HGPRT and PNP: Recombinant Enzymes from Schistosoma mansoni and Their Role in Immunotherapy during Experimental Murine Schistosomiasis.

Schistosomiasis is a parasitic infection caused by trematode worms (also called blood flukes) of the genus Schistosoma sp., which affects over 230 million people worldwide, causing 200,000 deaths annually. There is no vaccine or new drugs available, which represents a worrying aspect, since there is loss of sensitivity of the parasite to the medication recommended by the World Health Organization, Praziquantel. The present study evaluated the effects of the recombinant enzymes of S. mansoni Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT), Purine Nucleoside Phosphorylase (PNP) and the MIX of both enzymes in the immunotherapy of schistosomiasis in murine model. These enzymes are part of the purine salvage pathway, the only metabolic pathway present in the parasite for this purpose, being essential for the synthesis of DNA and RNA. Female mice of Swiss and BALB/c strains were infected with cercariae and treated, intraperitoneally, with three doses of 100 µg of enzymes. After the immunotherapy, the eggs and adult worms were counted in the feces; the number of eosinophils from the fluid in the peritoneal cavity and peripheral blood was observed; and the quantification of the cytokine IL-4 and the production of antibodies IgE was analyzed. The evaluation of the number of granulomas and collagen deposition via histological slides of the liver was performed. The results demonstrate that immunotherapy with the enzyme HGPRT seems to stimulate the production of IL-4 and promoted a significant reduction of granulomas in the liver in treated animals. The treatment with the enzyme PNP and the MIX was able to reduce the number of worms in the liver and in the mesenteric vessels of the intestine, to reduce the number of eggs in the feces and to negatively modulate the number of eosinophils. Therefore, immunotherapy with the recombinant enzymes of S. mansoni HGPRT and PNP might contribute to the control and reduction of the pathophysiological aspects of schistosomiasis, helping to decrease the morbidity associated with the infection in murine model.

Cytotoxic Effects Caused by Functionalized Carbon Nanotube in Murine Macrophages.

BACKGROUND/AIMS: The development of new nanomaterials has been growing in recent decades to bring benefits in several areas, especially carbon-based nanoparticles, which have unique physical-chemical properties and allow to take on several applications. Consequently, the use of new nanomaterials without previous toxicological studies raises concern about possible harmful health effects. The aim of this study was to investigate the cytotoxic profile of a new multi-walled carbon nanotube (MWCNT) functionalized with tetraethylenepentamine called OCNT-TEPA using in vitro assays in murine macrophage cells linage J774 A.1. METHODS: OCNT-TEPA was characterized by transmission electron microscopy (TEM) and high resolution TEM (HR-TEM), scanning electron microscopy (SEM), zeta potential and dynamic light scattering (DLS), and its cytotoxic effects were evaluated at 24 and 48 hours by cell viability assays (MTT and NR), morphology and cell recovery (optic microscopy and clonogenic assay), formation of reactive oxygen (ROS) and nitric oxide (NO) species, inflammatory profile (IL-6 and TNF cytokines), mitochondrial membrane potential analysis (MMP), activation of the caspase 3 pathway and cell death (flow cytometry). RESULTS: The data showed a significant decrease in cell viability, increased production of ROS and NO, alteration of mitochondrial membrane potential, increased levels of inflammatory cytokines, alteration of cell morphology, activation of the Caspase 3 pathway and consequently cell death, in the highest concentrations of OCNT-TEPA tested in the periods of 24 and 48 hours. CONCLUSION: The analyses showed that OCNT-TEPA has a dose-dependent cytotoxic profile, which may be harmful to murine macrophages (J774 A.1) and may represent a health risk.

Bactericidal activity of Ag4V2O7/ß-AgVO3 heterostructures against antibiotic-resistant Klebsiella pneumoniae.

Although Ag-based materials are efficient against antibiotic-resistant bacteria, their high toxicity to living organisms represents a major challenge for obtaining useful products. In this work, we report the bactericidal activity of Ag4V2O7/ß-AgVO3 heterostructures, which proved to be effective against Klebsiella pneumoniae (ATCC 1706, a standard strain; A54970, a multidrug-resistant carbapenemase (KPC)-producing strain; A34057, a multidrug-resistant strain capable of producing extended spectrum beta-lactamases (ESBL); and a community-isolated strain, A58240) at minimum inhibitory concentrations (MIC) as low as 62.5 µg/mL. This activity is higher than that reported for the individual silver vanadates (Ag4V2O7 or ß-AgVO3) owing to the synergistic interactions between both semiconductors. However, the most efficient heterostructure was found to be toxic to mouse 3 T3 fibroblasts and to L. sativa and C. sativus seeds, as indicated by MTT ((4,5 - dimethylthiazol -2yl) 2,5 -diphenylbromide), neutral red assays and germination index measurements. The antimicrobial, phytotoxic and cytotoxic activities were all associated with an efficient generation of reactive oxygen species (ROS) in the heterostructure, especially OH and O2- radicals. The ROS production by Ag4V2O7/ß-AgVO3 heterostructures was measured through photodegradation studies with Rhodamine B. While the bactericidal activity of the heterostructures is promising, especially when compared to Ag-based materials, their use in practical applications will require encapsulation either to avoid leaching or to mitigate their toxicity to humans, animals and plants.

Toxicological effects of the mixed iron oxide nanoparticle (Fe3O4 NP) on murine fibroblasts LA-9.

The increase in large-scale production of magnetic nanoparticles (NP) associated with the incomplete comprehensive knowledge regarding the potential risks of their use on environmental and human health makes it necessary to study the biological effects of these particles on organisms at the cellular level. The aim of this study to examine the cellular effects on fibroblast lineage LA-9 after exposure to mixed iron oxide NP (Fe3O4 NP). The following analyses were performed: field emission gun-scanning electron microscopy (SEM-FEG), dynamic light scattering (DLS), zeta potential, ultraviolet/visible region spectroscopy (UV/VIS), and attenuated total reactance-Fourier transform infrared (ATR-FTIR) spectroscopy analyses for characterization of the NP. The assays included cell viability, morphology, clonogenic potential, oxidative stress as measurement of reactive oxygen species (ROS) and nitric oxide (NO) levels, cytokines quantification interleukin 6 (IL-6) and tumor necrosis factor (TNF), NP uptake, and cell death. The size of Fe3O4 NP was 26.3 nm when evaluated in water through DLS. Fe3O4 NP did not reduce fibroblast cell viability until the highest concentration tested (250 µg/ml), which showed a decrease in clonogenic potential as well as small morphological changes after exposure for 48 and 72 hr. The NP concentration of 250 µg/ml induced enhanced ROS and NO production after 24 hr treatment. The uptake assay exhibited time-dependent Fe3O4 NP internalization at all concentrations tested with no significant cell death. Hence, exposure of fibroblasts to Fe3O4 NP-induced oxidative stress but not reduced cell viability or death. However, the decrease in the clonogenic potential at the highest concentration demonstrates cytotoxic effects attributed to Fe3O4 NP which occurred on the 7th day after exposure.

Functionalized Titanium Nanoparticles Induce Oxidative Stress and Cell Death in Human Skin Cells.

Purpose: Nanoparticles are resources of advanced nanotechnology being present in several products. Titanium dioxide nanoparticles are among the five most widely used NP currently expanding their benefits from the oil industry to the areas of diagnostic medicine due to their properties and small size. However, its impact on human health is still controversial in the literature. We aimed to evaluate the cytotoxicity of a new titanium NP functionalized with sodium carboxylic ligand (COOH-Na+) in human keratinocytes (HaCaT) and human fibroblasts (HDFn). Methods: The physical-chemical characterization was performed by the transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential techniques, respectively. MTT and LDH assays were used to assess cytotoxicity and cell membrane damage respectively, ELISA to identify the inflammatory profile and, reactive oxygen species assay and cytometry to detect reactive oxygen species and their relationship with apoptosis/necrosis mechanisms. Results: The results demonstrated a decrease in cell viability at the highest concentrations tested for both cell lines, but no change in LDH release was detected for the HaCaT. The cell membrane damage was found only at 100.0 µg/mL for the HDFn. It was demonstrated that cytotoxicity in the highest concentrations evaluated for both cell lines for the 72 h period. The HDFn showed damage to the cell membrane at a concentration of 100 µg/mL followed by a significant increase in reactive oxygen species production. No inflammatory profile was detected. The HaCaT showed apoptosis when exposed to the highest concentration evaluated and HDFn showed both apoptosis and necrosis for the same concentration. Conclusion: Thus, it is possible to conclude that the cytotoxicity mechanism differs according to the cell type evaluated, with HDFn being the most sensitive line in this case, and this mechanism can be defined in a dose and time dependent manner, since the highest concentrations also triggered death cell.

Analysis of cytotoxicity and genotoxicity in a short-term dependent manner induced by a new titanium dioxide nanoparticle in murine fibroblast cells.

The extensive use of titanium dioxide nanoparticles (TiO2 NPs) in cosmetics, food, personal care products, and industries brought concerns about their possible harmful effects. Nowadays it has become important to assess TiO2 NPs toxic effects as a way to understand their primary risks. In the cellular environment, after cell uptake, TiO2 NPs were described to induce reactive oxygen species (ROS) production, unbalance oxidative state, and activate apoptosis in several cell lines. Therefore, we aimed to evaluate the cytotoxicity and genotoxicity of a new TiO2 NP surface-functionalized with sodium carboxylic ligands in a murine fibroblast cell line (LA-9). TEM and DLS analyses were performed to define nanoparticle physicochemical characteristics. We evaluated the metabolic activity and LDH released after 24 h exposition to determine cytotoxic effects. Also, we evaluated DNA damage, intracellular reactive oxygen species (ROS) production, and apoptosis induction after 24 h exposure. The TiO2 NP impaired the cell membrane integrity at 1000 µg/mL, induced intracellular ROS production and late apoptosis at 24 h. The genotoxic effects were observed at all conditions tested at 24 h. Indeed, in fibroblasts exposed at 100 µg/mL was observed early apoptosis cells. The intracellular ROS content was increased in a dose-dependent manner. Thus, short-term exposure to TiO2 NP promoted cytotoxicity, genotoxicity and activated apoptosis pathways based on the potential role of oxygen species in the fibroblasts cell line.

Antitumor Effect of IL-2 and TRAIL Proteins Expressed by Recombinant Salmonella in Murine Bladder Cancer Cells.

BACKGROUND/AIMS: Cancer is the second most deadly disease in the world. The bladder cancer is one of the most aggressive types and shows a continuous increase in the number of cases. The use of bacteria as live vectors to deliver molecules directly to the tumor is a promising tool and has been used as an adjuvant treatment against several types of cancer. The aim of this study was to investigate the antitumor effect of Interleukin 2 (IL-2), TNF-related apoptosis-inducing ligand (TRAIL) and protein MIX against murine bladder cancer cells, lineage MB49. METHODS: The attenuated Salmonella strain SL3261 was transformed by inserting the IL-2 and TRAIL genes. The effects of proteins on cell viability (MTT method), cell morphology (optical microscopy), cell recovery (clonogenic assay), cell membrane (lactate dehydrogenase release - LDH), on oxidative stress pathway (levels of nitric oxide, NO) and apoptosis (flow cytometry and high resolution epifluorescence images) were evaluated at intervals of 24 and 48 hours of action. RESULTS: The results showed that there was a decrease in cell viability via damage to the cell membrane, alteration of cell morphology, non-recovery of cells, increase in the production of NO and incubate for of cells in the state of apoptosis in the two periods analyzed. CONCLUSION: The data presented suggest that IL-2, TRAIL and their MIX proteins in MB49 cells have cytotoxic potential and that this is associated with oxidative stress and apoptosis pathways. These results may contribute to the development of new therapeutic strategies for bladder cancer.

Apoptosis and Oxidative Stress Triggered by Carbon Black Nanoparticle in the LA-9 Fibroblast.

BACKGROUND/AIMS: A new type of nanoparticle, called NP CB-EDA (Black Carbon modified with ethylenediamine), is commonly used in the oil industry. In the literature, few studies are found in biological models, making NP-EDA potential cytotoxicity in organisms unclear. As its large surface area is capable of interacting with the biological system, that interaction could lead to factors harmful to health. The objective of this study was to investigate the cytotoxic effect of NP CB-EDA on fibroblasts LA-9 at 24 and 48 hours, at different concentrations of the nanoparticle (1, 50, 250, 500 and 1000 µg/ml). METHODS: NP CB-EDA was characterized by TEM microscopy and its effect on cell viability (MTT method), cell morphology (optical microscopy), cell membrane (lactate dehydrogenase release - LDH), oxidative stress pathways (species levels reactive oxygen, ROS and nitrogen, NOS) and apoptosis/necrosis (flow cytometry) were evaluated. RESULTS: The results show that NP CB-EDA at concentrations of 500 and 1000 µg/ml form clusters. The nanoparticle can be absorbed by cells decreasing cell viability. There was damage to the cell membrane of fibroblasts LA 9, an increase in the production of ROS, NOS and pro-inflammatory interleukins TNF-α and IL-6; it was also observed an increase in % of cells in the state of apoptosis in the two periods analyzed, being this response more significant in 24 hours, and concentrations of 250, 500 and 1000 µg/ml presenting higher cytotoxicity. CONCLUSION: The data suggest that NP CB-EDA in fibroblasts LA9 presents cytotoxic potential, which is associated with oxidative stress and apoptosis.

New Multi-Walled carbon nanotube of industrial interest induce cell death in murine fibroblast cells.

The search for new nanomaterials has brought to the multifactorial industry several opportunities for use and applications for existing materials. Carbon nanotubes (CNT), for example, present excellent properties which allow us to assume a series of applications, however there is concern in the industrial scope about possible adverse health effects related to constant exposure for inhalation or direct skin contact. Thus, using cell models is the fastest and safest way to assess the effects of a new material. The aim of this study was to investigate the cytotoxic profile in LA9 murine fibroblast lineage, of a new multi-walled carbon nanotube (MWCNT) that was functionalized with tetraethylenepentamine (TEPA) to obtain better physical-chemical characteristics for industrial use. The modifications presented in the CNT cause concern, as they can change its initial characteristics, making this nanomaterial harmful. HR-TEM, FE-SEM and zeta potential were used for the characterization. Cytotoxicity and cell proliferation tests, oxidative and nitrosative stress analyzes and inflammatory cytokine assay (TNF-α) were performed. The main findings demonstrated a reduction in cell viability, increased release of intracellular ROS, accompanied by an increase in TNF-α, indicating an important inflammatory profile. Confirmation of the data was performed by flow cytometry and ImageXpress with apoptosis/necrosis markers. These data provide initial evidence that OCNT-TEPA has a cytotoxic profile dependent on the concentration of LA9 fibroblasts, since there was an increase in free radicals, inflammation induction and cell death, suggesting that continuous exposure to this nanoparticle can cause damage to different tissues in the organism.