS) from Escherichia coli were analysed. Viability and differential counts of milk cells between high and low somatic cell count (SCC) quarters, and cisternal and alveolar milk with and without LPS stimulation were compared. Incubation and preparation of cells caused a cell loss which further increased with time independently of SCC and milk fraction. The viability of these cells was stable until 3 h post incubation and decreased until 6 h. Cell populations differed between both investigations, but did not change during the course of the experiment. mRNA expression of immune and apoptosis factors of the cells, measured by qPCR, did not change substantially: mRNA expression of caspase 3, Toll like receptor 4, and GM-CSF did not change, whereas the expression of the death receptor Fas/APO-1 (CD95), lactoferrin and lysozyme was decreased at 6 h. Cyclooxygenase-2 and TNF-alpha mRNA expression were decreased after 6 h of LPS treatment. In comparison with other studies in vivo or in vitro (in cell culture), in this study where cells are studied ex vivo (removed from the udder but kept in their natural environment, the milk) resident milk cells seem to be more vulnerable, less viable, less able to respond to stimulation, and thus less immune competent compared with cells that have freshly migrated from blood into milk after pathogen stimulation. The cell viability and differential cell count differed between high- and low-SCC milk and between cisternal and alveolar milk depending on the individual cow. In conclusion, the results support the view that for a most effective defence against invading pathogens the mammary gland is reliant on the recruitment of fresh immune cells from the blood.

Effect of Allium sativum (garlic) methanol extract on viability and apoptosis of human leukemic cell lines

Purpose: To investigate the effect of Allium sativum (garlic) methanol extract on viability and apoptosis of human leukemic cells. Methods: Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ug/mL of Allium sativum extract following 48-h treatment on U-937, Jurkat Clone E6-1 and K-562 cell lines. The mode of cell death was determined by Annexin V-FITC staining and analyzed by flow cytometry. Results: The results show that the half-maximal inhibitory concentration (IC50) of A. sativum on U-937, Jurkat Clone E6-1, K-562 cell lines was 105 ± 2.21, 489 ± 4.51 and 455 ± 3.13 μg/mL, respectively, compared with negative control, while apoptosis was 17.93 ± 0.95 % for U-937 cells (p ≤ 0.05), 38.37 ± 1.88 % for Jurkat Clone E6-1 cells (p ≤ 0.001) and 16.37 ± 1.10 % for K-562 cells. A majority of the cells were inhibited by the extract via apoptosis. Only U-937 cells (6.87 ± 0.65 %) showed significant necrosis compared to negative control (p ≤ 0.05). Conclusion: K-562 cells are the most resistant against garlic extract, in contrast to Jurkat Clone E6-1 cells. Garlic extract does not induce necrosis in Jurkat Clone E6-1 and K-562 cells.

Carbon Nanoparticles For Gene Transfection In Eukaryotic Cell Lines.

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

Nek5 Interacts With Mitochondrial Proteins And Interferes Negatively In Mitochondrial Mediated Cell Death And Respiration.

Mitochondria are involved in energy supply, signaling, cell death and cellular differentiation and have been implicated in several human diseases. Neks (NIMA-related kinases) represent a family of mammal protein kinases that play essential roles in cell-cycle progression, but other functions have recently been related. A yeast two-hybrid (Y2H) screen was performed to identify and characterize Nek5 interaction partners and the mitochondrial proteins Cox11, MTX-2 and BCLAF1 were retrieved. Apoptosis assay showed protective effects of stable hNek5 expression from Hek293-T's cell death after thapsigargin treatment (2μM). Nek5 silenced cells as well as cells expressing a kinase dead version of Nek5, displayed an increase in ROS formation after 4h of thapsigargin treatment. Mitochondrial respiratory chain activity was found decreased upon stable hNek5expression. Cells silenced for hNek5 on the other hand presented 1.7 fold increased basal rates of respiration, especially at the electrons transfer steps from TMPD to cytochrome c and at the complex II. In conclusion, our data suggest for the first time mitochondrial localization and functions for Nek5 and its participation in cell death and cell respiration regulation. Stable expression of hNek5 in Hek293T cells resulted in enhanced cell viability, decreased cell death and drug resistance, while depletion of hNek5by shRNA overcame cancer cell drug resistance and induced apoptosis in vitro. Stable expression of hNek5 also inhibits thapsigargin promoted apoptosis and the respiratory chain complex IV in HEK293T cells.

Viability of fibroblasts cultured under nutritional stress irradiated with red laser, infrared laser, and red light-emitting diode

Phototherapy is noninvasive, painless and has no known side effect. However, for its incorporation into clinical practice, more well-designed studies are necessary to define optimal parameters for its application. The viability of fibroblasts cultured under nutritional stress irradiated with either a red laser, an infrared laser, or a red light-emitting diode (LED) was analyzed. Irradiation parameters were: red laser (660 nm, 40 mW, 1 W/cm(2)), infrared laser (780 nm, 40 mW, 1 W/cm(2)), and red LED (637 +/- 15 nm, 40 mW, 1 W/cm(2)). All applications were punctual and performed with a spot with 0.4 mm(2) of diameter for 4 or 8 s. The Kruskal-Wallis test and analysis of variance of the general linear model (p <= 0.05) were used for statistical analysis. After 72 h, phototherapy with low-intensity laser and LED showed no toxicity at the cellular level. It even stimulated methylthiazol tetrazolium assay (MTT) conversion and neutral red uptake of fibroblasts cultured under nutritional stress, especially in the group irradiated with infrared laser (p = 0.004 for MTT conversion and p < 0.001 for neutral red uptake). Considering the parameters and protocol of phototherapy used, it can be concluded that phototherapy stimulated the viability of fibroblasts cultured under nutritional deficit resembling those found in traumatized tissue in which cell viability is reduced. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3602850]