Genotoxic mixtures and dissimilar action: Concepts for prediction and assessment

This article has been made available through the Brunel Open Access Publishing Fund. This article is distributed under the terms of the creative commons Attribution license which permits any use, distribution, and reproduction in any medium, provided the original author(s)and the source are credited.Combinations of genotoxic agents have frequently been assessed without clear assumptions regarding their expected (additive) mixture effects, often leading to claims of synergisms that might in fact be compatible with additivity. We have shown earlier that the combined effects of chemicals, which induce micronuclei (MN) in the cytokinesis-block micronucleus assay in Chinese hamster ovary-K1 cells by a similar mechanism, were additive according to the concept of concentration addition (CA). Here, we extended these studies and investigated for the first time whether valid additivity expectations can be formulated for MN-inducing chemicals that operate through a variety of mechanisms, including aneugens and clastogens (DNA cross-linkers, topoisomerase II inhibitors, minor groove binders). We expected that their effects should follow the additivity principles of independent action (IA). With two mixtures, one composed of various aneugens (colchicine, flubendazole, vinblastine sulphate, griseofulvin, paclitaxel), and another composed of aneugens and clastogens (flubendazole, doxorubicin, etoposide, melphalan and mitomycin C), we observed mixture effects that fell between the additivity predictions derived from CA and IA. We achieved better agreement between observation and prediction by grouping the chemicals into common assessment groups and using hybrid CA/IA prediction models. The combined effects of four dissimilarly acting compounds (flubendazole, paclitaxel, doxorubicin and melphalan) also fell within CA and IA. Two binary mixtures (flubendazole/paclitaxel and flubendazole/doxorubicin) showed effects in reasonable agreement with IA additivity. Our studies provide a systematic basis for the investigation of mixtures that affect endpoints of relevance to genotoxicity and show that their effects are largely additive.UK Food Standards Agenc

The biocompatibility of titanium in a buffer solution: compared effects of a thin film of TiO2 deposited by MOCVD and of collagen deposited from a gel

This study aims at evaluating the biocompatibility of titanium surfaces modified according two different ways: (i) deposition of a bio-inert, thin film of rutile TiO2 by chemical vapour deposition (MOCVD), and (ii) biochemical treatment with collagen gel, in order to obtain a bio-interactive coating. Behind the comparison is the idea that either the bio-inert or the bio-active coating has specific advantages when applied to implant treatment, such as the low price of the collagen treatment for instance. The stability in buffer solution was evaluated by open circuit potential (OCP) for medium time and cyclic voltametry. The OCP stabilized after 5104 min for all the specimens except the collagen treated sample which presented a stable OCP from the first minutes. MOCVD treated samples stabilized to more electropositive values. Numeric results were statistically analysed to obtain the regression equations for long time predictable evolution. The corrosion parameters determined from cyclic curves revealed that the MOCVD treatment is an efficient way to improve corrosion resistance. Human dermal fibroblasts were selected for cell culture tests, taking into account that these cells are present in all bio-interfaces, being the main cellular type of connective tissue. The cells grew on either type of surface without phenotype modification. From the reduction of yellow, water-soluble 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT cytotoxicity test), MOCVD treated samples offer better viability than mechanically polished Ti and collagen treated samples as well. Cell spreading, as evaluated from microscope images processed by the program Sigma Scan, showed also enhancement upon surface modification. Depending on the experimental conditions, MOCVD deposited TiO2 exhibits different nanostructures that may influence biological behaviour. The results demonstrate the capacity of integration in simulated physiologic liquids for an implant pretreated by either method

Human surfactant protein D alters oxidative stress and HMGA1 expression to induce p53 apoptotic pathway in eosinophil leukemic cell line

This article is made available through the Brunel Open Access Publishing Fund. Copyright: © 2013 Mahajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Surfactant protein D (SP-D), an innate immune molecule, has an indispensable role in host defense and regulation of inflammation. Immune related functions regulated by SP-D include agglutination of pathogens, phagocytosis, oxidative burst, antigen presentation, T lymphocyte proliferation, cytokine secretion, induction of apoptosis and clearance of apoptotic cells. The present study unravels a novel ability of SP-D to reduce the viability of leukemic cells (eosinophilic leukemic cell line, AML14.3D10; acute myeloid leukemia cell line, THP-1; acute lymphoid leukemia cell lines, Jurkat, Raji; and human breast epithelial cell line, MCF-7), and explains the underlying mechanisms. SP-D and a recombinant fragment of human SP-D (rhSP-D) induced G2/M phase cell cycle arrest, and dose and timedependent apoptosis in the AML14.3D10 eosinophilic leukemia cell line. Levels of various apoptotic markers viz. activated p53, cleaved caspase-9 and PARP, along with G2/M checkpoints (p21 and Tyr15 phosphorylation of cdc2) showed significant increase in these cells. We further attempted to elucidate the underlying mechanisms of rhSP-D induced apoptosis using proteomic analysis. This approach identified large scale molecular changes initiated by SPD in a human cell for the first time. Among others, the proteomics analysis highlighted a decreased expression of survival related proteins such as HMGA1, overexpression of proteins to protect the cells from oxidative burst, while a drastic decrease in mitochondrial antioxidant defense system. rhSP-D mediated enhanced oxidative burst in AML14.3D10 cells was confirmed, while antioxidant, N-acetyl-L-cysteine, abrogated the rhSP-D induced apoptosis. The rhSP-D mediated reduced viability was specific to the cancer cell lines and viability of human PBMCs from healthy controls was not affected. The study suggests involvement of SP-D in host’s immunosurveillance and therapeutic potential of rhSP-D in the eosinophilic leukemia and cancers of other origins.Department of Biotechnology, Indi

Immunoglobulin G; structure and functional implications of different subclass modifications in initiation and resolution of allergy.

IgE and not IgG is usually associated with allergy. IgE lodged on mast cells in skin or gut and basophils in the blood allows for the prolonged duration of allergy through the persistent expression of high affinity IgE receptors. However, many allergic reactions are not dependent on IgE and are generated in the absence of allergen specific and even total IgE. Instead, IgG plasma cells are involved in induction of, and for much of the pathogenesis of, allergic diseases. The pattern of IgG producing plasma cells in atopic children and the tendency for direct or further class switching to IgE are the principle factors responsible for long-lasting sensitization of mast cells in allergic children. Indirect class switching from IgG producing plasma cells has been shown to be the predominant pathway for production of IgE while a Th2 microenvironment, genetic predisposition, and the concentration and nature of allergens together act on IgG plasma cells in the atopic tendency to undergo further immunoglobulin gene recombination. The seminal involvement of IgG in allergy is further indicated by the principal role of IgG4 in the natural resolution of allergy and as the favourable immunological response to immunotherapy. This paper will look at allergy through the role of different antibodies than IgE and give current knowledge of the nature and role of IgG antibodies in the start, maintenance and resolution of allergy

Simvastatin inhibits TLR8 signaling in primary human monocytes and spontaneous TNF production from rheumatoid synovial membrane cultures

Simvastatin has been shown to have anti-inflammatory effects that are independent of its serum cholesterol lowering action, but the mechanisms by which these anti-inflammatory effects are mediated have not been elucidated. To explore the mechanism involved, the effect of simvastatin on Toll-like receptor (TLR) signalling in primary human monocytes was investigated. A short pre-treatment with simvastatin dose-dependently inhibited the production of tumor necrosis factor-α (TNF) in response to TLR8 (but not TLRs 2, 4, or 5) activation. Statins are known inhibitors of the cholesterol biosynthetic pathway, but intriguingly TLR8 inhibition could not be reversed by addition of mevalonate or geranylgeranyl pyrophosphate; downstream products of cholesterol biosynthesis. TLR8 signalling was examined in HEK 293 cells stably expressing TLR8, where simvastatin inhibited IKKα/β phosphorylation and subsequent NF-κB activation without affecting the pathway to AP-1. Since simvastatin has been reported to have anti-inflammatory effects in RA patients and TLR8 signalling contributes to TNF production in human RA synovial tissue in culture, simvastatin was tested in these cultures. Simvastatin significantly inhibited the spontaneous release of TNF in this model which was not reversed by mevalonate. Together, these results demonstrate a hitherto unrecognized mechanism of simvastatin inhibition of TLR8 signalling that may in part explain its beneficial anti-inflammatory effects

Rheumatoid synovial fluid interleukin-17-producing CD4 T cells have abundant tumor necrosis factor-alpha co-expression, but little interleukin-22 and interleukin-23R expression

Introduction\ud Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to systematically analyse the phenotype, cytokine profile and frequency of interleukin-17 (IL-17) producing CD4-positive T cells in mononuclear cells isolated from peripheral blood, synovial fluid and synovial tissue of RA patients with established disease, and to correlate cell frequencies with disease activity. \ud \ud Methods\ud Flow cytometry was used to analyse the phenotype and cytokine production of mononuclear cells isolated from peripheral blood (PBMC) (n = 44), synovial fluid (SFMC) (n = 14) and synovium (SVMC) (n = 10) of RA patients and PBMC of healthy controls (n = 13). \ud \ud Results\ud The frequency of IL-17-producing CD4 T cells was elevated in RA SFMC compared with RA PBMC (P = 0.04). However, the frequency of this population in RA SVMC was comparable to that in paired RA PBMC. The percentage of IL-17-producing CD4 T cells coexpressing tumor necrosis factor alpha (TNFα) was significantly increased in SFMC (P = 0.0068). The frequency of IFNγ-producing CD4 T cells was also significantly higher in SFMC than paired PBMC (P = 0.042). The majority of IL-17-producing CD4 T cells coexpressed IFNγ. IL-17-producing CD4 T cells in RA PBMC and SFMC exhibited very little IL-22 or IL-23R coexpression. \ud \ud Conclusions\ud These findings demonstrate a modest enrichment of IL-17-producing CD4 T cells in RA SFMC compared to PBMC. Th17 cells in SFMC produce more TNFα than their PBMC counterparts, but are not a significant source of IL-22 and do not express IL-23R. However, the percentage of CD4 T cells which produce IL-17 in the rheumatoid joint is low, suggesting that other cells may be alternative sources of IL-17 within the joints of RA patients. \ud \u

Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro

Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration–viability curve. Results show that cell survival is significantly higher in cooled cells (T < 22° C) than in non-cooled cells (T = 37° C), but no significant differences are visible between T = 10° C and T = 22° C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided

An improved microtiter assay for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

BACKGROUND: The anti-HIV-1 neutralizing antibody assay is widely used in AIDS vaccine research and other experimental and clinical studies. The vital dye staining method applied in the detection of anti-HIV-1 neutralizing antibody has been used in many laboratories. However, the unknown factor(s) in sera or plasma affected cell growth and caused protection when the tested sera or plasma was continuously maintained in cell culture. In addition, the poor solubility of neutral red in medium (such as RPMI-1640) also limited the use of this assay. METHODS: In this study, human T cell line C8166 was used as host cells, and 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) instead of neutral red was used as vital dye. In order to avoid the effect of the unknown factor(s), the tested sera or plasma was removed by a washout procedure after initial 3–6 h culture in the assay. RESULT: This new assay eliminated the effect of the tested sera or plasma on cell growth, improved the reliability of detection of anti-HIV-1 neutralizing antibody, and showed excellent agreement with the p24 antigen method. CONCLUSION: The results suggest that the improved assay is relatively simple, highly duplicable, cost-effective, and well reliable for evaluating anti-HIV-1 neutralizing antibodies from sera or plasma

Comparison of Enzymatic and Non-Enzymatic Means of Dissociating Adherent Monolayers of Mesenchymal Stem Cells

The dissociation of adherent mesenchymal stem cell (MSC) monolayers with trypsin and enzyme-free dissociation buffer was compared. A significantly lower proportion of viable cells were obtained with enzyme-free dissociation buffers compared to trypsin. Subsequently, the dissociated cells were re-seeded on new cell culture dishes and were subjected to the MTT assay 24 h later. The proportion of viable cells that reattached was significantly lower for cells obtained by dissociation with enzyme-free dissociation buffer compared to trypsin. Frozen–thawed MSC displayed a similar trend, yielding consistently higher cell viability and reattachment rates when dissociated with trypsin compared to enzyme-free dissociation buffer. It was also demonstrated that exposure of trypsin-dissociated MSC to enzyme-free dissociation buffer for 1 h had no significant detrimental effect on cell viability