Different Sensitivity of Macrophages to Phospholipidosis Induction by Amphiphilic Cationic Drugs.

Phospholipidosis (PLD), the intracellular accumulation of phospholipids, is an adaptive response to toxic stimuli and serves as an important parameter in the biological assessment of compounds. Cationic amphiphilic drugs are the main inducers of PLD and may impair the function of alveolar macrophages. In vivo and in vitro models are used for PLD screening but the choice of the cellular model may be important because PLD develops in a cell- and species-specific manner. In this study, a panel of different staining (LysoSensor, Acridine Orange, Nile Red, HCS LipidTOX, LysoID) was evaluated in murine (DMBM-2, J774, RAW264.7) and human (THP-1, monocyte-derived macrophages from peripheral blood) cells to identify the most sensitive and easy to analyze staining method and to detect species-specific differences in the reaction pattern. Amiodarone and chloroquine served as inducers of PLD. High content screening was used to compare number, area, and intensity of the staining. Due to the fast staining protocol and the sensitivity of the detection, LysoID proved to be the most suitable dye of the testing. The lower induction of PLD by chloroquine reported in vivo was also seen in this study. THP-1 macrophages, followed by DMBM-2 cells, produced the most similar reaction pattern to human monocyte-derived macrophages.

Nanoparticle physicochemical properties determine the activation of intracellular complement.

The complement system plays an essential role in both innate and adaptive immunity. The traditional understanding of this system comes from studies investigating complement proteins produced by the liver and present in plasma to "complement" the immune cell-mediated response to invading pathogens. Recently, it has been reported that immune cells including, but not limited to, T-cells and monocytes, express complement proteins. This complement is referred to as intracellular (IC) and implicated in the regulation of T-cell activation. The mechanisms and the structure-activity relationship between nanomaterials and IC, however, are currently unknown. Herein, we describe a structure-activity relationship study demonstrating that under in vitro conditions, only polymeric materials with cationic surfaces activate IC in T-cells. The effect also depends on particle size and occurs through a mechanism involving membrane damage, thereby IC on the cell surface serves as a self-opsonization marker in response to the nanoparticle-triggered danger affecting the cell integrity.

Types of antiseptics, presentations and rules of use. / Tipos de antisépticos, presentaciones y normas de uso.

Antiseptics are chemical substances that when applied topically onto intact skin, mucous membranes or wounds partially or completely reduces the population of living microorganisms in those tissues. Different types of antiseptics are available - those most commonly used in clinical practice being alcohols, iodinated compounds and chlorhexidine. When using an antiseptic, consideration is required of its spectrum of antimicrobial activity, latency, residual effects, possible interferences of the presence of organic material with the activity of the antiseptic, its side effects, compatibility with other antiseptics, and cost. This article is part of a supplement entitled "Antisepsis in the critical patient", which is sponsored by Becton Dickinson.

Differential Effect of Cobalt and Chromium Ions as Well as CoCr Particles on the Expression of Osteogenic Markers and Osteoblast Function.

The balance of bone formation and resorption is the result of a regulated crosstalk between osteoblasts, osteoclasts, and osteocytes. Inflammation, mechanical load, and external stimuli modulate this system. Exposure of bone cells to metal ions or wear particles are thought to cause osteolysis via activation of osteoclasts and inhibition of osteoblast activity. Co2+ ions have been shown to impair osteoblast function and the expression of the three transforming growth factor (TGF)-ß isoforms. The current study was performed to analyze how Co2+ and Cr3+ influence the expression, proliferation, and migration profile of osteoblast-like cells. The influence of Co2+, Cr3+, and CoCr particles on gene expression was analyzed using an osteogenesis PCR Array. The expression of different members of the TGF-ß signaling cascade were down-regulated by Co2+, as well as several TGF-ß regulated collagens, however, Cr3+ had no effect. CoCr particles partially affected similar genes as the Co2+treatment. Total collagen production of Co2+ treated osteoblasts was reduced, which can be explained by the reduced expression levels of various collagens. While proliferation of MG63 cells appears unaffected by Co2+, the migration capacity was impaired. Our data may improve the knowledge of changes in gene expression patterns, and the proliferation and migration effects caused by artificial materials.

Integrative proteomics and metabolomics analysis reveals the toxicity of cationic liposomes to human normal hepatocyte cell line L02.

Cationic liposomes (CLs) are vital nonviral vectors with a wide range of applications. Although the toxicity of CLs is far lower than that of viral vectors, increasing evidence suggests that there are limited clinical applications of CLs because of their potential toxicity. In the present study, the toxicity of CLs toward L02 cells was investigated and comprehensively analyzed based on proteomics and metabolomics data. Using quantitative iTRAQ-LC-MS/MS proteomics coupled with UHPLC-Q-TOF-MS based metabolomics, we determined that exposure to CLs generated 90 significantly altered proteins and 65 altered metabolites in cells. Metabolomic analysis also showed significant alterations in metabolic pathways, including small molecules involved in energy and lipid metabolism. Proteomics revealed that exposure to CLs significantly influenced multiple proteins, including those involved in the folding of proteins and metabolism. Furthermore, the proteins participated in oxidative stress, which also influenced lipid metabolism. Overall, our findings indicate that high-throughput metabolomics and proteomics can provide insight into the toxicological mechanisms of CLs using high-resolution mass spectrometry. To our knowledge, this is the first study combining proteomics and metabolomics to investigate the potential effects of CLs on any cells. Specifically, we integrated quantitative iTRAQ-based proteomics with UHPLC-Q-TOF-MS-based metabolomics datasets to comprehensively assess the potential mechanisms of CL toxicity towards L02 cells.

In vivo toxicity of cationic micelles and liposomes.

This study investigated toxicity of nanocarriers comprised of cationic polymer and lipid components often used in gene and drug delivery, formulated as cationic micelles and liposomes. Rats were injected intravenously with 10, 25 or 100 mg/kg and sacrificed after 24 or 48 h, or 24 h after the last of three intravenous injections of 100 mg/kg every other day. Histological evaluation of liver, lung and spleen, clinical chemistry parameters, and hematology indicated little effect of treatment. DNA strand breaks were increased in the lung and spleen. Further, in the dose response study we found unaltered expression levels of genes in the antioxidant response (HMOX1) and repair of oxidized nucleobases (OGG1), whereas expression levels of cytokines (IL6, CXCL2 and CCL2) were elevated in lung, spleen or liver. The results indicate that assessment of genotoxicity and gene expression add information on toxicity of nanocarriers, which is not obtained by histology and hematology. FROM THE CLINICAL EDITOR: This study investigates the toxicity of cationic micelles and liposomes utilized as nanocarriers in gene and drug delivery, demonstrating its effects on the lungs, spleen and liver.

Evaluating the risk of phospholipidosis using a new multidisciplinary pipeline approach.

Phospholipidosis (PLD) is an undesirable potential side-effect of drugs, and cationic amphiphilic drugs (CADs) represent the main class of PLD inducers. A CADs toxicophore has been recently proposed, although the CADs definition is far from being trivial. In this work we derive a three-dimensional CADs toxicophore (here named PLD-phore) using a molecular interaction field approach, and test its suitability to discriminate between PLD inducers and non-inducers in a virtual screening approach. Ten commercially available compounds predicted to be PLD inducers and non-inducers based on their similarity to the PLD-phore were experimentally tested for PLD induction using two cell-based in vitro assays (fluorescent lipid uptake, activity of secreted lysosomal ß-hexosaminidase). When a positive effect was observed, the PLD induction was also confirmed by transmission electron microscopy. Two exceptions to the general statement about CADs and PLD induction were detected and discussed, and for one compound the cell-based in-vitro assays lead to different outcomes.

The biomolecular corona is retained during nanoparticle uptake and protects the cells from the damage induced by cationic nanoparticles until degraded in the lysosomes.

Nanoparticles have unique capacities of interacting with the cellular machinery and entering cells. To be able to exploit this potential, it is essential to understand what controls the interactions at the interface between nanoparticles and cells: it is now established that nanoparticles in biological media are covered by proteins and other biomolecules forming a "corona" on the nanoparticle surface, which confers a new identity to the nanoparticles. By labelling the proteins of the serum, using positively-charged polystyrene, we now show that this adsorbed layer is strong enough to be retained on the nanoparticles as they enter cells and is trafficked to the lysosomes on the nanoparticles. There, the corona is degraded and this is followed by lysosomal damage, leading to cytosolic release of lysosomal content, and ultimately apoptosis. Thus the corona protects the cells from the damage induced by the bare nanoparticle surface until enzymatically cleared in the lysosomes. FROM THE CLINICAL EDITOR: This study investigates the effects of protein corona that normally forms on the surface of nanoparticles during in vivo use, describing the steps of intracellular processing of such particles, to enhance our understanding of how these particles interact with the cellular machinery.

In vitro cytotoxicity of Cu²âº, Zn²âº, Ag⁺ and their mixtures on primary human endometrial epithelial cells.

BACKGROUND: To avoid the inherent disadvantages of copper-containing intrauterine device (Cu-IUD) induced by free Cu²âº, two other well-performing metal ions, namely, Ag⁺, with long-effective antimicrobial properties, and Zn²âº, as an essential trace element, are being considered for use in the future as multifunctional IUDs. The purpose of this study was to assess the cytotoxicity of these metal ions and their mixtures on primary human endometrial epithelial cells (HEECs) cultured in vitro and to provide several choices of alternative potential materials for creating excellent IUDs in the future. STUDY DESIGN: With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-formazan (MTT-f) production, the cytotoxic effects of single metal ions (Cu²âº, Zn²âº, Ag⁺) on HEECs after exposure for 24, 48 or 72 h were investigated, and the synergistic and antagonistic effects of two ions applied simultaneously were also assessed. RESULTS: The cytotoxicity of the metal ions on HEECs ranked as follows: Ag⁺>Cu²âº>Zn²âº. All combinations of those tested indicated that the Cu²âº+Zn²âº system exhibited an antagonistic effect absolutely, the Zn²âº+Ag⁺ system showed both antagonism and slight synergism, and asynergistic effect was observed in the Cu²âº+Ag⁺ system. CONCLUSION: From a perspective of favorable biocompatibility, Zn²âº and the Cu²âº+Zn²âº mixture showed evidence of potential components for use in future IUDs. Although having strong cytotoxicity, Ag⁺ with its low release rate and broad-spectrum antibiotic activity may also be considered. The study also demonstrated the relative stability of Cu²âº as a classic material of IUD.

Effects of very low fluences of high-energy protons or iron ions on irradiated and bystander cells.

In space, astronauts are exposed to radiation fields consisting of energetic protons and high atomic number, high-energy (HZE) particles at very low dose rates or fluences. Under these conditions, it is likely that, in addition to cells in an astronaut's body being traversed by ionizing radiation particles, unirradiated cells can also receive intercellular bystander signals from irradiated cells. Thus this study was designed to determine the dependence of DNA damage induction on dose at very low fluences of charged particles. Novel techniques to quantify particle fluence have been developed at the NASA Space Radiation Biology Laboratory (NSRL) at Brookhaven National Laboratory (BNL). The approach uses a large ionization chamber to visualize the radiation beam coupled with a scintillation counter to measure fluence. This development has allowed us to irradiate cells with 1 GeV/nucleon protons and iron ions at particle fluences as low as 200 particles/cm(2) and quantify biological responses. Our results show an increased fraction of cells with DNA damage in both the irradiated population and bystander cells sharing medium with irradiated cells after low fluences. The fraction of cells with damage, manifest as micronucleus formation and 53BP1 focus induction, is about 2-fold higher than background at doses as low as ∼0.47 mGy iron ions (∼0.02 iron ions/cell) or ∼70 µGy protons (∼2 protons/cell). In the irradiated population, irrespective of radiation type, the fraction of damaged cells is constant from the lowest damaging fluence to about 1 cGy, above which the fraction of damaged cells increases with dose. In the bystander population, the level of damage is the same as in the irradiated population up to 1 cGy, but it does not increase above that plateau level with increasing dose. The data suggest that at fluences of high-energy protons or iron ions less than about 5 cGy, the response in irradiated cell populations may be dominated by the bystander response.

Synthesis and characterization of a novel cationic polymer gene delivery vector.

Non-viral vectors have been widely used in gene transfection. However, its drawbacks limit its applications. In this study, a novel cationic polymer was developed as a DNA condensing agent for systemic gene delivery. Its transfection efficiency, cytotoxicity, and biocompatibility were also evaluated. Sofast, novel cationic polymer of branched polyethlenimine, was constructed by chemical methods. Its diameter, zeta potential, nucleic acid binding ability, and anti-nuclease ability were detected by electron microscopy and gel electrophoresis. In vitro, the efficiency of transfection was measured by comparing it with other gene vectors in different cell lines. MTT assay was performed to determine cytotoxicity. The compatibility of Sofast gene vector in the serum and its stability were investigated. Mouse, guinea pig and rabbit were used to process the toxic, allergenic, and pyrogenic properties of the vector in vivo. The in vivo expression was performed in the guinea pig. The results from an in vitro assay proved that the Sofast gene vector had a higher transfection efficiency than other gene vectors in a variety of primary cell cultures and transformed cell lines. The cytotoxicity assay showed a lower cytotoxicity and the cellular survival rate was >90%. The Sofast gene vector possessed compatibility with the serum and was fit to be transported at normal temperature. The results from in vivo tests indicated that the Sofast gene vector had greatly lower cytotoxicity, better biocompatibility, and higher transfection efficiency compared with other gene vectors. Because the Sofast gene vector had higher transfection efficiency, lower cytotoxicity and better compatibility than other gene vectors, it could be used for gene transfection both in vitro and in vivo.

Drug labeling; orally ingested over-the-counter drug products containing calcium, magnesium, and potassium. Final rule.

The Food and Drug Administration (FDA) is amending the general labeling provisions for over-the-counter (OTC) drug products to require that the labeling of all OTC drug products intended for oral ingestion include: The calcium content per dosage unit when the product contains 20 milligrams (mg) or more per single dose; a warning statement that persons with kidney stones and persons on a calcium-restricted diet should ask a doctor before using when the product contains more than 3.2 grams (g) of calcium in the labeled maximum daily dose; the magnesium content per dosage unit when the product contains 8 mg or more per single dose; a warning statement that persons with kidney disease and persons on a magnesium-restricted diet should ask a doctor before using if the product contains more than 600 mg magnesium in the labeled maximum daily dose; the potassium content per dosage unit when the product contains 5 mg or more per single dose; and a warning statement that persons with kidney disease and persons on a potassium restricted diet should ask a doctor before using if the product contains more than 975 mg potassium in the labeled maximum daily dose. FDA is issuing this final rule in order to provide uniform calcium, magnesium, and potassium content and warning labeling for all OTC drug products intended for oral ingestion whether marketed under an OTC drug monograph, the ongoing OTC drug review, a new drug application (NDA) or abbreviated new drug application (ANDA), or no application.

Self-assembled ternary complex of cationic dendrimer, cucurbituril, and DNA: noncovalent strategy in developing a gene delivery carrier.

A ternary complex of PPI-DAB dendrimer [(1,4-diaminobutane); Gen = N; dendri-poly(propyleneimine); -[NHC(=O)CH(2)NH(2)(+)(CH(2))(4)NH(3)(+)](z)()], DNA, and cucurbituril (CB) was evaluated as an example of a totally self-assembled gene delivery carrier. The complex was formed in a noncovalent way in which DNA interacts with PPI-DAB electrostatistically and CB with PPI-DAB through multiple noncovalent interactions. Dynamic light scattering data indicated that the diameter and size distributions of the complexes were dependent upon the sequence of mixing of each component with unimodal distribution ranging from 150.8 to 210.2 nm under favorable conditions. Fluorescence studies showed the quantitative binding of CB to PPI-DAB after ternary complex formation. The complex was able to transfect mammalian cells with high efficiency and the cytotoxicity of the PPI-DAB/CB complex was relatively low.

A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis.

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Cationic liposome-mediated gene delivery to the liver and to hepatocellular carcinomas in mice.

The potential of cationic liposomes as nonviral vectors for in vivo gene delivery to the liver and to intrahepatic hepatocellular carcinoma (HCC) was investigated. Mice were injected via the tail vein or portal vein with a cationic lipid complexed to plasmid DNA (pDNA) encoding the chloramphenicol acetyltransferase (CAT) reporter gene at various cationic lipid:pDNA molar ratios to analyze the efficiency of gene delivery after intravenous administration. Tail vein injection resulted in high CAT expression levels in lung and spleen and low levels in the liver. Portal vein injection, by comparison, significantly enhanced hepatic reporter gene expression but also resulted in pronounced hepatic toxicity. Gene delivery to intrahepatic tumors produced by intrahepatic injection of human HCC cells was analyzed in nude mice. Tail vein injection as well as portal vein injection resulted in low levels of gene expression in intrahepatic tumors. By comparison, high levels of gene expression were achieved by direct, intratumoral injection of liposome-pDNA complexes, with only minimal expression in the surrounding normal liver. Therefore, direct liposome-pDNA complex injection appears far superior to systemic or portal intravenous administration for gene therapy of localized intrahepatic tumors, and may be a useful adjunct in the treatment of human HCCs.

Flame atomic absorption spectrometric determination of trace chromium in various standard samples after preconcentration with 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol.

Chromium can be quantitatively retained as 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP)-tetra-phenylborate(TPB) complex onto microcrystalline naphthalene in the pH range 4.8-5.9 from a large volume of aqueous solutions of various standard samples. After filtration, the solid mass consisting of the chromium complex and naphthalene was dissolved with 5 mL of dimethylformamide and the metal was determined by air-acetylene FAAS. A detection limit of 4 ng/mL for chromium was established. The interference of a large number of anions and cations has been studied and the optimized conditions developed were utilized for the trace determination of chromium in various standard alloys and biological samples.

Preparation and characterization of cationic microspheres for gene delivery.

The production and characterization of cationic microparticles based on Eudragit RS and cationic agents (i.e. a cationic acrylic polymer and three different cationic surfactants) for the delivery of nucleic acids is here described. It was found that morphological and dimensional characteristics of microparticles were influenced by the type and concentration of cationic agent employed and by some experimental parameters such as stirring speed, emulsifying agent and type of rotor. The desoxiribonucleotide Defibrotide (DFT) was associated with positively charged microparticles and its in vitro release kinetics from microparticles were determined. A study of the in vitro toxicity of cationic microparticles on cultured human cell line K562 was also performed, demonstrating that DDAB(18) microparticles display very low cytotoxicity.

Modification of human LDL by in vitro incubation with cigarette smoke or copper ions: implications for allergies, asthma and atherosclerosis.

Cigarette smoking has been linked to a higher risk of not only atherosclerosis and related diseases, but asthma and allergies as well. The mechanisms linking smoking to these diseases may be due in part to increased low density lipoprotein (LDL) modification. In the current study, we compared the modification in vitro of LDL isolated from healthy volunteers that had been exposed to either the gas phase of cigarette smoke or copper ion mediated oxidation. The study used as measures of modification/damage the levels of protein carbonyl groups, changes in electrophoretic mobility on agarose gel electrophoresis and levels of thiobarbituric-reacting substances. Other measures used to assess other aspects of LDL modification included SDS PAGE and immunoblotting. Both copper ion or exposure to the gas phase of cigarette smoke increased electrophoretic mobility of LDL but the increase was greater in the gas phase smoke group. In contrast, thiobarbituric reacting substance levels were increased primarily in copper oxidized LDL. Protein carbonyl levels were increased to a similar extent in both copper ion and smoke exposed samples. Addition of EDTA prevented the modifications found upon copper mediated oxidation of LDL, but EDTA did not prevent the modification of the gas phase cigarette smoke exposed LDL. In summary, the results indicate that protein carbonyl formation can be used as a measure of the modification of LDL particles and, using several different assessment techniques, there are distinct differences in the modified LDL produced by in vitro incubation with gas phase cigarette smoke relative to that found upon incubation of LDL with copper ion. The in vitro smoking-produced LDL modifications may potentially be relevant to the process of lipoprotein modification in vivo and to the subsequent biological effects of these modified lipoproteins on processes affected by the immune system involvement, such as atherosclerosis and allergy/asthma.

Cations in the didanosine tablet reduce ciprofloxacin bioavailability.

The effect of the magnesium-aluminum cations contained in didanosine chewable tablets on ciprofloxacin pharmacokinetics was evaluated in 12 healthy volunteers. The study was designed as a randomized, balanced, open, two-period, two-treatment crossover trial with a 7-day washout period between treatments. In one phase, subjects received a single 750 mg ciprofloxacin tablet alone. In the didanosinecation regimen, subjects received two didanosine-placebo tablets every 12 hours for two doses. On day 2, they received two didanosine-placebo tablets immediately followed by a single 750 mg ciprofloxacin tablet. Serial blood samples were collected for 24 hours after administration of each ciprofloxacin dose. The average maximum concentration of ciprofloxacin alone was 3.38 +/- 0.63 (SD) micrograms/ml compared with 0.25 +/- 0.21 (SD) micrograms/ml when ciprofloxacin was administered with the didanosine placebo (p < 0.0001). The mean (+/- SD) area under the plasma drug concentration-time curve from time 0 to the last measurable concentration for ciprofloxacin alone was 15.50 +/- 2.69 micrograms.hr/ml compared with 0.26 +/- 0.21 micrograms.hr/ml when ciprofloxacin was coadministered with the didanosine-placebo (p < 0.0001). The mean time to maximum concentration of ciprofloxacin alone decreased from 1.56 +/- 0.62 to 0.75 +/- 0.38 hours with buffer administration (p = 0.0012). The simultaneous administration of ciprofloxacin and didanosine should be avoided.

Cations and anions in drinking water as putative contributory factors to endemic goitre in Plateau State, Nigeria.

The prevalence of endemic goitre in Plateau State, Nigeria was established and an attempt was made to identify some of the possible environmental goitrogenic agents in the region to establish their likely relationship with the goitre endemicity. Iodine deficiency appears to be a major aetiological factor for the disease as indicated by low iodine levels observed in portable drinking water and in daily urinary excretion. The carbonate (CO3-) content of drinking water supply was found to bear a significant positive correlation with the goitre rate for the entire state (p less than 0.005). The calcium (Ca++) and magnesium (Mg++) levels of the drinking water also exhibited relatively good linear direct correlations with the percentage goitre distribution in a region, nearly 2/3 of the state. It is concluded that there is possibly an interplay of several factors and in particular the carbonate content of drinking water which, in association with a state of iodine deficiency, may be regarded as responsible for the goitre endemic seen in this part of the Continental Africa.