Avaliação in vitro da citotoxicidade de monômeros, plastificante e produtos de degradação liberados a partir de resinas para reembasamento imediato

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito do tratamento térmico em microondas e do tempo de armazenamento em água sobre a citotoxicidade de resinas acrílicas para base e reembasamento de próteses

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ras signaling in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is important in immunologic cytotoxicity, autoimmunity, sepsis, normal embryonic development, and wound healing. TNF exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. We found that enforced expression of an activated H-ras oncogene converted the non-tumorigenic TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells (10TEJ) that also became very sensitive to TNF-induced apoptosis. This finding suggested that the oncogenic form of H-Ras, in which the p21 is locked in the GTP-bound form, could play a role in TNF-induced apoptosis of these cells. To investigate whether Ras activation is an obligatory step in TNF-induced apoptosis, we introduced two different molecular antagonists of Ras, namely the Rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras transformed 10TEJ cells. Expression of either Rap1A or RasN17 in 10TEJ cells resulted in abrogation of TNF-induced apoptosis. Similar results were obtained by expression of either Ras antagonist in L929 cells, a fibroblast cell line that is sensitive to TNF-induced apoptosis but does not have a ras mutation. The effects of Rap-1A and RasN17 appear to be specific to TNF, since cytotoxicity induced by doxorubicin and thapsigargin are unaffected. Additionally, constitutive apoptosis sensitivity in isolated nuclei, as measured by activation of Ca$\sp{2+}$-dependent endogenous endonuclease, is not affected by Rap-1A or RasN17. Moreover, TNF treatment of L929 cells increased Ras-bound GTP, indicating that Ras activation is triggered by TNF. Thus, Ras activation is required for TNF-induced apoptosis in mouse cells. ^

Chemotherapy pretreatment sensitizes solid tumor-derived cell lines to Vα24+ NKT cell-mediated cytotoxicity

There is an increasing awareness of the therapeutic potential for combining immune-based therapies with chemotherapy in the treatment of malignant diseases, but few published studies evaluate possible cytotoxic synergies between chemotherapy and cytotoxic immune cells. Human Vα24 +/Vβ11+ NKT cells are being evaluated for use in cell-based immunotherapy of malignancy because of their immune regulatory functions and potent cytotoxic potential. In this study, we evaluated the cytotoxicity of combinations of chemotherapy and NKT cells to determine whether there is a potential to combine these treatment modalities for human cancer therapy. The cytotoxicity of NKT cells was tested against solid-tumor derived cell lines NCI-H358, DLD-1, HT-29, DU-145, TSU-Pr1 and MDA-MB231, with or without prior treatment of these target cells, with a range of chemotherapy agents. Low concentrations of chemotherapeutic agents led to sensitization of cell lines to NKT-mediated cytotoxicity, with the greatest effect being observed for prostate cancer cells. Synergistic cytotoxicity occurred in an NKT cell in a dose-dependent manner. Chemotherapy agents induced upregulation of cell surface TRAIL-R2 (DR5) and Fas (CD95) expression, increasing the capacity for NKT cells to recognize and kill via TRAIL- and FasL-mediated pathways. We conclude that administration of cytotoxic immune cells after chemotherapy may increase antitumor activities in comparison with the use of either treatment alone.

Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies.

Cytotoxicity of topical antimicrobial agents used in burn wounds in Australasia

BACKGROUND: Burn sepsis is a leading cause of mortality and morbidity in patients with major burns. The use of topical antimicrobial agents has helped improve the survival of these patients. Silvazine (Sigma Pharmaceuticals, Melbourne, Australia) (1% silver sulphadiazine and 0.2% chlorhexidine digluconate) is used exclusively in Australasia, and there is no published study on its cytotoxicity. This study compared the relative cytotoxicity of Silvazine with 1% silver sulphadiazine (Flamazine (Smith & Nephew Healthcare, Hull, UK)) and a silver-based dressing (Acticoat (Smith & Nephew Healthcare, Hull, UK)). METHODS: Dressings were applied to the centre of culture plates that were then seeded with keratinocytes at an estimated 25% confluence. The plates were incubated for 72 h and culture medium and dressings then removed. Toluidine blue was added to stain the remaining keratinocytes. Following removal of the dye, the plates were photographed under standard conditions and these digital images were analysed using image analysis software. Data was analysed using Student's t-test. RESULTS: In the present study, Silvazine is the most cytotoxic agent. Seventy-two hour exposure to Silvazine in the present study results in almost no keratinocyte survival at all and a highly statistically significant reduction in cell survival relative to control, Acticoat and Flamazine (P<0.001, P<0.01, P<0.01, respectively). Flamazine is associated with a statistically significant reduction in cell numbers relative to control (P<0.05), but is much less cytotoxic than Silvazine (P<0.005). CONCLUSION: In this in-vitro study comparing Acticoat, Silvazine and Flamazine, Silvazine shows an increased cytotoxic effect, relative to control, Flamazine and Acticoat. An in-vivo study is required to determine whether this effect is carried into the clinical setting.

Cytotoxicity testing of burn wound dressings, ointments and creams : a method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). METHODS: We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. RESULTS: Seventeen products were tested. The least cytotoxic products were Melolite, White soft Paraffin and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet, Mepitel((R)), PolyMem((R)), DuoDerm((R)) and Xeroform. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream a moisturizer which contains the preservative Chlorocresol. CONCLUSION: This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Hydrogels containing silver nanoparticles for burn wounds show antimicrobial activity without cytotoxicity

This research introduces a novel dressing for burn wounds, containing silver nanoparticles in hydrogels for infected burn care. The 2-acrylamido-2-methylpropane sulfonic acid sodium salt hydrogels containing silver nanoparticles have been prepared via ultraviolet radiation. The formation of silver nanoparticles was monitored by surface plasmon bands and transmission electron microscopy. The concentration of silver nitrate loaded in the solutions slightly affected the physical properties and mechanical properties of the neat hydrogel. An indirect cytotoxicity study found that none of the hydrogels were toxic to tested cell lines. The measurement of cumulative release of silver indicated that 70%–82% of silver was released within 72 hr. The antibacterial activities of the hydrogels against common burn pathogens were studied and the results showed that 5 mM silver hydrogel had the greatest inhibitory activity. The results support its use as a potential burn wound dressing.

Myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones include double-positive CD8+CD4+ T cells : evidence from myeloma-bearing mouse model

The graft-versus-myeloma (GVM) effect represents a powerful form of immune attack exerted by alloreactive T cells against multiple myeloma cells, which leads to clinical responses in multiple myeloma transplant recipients. Whether myeloma cells are themselves able to induce alloreactive T cells capable of the GVM effect is not defined. Using adoptive transfer of T naive cells into myeloma-bearing mice (established by transplantation of human RPMI8226-TGL myeloma cells into CD122(+) cell-depleted NOD/SCID hosts), we found that myeloma cells induced alloreactive T cells that suppressed myeloma growth and prolonged survival of T cell recipients. Myeloma-induced alloreactive T cells arising in the myeloma-infiltrated bones exerted cytotoxic activity against resident myeloma cells, but limited activity against control myeloma cells obtained from myeloma-bearing mice that did not receive T naive cells. These myeloma-induced alloreactive T cells were derived through multiple CD8(+) T cell divisions and enriched in double-positive (DP) T cells coexpressing the CD8alphaalpha and CD4 coreceptors. MHC class I expression on myeloma cells and contact with T cells were required for CD8(+) T cell divisions and DP-T cell development. DP-T cells present in myeloma-infiltrated bones contained a higher proportion of cells expressing cytotoxic mediators IFN-gamma and/or perforin compared with single-positive CD8(+) T cells, acquired the capacity to degranulate as measured by CD107 expression, and contributed to an elevated perforin level seen in the myeloma-infiltrated bones. These observations suggest that myeloma-induced alloreactive T cells arising in myeloma-infiltrated bones are enriched with DP-T cells equipped with cytotoxic effector functions that are likely to be involved in the GVM effect.

Isolation, structure determination and cytotoxicity studies of tryptophan alkaloids from an Australian marine sponge Hyrtios sp

Mass-guided fractionation of the MeOH extract from a specimen of the Australian marine sponge Hyrtios sp. resulted in the isolation of two new tryptophan alkaloids, 6-oxofascaplysin (2), and secofascaplysic acid (3), in addition to the known metabolites fascaplysin (1) and reticulatate (4). The structures of all molecules were determined following NMR and MS data analysis. Structural ambiguities in 2 were addressed through comparison of experimental and DFT-generated theoretical NMR spectral values. Compounds 1–4 were evaluated for their cytotoxicity against a prostate cancer cell line (LNCaP) and were shown to display IC50 values ranging from 0.54 to 44.9 μM.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver® and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver® showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

On the cytotoxicity of carbon nanotubes

The cytotoxicity of carbon nanotubes (CNTs) is a major concern today well before its unusual physicochemical, mechanical, and electrical properties are fully exploited for commercial interests and subsequent mass production leading to greater possibilities for its exposure to humans and the environment. Contradictory reports on cytotoxicity of CNTs often appear in the literature and a mechanistic explanation of the reported toxicity remains obscure. We review here the conflicting results to focus categorically on an array of issues in CNT cytotoxicity. They include dispersion, aggregation status, coating or functionalization and immobilization, cellular uptake or internalization, purity in terms of metal catalyst contaminants, size and size distribution, surface area, surface chemistry and surface reactivity, cell types selected for experimentation as well as bioassay of nanotoxicity itself attesting as an issue in cytotoxicity. Recently a general agreement has emerged towards the potential toxicity of CNTs, although various paradigms explaining the mechanisms of CNT cytotoxicity continue to be elusive in the literature. A lack of synergy among various issues while studying cytotoxicity and most developed paradigms for the mechanism of CNT toxicity is highlighted.

Synthesis of 2-(5-((5-(4-chlorophenyl)furan 2-yl)methylene)-4-oxo-2-thioxo-thiazolidi n-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells

In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a-1) were synthesized. Variation in the functional group at C-terminal of the thiazolidinone led to set of compounds bearing amide moiety. Their chemical structures were confirmed by H-1 NMR, IR and Mass Spectra analysis. These thiazolidinone compounds containing furan moiety exhibits moderate to strong antiproliferative activity in a cell cycle stage-dependent and dose dependent manner in two different human leukemia cell lines. The importance of the electron donating groups on thiazolidinone moiety was confirmed by MTT and Trypan blue assays and it was concluded that the 4th position of the substituted aryl ring plays a dominant role for its anticancer property. Among the synthesized compounds, 5e and 5f have shown potent anticancer activity on both the cell lines tested. To rationalize the role of electron donating group in the induction of cytotoxicity we have chosen two molecules (5e and 5k) having different electron donating group at different positions. LDH assay, Flow cytometric analysis and DNA fragmentation suggest that 5e is more cytotoxic and able to induce the apoptosis. (C) 2009 Elsevier Ltd. All rights reserved.

Immunologic studies on nucleic acids and their components. I. An analysis of the specificity of anti-deoxyadenylate antibodies by a membrane-binding technique

Anti-deoxyadenylate antibodies were produced in rabbits by injecting a conjugate of deoxyadenosine 5′-phosphate with bovine serum albumin. The antisera, as analyzed by double diffusion in agar and the quantitative precipitin reaction, showed hapten-specific antibodies. The specific interaction between [3H]deoxyadenylate and antiserum was studied by a sensitive nitrocellulose membrane-binding assay. The specificity of the antibodies was analyzed by measuring the effectiveness of other nucleotides or derivatives to inhibit the hapten-antibody binding. The requirements for recognition by the antibody sites were studied by using a series of naturally occurring nucleic acid components as well as some synthetic derivatives as inhibitors. The antibodies were found to show a high degree of specificity for the whole nucleotide, the base, sugar and phosphate playing almost equally important roles. There was cross reactivity with other mononucleotides, although of a low order. The antibodies were able to react with DNA and tRNA.