Recognition of chemical compounds in contaminated water using time-dependent multiple dose cellular responses.

An early determination of toxicant compounds of water contaminations can gain critical time to protect citizens' health and save substantial amounts of medical costs. To determine toxins in real time, a multi-dose classification algorithm using cellular state variable identification (CSVID) is developed in this paper. First, the dynamic cytotoxicity response profiles of living cells are measured using a real-time cell electronic sensing (RT-CES) system. Changes in cell number expressed as cell index (CI) are recorded on-line as time series. Then CSVID, which reflects the cell killing, cell lysis and certain cellular pathological changes, is extracted from those dynamic cellular responses. Finally, a support vector machine (SVM) algorithm based on CSVID is employed to classify chemical compounds and determine their analogous cellular response pathway. In order to increase the classification accuracy, a majority vote of the class labels is also proposed. Several validation studies demonstrate that CSVID-based classification algorithm has great potential in distinguishing the cytotoxicity response of the cells in the presence of toxins.

Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory CD4+CD25+ T lymphocytes in the peripheral blood of patients with advanced renal carcinoma.

OBJECTIVE: To analyze the effect of Argon-Helium cryosurgery (AHCS) combined with transcatheter renal arterial embolization (TRAE) on the differentiation of regulatory CD4+ CD25+ T cell (Treg) and its implication in patients with renal carcinoma. METHODS: Seventy seven patients are included in the study, and divided into two groups: TRAE group (n=45, receiving TRAE only) and TRAE+cryoablation group (n=32, receiving cryoablation 2-3 weeks after TRAE). The percentage of Treg cells and T lymphocyte subsets (CD4+T, CD8+T, and CD4+T/CD8+T) in the peripheral blood is measured by flow cytometry previous to the therapy and 3 months after therapy. Meanwhile, the extent of tumor necrosis is measured by MRI or CT 1 month after therapy. RESULTS: The percentages of Treg cells of patients in TRAE + cryoablation group decrease from (6.65±1.22)% to (3.93±1.16)%, (t=42.768, P<0.01), and the percentages of CD4+T and CD4+T/CD8+T increase significantly (P<0.01). However, the results of patients in TRAE group show that the percentages of Treg, CD4+T, CD8+T and CD4+T/CD8+T increase slightly although the differences had no statistical significance (P>0.05). The tumor necrosis rate of TRAE+cryoablation group is 57.5%, significantly higher than those of TRAE group, which shows 31.6% (t=6.784, P<0.01). The median survival duration of the TRAE+cryoablation group is 20 months, significantly longer than that of the TRAE group (χ² = 7.368, P<0.01). The decreasing extent of Treg cells is correlated with tumor necrosis rates (r=0.90, P<0.01) and life time (r=0.67, P<0.01). CONCLUSION: The therapy of TRAE combined with cryoablation contributes to reduce the percentage of Treg cells and improve the immune situation of patients with renal cell carcinoma, which consequently increase tumor necrosis rate and prolong the patients' survival duration.

Microelectronic-sensing assay to detect presence of Verotoxins in human faecal samples.

AIMS: To develop a novel Vero cell assay that implements a real-time cell electronic sensing (RT-CES) system for the determination of the presence of verotoxin-producing Escherichia coli (VTEC). The assay overcomes the major drawbacks in conventional Vero cell assay, for example, labour-intensive and time-consuming. METHODS AND RESULTS: Cells were grown onto the surfaces of microelectronic sensors that are integrated into the bottom surfaces of the microtiter plate. Cellular viability was monitored in real-time and quantified based on changes in the sensor's electrical impedance. For cell viability measurement, the data generated on the RT-CES system correlated well with those obtained by the Vero cell assay for Verotoxins. To assess cytotoxicity, test cells growing on microelectronic sensors were treated with either supernatant from pure cultures, or stool samples. The specific neutralizing antibodies of VT1 and VT2 were used to identify specific toxins in the samples. CONCLUSIONS: The RT-CES assay provides a sensitive measurement comparable to conventional crystal violet assay. The assay has been successfully and specifically used to identify VTEC in human faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The RT-CES assay significantly shortens the testing time from 48 to 72 h required by the crystal violet assay to only 15 h with automated operation.

Cytogenetic effects induced by accelerated carbon ions with shielding.

Our work aims to understand the effects of shielding on the induction of biological damage by heavy charged particles and to compare the shielding effects of different materials at the same LET from two aspects: the biological effectiveness including or not including secondary particles emitted at large angles and the biological effectiveness at different angles with respect to the beam direction. We designed and conducted biological experiments to determine the biological effectiveness of 200 MeV/u carbon ions after traversing different shielding materials (Lucite and aluminium). Whole blood samples, which were either attached to the shielding material (48 mm Lucite or 29 mm aluminium)or positioned at 300 cm away from it at different angles with respect to the beam axis, were exposed to carbon ion beams. For comparison, whole blood samples were exposed directly to 200 MeV/u carbon ions. Chromosomal aberrations in lymphocytes were scored. The results indicated that the biological effectiveness per unit dose was not significantly changed by 48 mm Lucite or 29 mm aluminium, and no significant differences were observed in lymphocytes attached to the target and in lymphocytes positioned at a distance of 300 cm away from the target, at 0º angle of the beam axis. However, when plotted as a function of the number of ions hitting the shielding target, the curves are separated and the shield increases the effectiveness per unit ion. The frequency of chromosomal aberrations at tilted angles behind 29 mm Al and 48 mm Lucite was almost the same. These lesions were considered to be caused by secondary particles due to the passage of particles through the shielding materials.

Data-based modeling and prediction of cytotoxicity induced by contaminants in water resources.

This paper is concerned with dynamic modeling, prediction and analysis of cell cytotoxicity induced by water contaminants. A real-time cell electronic sensing (RT-CES) system has been used for continuously monitoring dynamic cytotoxicity responses of living cells. Cells are grown onto the surfaces of the microelectronic sensors. Changes in cell number expressed as cell index (CI) have been recorded on-line as time series. The CI data are used to develop dynamic prediction models for cell cytotoxicity process. We consider support vector regression (SVR) algorithm to implement data-based system identification for dynamic modeling and prediction of cytotoxicity. Through several validation studies, multi-step-ahead predictions are calculated and compared with the actual CI obtained from experiments. It is shown that SVR-based dynamic modeling has great potential in predicting the cytotoxicity response of the cells in the presence of toxicant.

Neuroprotective effects of R,R-tetrahydrochrysene against glutamate-induced cell death through anti-excitotoxic and antioxidant actions involving estrogen receptor-dependent and -independent pathways.

Glutamate-induced neural cell death is mediated by excitotoxicity and oxidative stress. Treatment of glutamate toxicity with estrogen and its related compounds for neuroprotection remains controversial. In this study, we examined the effects of selective estrogen receptor (ER) ligands on glutamate toxicity and found that R,R-tetrahydrochrysene (R,R-THC), an antagonist of ERbeta and agonist of ERalpha, has neuroprotective effects against glutamate-induced death in primary rat cortical cells and mouse N29/4 hypothalamic cells. The protective effect of R,R-THC was dose-dependent and was maintained even when added several hours after the initial glutamate exposure. R,R-THC blocked glutamate-induced depletion of intracellular glutathione, increased superoxide dismutase activity, and protected cells from hydrogen peroxide-induced death. R,R-THC also prevented glutamate-induced nuclear translocation of apoptotic inducing factor and release of mitochondrial cytochrome c. The protective effect of R,R-THC was blocked by methyl-piperidino-pyrazole (MPP; an ERalpha antagonist) in glutamate-treated cortical cells, and pretreatment with MK-801 (an NMDA receptor antagonist) but not CNQX (an AMPA/kainate receptor antagonist) increased cell survival. On the other hand, MPP did not block the protective effect of R,R-THC in glutamate-treated N29/4 cells, and neither MK-801 nor CNQX conferred protection. Activation of ERalpha and/or ERbeta with 17beta-estradiol (E2), propyl-pyrazole-triol or diarylpropionitrile did not provide effective neuroprotection, and pretreatment with ICI 182,780 did not inhibit the protective effect of R,R-THC in either type of cell. These results suggest that the use of ER agonists (including E2) has limited beneficial effects when both excitotoxicity and oxidative stress occur. In contrast to agonists of ERs, R,R-THC, which possesses anti-excitotoxic and antioxidant actions via ER-dependent and -independent pathways, provides significant neuroprotection.

Factors influencing the removal of thymine glycol from DNA in gamma-irradiated human cells.

The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is > 80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before repair commences. However, if cells are irradiated with 0.25 Gy 4 h prior to the 2-Gy dose, removal of the thymine glycols commences immediately after the second irradiation, suggesting that repair of thymine glycol is inducible. Our current studies are directed at two aspects of the repair process, (1) factors involved in the repair process leading up to and including glycosylase-mediated removal of thymine glycol and (2) the control of the inducible response. We have observed that mutation of the XPG gene drastically reduced the level and rate of global removal of thymine glycol (induced by 2-Gy irradiation), and there was no evidence for an inducible response. Similar results were seen with a Cockayne syndrome B (CSB) cell line. We have also examined repair in quiescent and phytohemagglutinin-stimulated human lymphocytes. Both show similar kinetics for the rate of removal of thymine glycol under induced and noninduced conditions.

Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection.

Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. Sensitive techniques are required for measuring cellular levels of DNA damage. We describe in detail a novel immunoassay that makes use of the resolving power of capillary electrophoresis and the sensitivity of laser-induced fluorescence detection. An example is given of the detection of thymine glycol in DNA produced by irradiation of human cells with a clinical dose of 2 Gy. A detection limit of approximately 10(-21) mol allowed us to monitor the repair of the lesion and to suggest that the cellular repair response may be inducible.

Competitive immunoassay for cyclosporine using capillary electrophoresis with laser induced fluorescence polarization detection.

Frequent monitoring of immunosuppressive drug cyclosporine A (CsA) in blood samples of tissue transplant patients is required in clinical practice because of the narrow therapeutic range between the immunosuppressive effect and the toxic effect of this drug. We describe a competitive immunoassay capillary electrophoresis (CE) with laser induced fluorescence polarization detection method, which is rapid and sensitive for the determination of CsA. The method is based on the competitive immunochemical reaction between the analyte and fluorescent hapten (CsA*) with the antibody, CE separation of the antibody bound and free fluorescent CsA*, followed by the laser induced fluorescence polarization detection (LIFP) of the fluorescent species. The method detection limit is governed by the stability of the antibody-CsA* complex rather than by the detector noise. The use of post-column sheath flow cuvette LIFP detection resulted in excellent detection limit, typically 0.9 nM (or 9.10(-19) mol for 1 nl injection) of CsA. CsA in whole blood samples from organ transplant patients were measured and results agreed well with those obtained by using a standard fluorescence polarization immunoassay. Each determination took less than 3 min. The CsA metabolites AM9 and AM19 were also determined by using this technique, and their cross-reactivities with the antibody were 13% and 2%, respectively.

Inducible repair of thymine glycol detected by an ultrasensitive assay for DNA damage.

An ultrasensitive assay for measuring DNA base damage is described that couples immunochemical recognition with capillary electrophoresis and laser-induced fluorescence detection. The method provides a detection limit of 3 x 10(-21) moles, an improvement of four to five orders of magnitude over current methods. Induction and repair of thymine glycols were studied in irradiated A549 cells (a human lung carcinoma cell line). Exposure of these cells to a low dose of radiation (0.25 Gray) 4 hours before a clinically relevant dose (2 Gray) enhanced removal of thymine glycols after the higher dose. These data provide evidence for an inducible repair response for radiation-induced damage to DNA bases.

Excipient interaction with cetylpyridinium chloride activity in tablet based lozenges.

PURPOSE: The purpose of the investigation was to determine the effect of tablet excipients on the activity of cetylpyridinium chloride (CPC) and the relative interaction between excipients and CPC. METHODS: An analytical assay was developed to evaluate the interaction between CPC and the excipients. In vivo activity was investigated using six volunteers by determining the reduction in colony forming units recoverable from the oropharynx after sucking each proprietary lozenge separately on different days. In vitro determinations investigated the relative antimicrobial activity of aqueous solutions of the lozenges and, the effect of pH and tablet base excipients on that activity against Staphylococcus aureus, Streptococcus pyogenes and Candida albicans. RESULTS: Both in vivo and in vitro results showed that the tablet based lozenges had markedly reduced antimicrobial activities compared with previous results with a candy based lozenge (in vivo and in vitro) or the same concentration of aqueous CPC (in vitro). Magnesium stearate suspensions in CPC 250 micrograms/ml indicated that magnesium stearate adsorbed CPC and at 0.4% lozenge weight and above significantly reduced the antimicrobial activity of CPC 250 micrograms/ml. CONCLUSIONS: The reduced activity of CPC in tablet based lozenges resulted from a decreased availability of CPC in solution due to an adsorption of CPC on magnesium stearate. To avoid this reduction in activity tablet based lozenges containing CPC 250 micrograms/ml, or similar concentrations, plus magnesium stearate should contain not more than 0.3% w/w lozenge weight of the lubricant.

Determination of peptidoglycan-associated protein in Escherichia coli NCIB 8545 by capillary zone electrophoresis.

An efficient reliable and sensitive capillary zone electrophoresis assay for the six major bacterial peptidoglycan-associated proteins of Escherichia coli NCIB 8545 is described. The method provides the facility to determine quantitatively the effect of antibacterials on bacterial peptidoglycan-associated protein synthesis and thus to further elucidate the mechanism of antibacterial action of such drugs as the antifolates which recently have been shown to adversely affect peptidoglycan synthesis.

Brodimoprim synergy against Enterococcus faecalis evaluated in vitro.

Combinations of either brodimoprim or trimethoprim plus either carbenicillin, gentamicin, ciprofloxacin or rifampicin showed synergy at sub-inhibitory concentrations against both Enterococcus faecalis NCTC 5957 and 775. Brodimoprim alone and in combination showed greater antibacterial activity against both strains of E. faecalis than trimethoprim. MBCs of brodimoprim and trimethoprim were 14.4 and 25.6 mg/L for E. faecalis NCTC 5957 and 7.2 and 12.8 mg/L for E. faecalis NCTC 775. Combinations of either brodimoprim or trimethoprim plus the other antibacterial agents, except gentamicin and dibromopropamidine isethionate, were bactericidal at achievable plasma concentrations. Viable count determinations of cultures of both test organisms in the presence of 3/4 of the MIC of each of the four antibiotics and the two antifolates alone and combinations of each antibiotic with either brodimoprim or trimethoprim indicated that only the combinations prevented recovery and regrowth of the cultures over 24 h. The ATP released from cultures of both strains of E. faecalis treated with brodimoprim and trimethoprim at the same concentrations was approximately 1.5 times greater with brodimoprim than with trimethoprim. Combinations of 3/4 of the MIC of each of the antibiotics in combination with 3/4 of the MIC of brodimoprim against cultures of both strains of E. faecalis resulted in greater release of ATP than occurred with equivalent trimethoprim combinations. It is postulated that the increased activities observed with the brodimoprim combinations resulted from an effect of brodimoprim on the bacterial cell permeability control. These results indicate that both brodimoprim and trimethoprim offer potential benefits for use with either carbenicillin, gentamicin, ciprofloxacin or rifampicin for the treatment of E. faecalis infections.

The effect of formulation on the antimicrobial activity of cetylpyridinium chloride in candy based lozenges.

PURPOSE: The purpose of this investigation was to determine the influence on the antimicrobial activity of cetylpyridinium chloride of the various components of the formulation of each of six candy based lozenges. METHODS: In vivo activity was investigated using six volunteers by determining the reduction in colony forming units recoverable from the oropharynx after sucking each lozenge separately on different days. In vitro determinations investigated the relative activity of aqueous solutions of the lozenges, the effect on activity of additional active ingredients, pH and lozenge base ingredients against separate inocula of each of the test organisms Staphylococcus aureus, Streptococcus pyogenes and Candida albicans. RESULTS: Both in vivo and in vitro results showed that the pH of the dissolved lozenge solution was the single most influential readily adjustable formulation parameter which significantly influenced the activity of cetylpyridinium chloride activity in candy based lozenges. CONCLUSIONS: Lozenges containing cetylpyridinium chloride as the active ingredient should be formulated at a pH greater than 5.5.

Mechanism of sulphadiazine enhancement of trimethoprim activity against sulphadiazine-resistant Enterococcus faecalis.

Sulphadiazine had little or no antibacterial effect against three strains of Enterococcus faecalis (MICs of above 3600 mg/L) but it caused approximately six-fold increases in bacterial uptake of trimethoprim, increased release of bacterial ATP and produced ultrastructural damage to both the trimethoprim resistant E. faecalis 463 and the trimethoprim sensitive NCTC 5957. MICs of trimethoprim were 0.6, 0.8 and 76.8 mg/L for E. faecalis NCTC 775, NCTC 5957 and 463 respectively. When log phase E. faecalis 463 and NCTC 5957 were grown for 4 h in trimethoprim 56 and 0.6 mg/L plus sulphadiazine 320 and 100 mg/L respectively there was an approximate ten-fold and nine-fold increase in uptake of sulphadiazine and a two-fold increase in leakage of ATP. Trimethoprim caused more damage to the cell wall and cytoplasmic membrane than sulphadiazine but the combination of sulphadiazine plus trimethoprim caused the most cell damage. The increased activity observed with the combination seems very likely to have resulted from the increased uptakes of the antibacterials, which in turn had resulted from the cell wall damage and consequent increased cell permeability caused by each antibacterial. It is proposed that a markedly subinhibitory concentration of sulphadiazine enhanced the antibacterial activity of trimethoprim against all three strains of E. faecalis by this mechanism.

Investigation of cell envelope damage to Pseudomonas aeruginosa and Enterobacter cloacae by dibromopropamidine isethionate.

Electron micrographs of log-phase Pseudomonas aeruginosa and Enterobacter cloacae cultured for 4 h in the presence of subinhibitory concentrations of dibromopropamidine isethionate indicate that this antibacterial agent can cause marked damage to the cell envelope structures of both species. This result provides an explanation of how dibromopropamidine can enhance the uptake and thus the activity of a second antibacterial agent used in combination with it.

An electronmicroscope study of the effect of sulphadiazine and trimethoprim on Enterobacter cloacae.

Electronmicroscopy of thin sections of log phase cells of Enterobacter cloacae NCTC 10005 grown for 4 h in the presence of sulphadiazine 250 micrograms/ml, trimethoprim 12.5 microliters/ml or the combination of sulphadiazine 250 micrograms/ml plus trimethoprim 12.5 micrograms/ml indicated that both agents caused marked morphological damage even though the MIC of sulphadiazine for the E. cloacae strain was > 3000 micrograms/ml. The damage took the form of electron-transparent areas devoid of ribosomes in the cytoplasm and detachment of the outer membrane. The latter was most marked with trimethoprim, which also caused damage to the cytoplasmic membrane. It is postulated that the synthesis of the peptidoglycan layer was affected by the antimetabolites since the morphological effects were strikingly similar to those caused by treatment of E. cloacae with disodium edetate plus lysozyme. Viable counts of cultures undergoing the same treatments as those prepared for electronmicroscopy indicated that although sulphadiazine merely partially inhibited growth it nevertheless enhanced the bactericidal action of trimethoprim over a 5-h period.

Simultaneous determination of some antibacterial drugs in isosensitest broth using high-performance liquid chromatography with solid-phase extraction.

A method is described for the simultaneous determination of combinations of some antibacterial drugs in a matrix of isosensitest broth. A double solid-phase extraction procedure is described in which trimethoprim and dibromopropamidine isethionate together with 4-chlorophenylbiguanide as internal standard are freed from endogenous components by a cation-exchange extraction cartridge and subsequently removed and individually separated by reversed-phase ion-pair chromatography. Sulfadiazine, sulfamerazine and p-aminobenzoic acid, unretained by ion exchange, are similarly isolated for chromatography by adsorption on a CH-bonded phase cartridge and individually assayed using the same chromatographic system. The rationale of the pre-treatment and chromatography is described and the quantitative aspects of the analyses of selected combinations of these drugs are reported.