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PubMed2004

[Immunosuppressive activity of exopolysaccharide from Paecilomyces gunnii].

OBJECTIVE: To investigate bioactivities of Paecilomyces gunnii exopolysaccharide (PGEP). METHODS: The effects of PGEP on mice spleen lymphocytes proliferation, mice peritoneal macrophage (PMphi) phagocytosis and cytotoxin T lymphocytes (CTL) activity were studied by MTT method, neutral red colorimetry, respectively. RESULTS: In vitro, mice spleen lymphocytes proliferation, mice peritoneal macrophage (PMphi) phagocytosis of neutral red and CTL activity were significant suppressed by PGEP, especially in 10 microg/ml. CONCLUSION: PGEP had the immunosuppressive activities.

Animals↗
PubMedDate not supplied

The effects of cytocentrifugation on differential cell counts in samples obtained by bronchoalveolar lavage.

Quantification of the differential cell count and total number of cells recovered from the lower respiratory tract by bronchoalveolar lavage is a valuable technique for the diagnostic study of interstitial lung diseases. To examine the effect on the cell counts of different methods of processing the lavage fluid, two comparisons were performed. First, two methods of differential cell counting were compared using 28 fluids. One count was performed in a Malassez hemocytometer after incubation of the living cells with neutral red for five minutes at room temperature; large cells and some small cells that had incorporated neutral red were identified as macrophages. Another count was performed on cytocentrifuge preparations made using the Shandon Cytospin I and Cytospin II and stained by the May-Grünwald-Giemsa method. The percentage of cells identified as lymphocytes was significantly lower on the cytocentrifuge preparations than with the Malassez hemocytometer. In the second study, the differential cell counts on smears prepared by the two types of cytocentrifuge (Cytospin I and Cytospin II) were compared for 32 bronchoalveolar lavage fluids. The percentage of small cells (especially lymphocytes) was lower on preparations made with the Cytospin I than on those made with the Cytospin II, but the difference was not significant. The results indicate that (1) cytocentrifugation of bronchoalveolar lavage fluids does result in a significant loss of small cells, especially lymphocytes, and (2) this loss is not significantly lessened by the use of the Cytospin II.

Bronchoalveolar Lavage Fluid↗
PubMed1996

Thapsigargin induces rapid, transient growth inhibition and c-fos expression followed by sustained growth stimulation in mouse keratinocyte cultures.

Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.

Animals↗
PubMed1981

Gastric mucosal blood flow and acid secretory changes in man with impromidine: a new specific histamine-H2-receptor agonist.

The effect of impromidine, a new histamine-H2-receptor agonist, on gastric mucosal blood flow (neutral red clearance) and acid secretion was studied in nine volunteers. Impromidine stimulated a dose-dependent increase in neutral red clearance and acid secretion. Simultaneous cimetidine in three doses caused a parallel shift to the right for both acid output and clearance with unchanged maxima compatible with simple surmountable antagonism. There were small cardiovascular changes: an increased heart rate with a decreased diastolic pressure during infusion of impromidine. These changes were antagonised by cimetidine.

Adult↗
PubMed1979

An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens.

A selective medium consisting of trypticase soy agar with 4% glycerol, 5 mg/l crystal violet, 50 mg/l neutral red and 4 mg/l of gentamicin was devised for isolation of Pseudomonas pseudomallei from clinical specimens. Absorption of neutral red was found to be suitable for differentiating this organism from other bacteria, while gentamicin was effective in selecting Ps. pseudomallei from organisms commonly found in clinical material. The medium was more suitable for screening clinical specimens than MacConkey's agar with colistin-S because it was more selective and allowed multiple specimens to be inoculated on a single plate. Eight thousand clinical specimens from an area endemic for melioidosis were screened on the selective medium. This resulted in the recovery of 8 isolates of Ps. pseudomallei that would not have been detected using routine culture media alone.

Antibodies, Bacterial↗
PubMed1993

Assessment of the relation between the initial viability and the attachment of freshly isolated rat hepatocytes used for the in vivo/in vitro DNA repair assay (UDS).

Crucial steps of the in vivo/in vitro DNA repair assay (UDS) are the hepatocyte isolation procedure and the establishment of the hepatocyte cultures. Since the attachment of the isolated hepatocytes on the surface of the culture vessel is an essential prerequisite for the in vitro part of this assay to yield scorable autoradiograms, we assessed the relation between the initial viabilities of hepatocyte preparations and the resulting attachment efficiencies from 286 rats. The initial viability was determined by means of the trypan blue dye exclusion assay. The actual cell number was corrected for the viability and a constant number of 2.5 x 10(5) viable cells were seeded into each well of gelatinized six-well dishes. The amount of adherent cells was determined after a 1.5-h attachment period using a recently described modification (Fautz et al., 1991) of the neutral red dye absorption assay. The attachment is described by the optical density at 540 nm obtained after the elution of neutral red from the adherent cells (OD540 value). To facilitate a comparison of the data we divided the 286 animals into eight arbitrary viability groups. The mean values of the viability groups were 53.1, 62.2, 66.3, 68.4, 70.9, 73.6, 76.9, and 84.0% living cells. Although there was a great interindividual variation, the resulting mean OD540 values were nearly uniform, about 0.5, in all eight groups, regardless of the initial viability of the hepatocytes. UDS data obtained from 46 animals treated with the positive control chemical 2-acetylaminofluorene demonstrated that there was no correlation between the in vitro DNA repair capacity and the initial viability or the attachment efficiency of the hepatocytes. Our results suggest that (i) great interindividual differences exist between the attachment of particular cell preparations with no regard to the initial viability, (ii) the correction of the cell number for viability leads to relatively uniform OD540 mean values and (iii) for an in vivo/in vitro UDS assay even cell suspensions with relatively low viabilities can be used since they will yield adherent cultures which are capable of DNA repair synthesis. The latter item often allows a reduction in the number of animals required for this in vivo assay because it is not necessary to perform repeated experiments because of low viability preparations.

Animals↗
PubMed2004

[The effect of ultrasonication on immuno-modulating activity of mycelial polysaccharide from Cordyceps gunnii].

OBJECTIVE: To investigate immuno-modulating activity change after mycelial polysaccharide from Cordyceps gunnii (MPCG) was depolymerized by ultrasonication. METHODS: The effects of MPCG and its depolymerizing mixture on mice spleen lymphocytes proliferation, mice peritoneal macrophage (PMphi) phagocytosis and cytotoxin T lymphocytes (CTL) activity were studied by MTT method, neutral red colorimetry, respectively. RESULTS: In vitro, MPCG depolymerized by ultrasonication could more significantly inhibite spleen lymphocytes proliferation, PMphi phagocytosis of neutral red and CTL activity in mice than MPCG, in particular at doses of 10 microg/ml and 100 microg/ml. Whereas, four kinds of depolymerized mixture depolymerized for 1h to 2h and 4h to 8h by ultrasonication, respectively, had not evident differences at three various doses. CONCLUSION: MPCG was suitable for depolymerizing for 1h to 2h and its depolymerized mixture had stronger immuno-modulating activity than MPCG.

Animals↗
PubMed1996

Synthesis and antitumor activity of some analogues of flavone acetic acid.

Some coumarin-, flavonol- and flavanon-acetic acids are described. The cytotoxicity of the synthesized compounds was determined on a human colon carcinoma cell line (LoVo) through evaluation of neutral red uptake, performed by the Riddel method. All tested derivatives were able to induce a statistically significant reduction of lysosomal neutral red uptake at 5 x 10(-5) M concentration. Some compounds were more active than the reference compound flavon-8-acetic acid.

Acetates↗
PubMed2002

The effect of hexaaza- and hexathia-macrocyclic ligands on transition metal cytotoxicity in human hepatoma-derived cultured cells.

The effect of macrocyclic ligands on cytotoxic concentrations of the transition metal ions of copper, zinc, and cadmium was investigated. For this purpose, a hexaaza- [3,6,9,17,20,23-hexaazatricyclo[23.3.1.1(11,15)] triaconta-1(29),11(30),12,14,25,27-hexaene (L2)] and hexathia-chelating ligand [1,4,7,10,13,16-hexathiacyclooctadecane (L3)] were used in the human hepatoma-derived HepG2 cell line. The cytotoxicity was measured by the neutral red uptake inhibition assay. First, the NI50 of the ligands, i.e., the concentration of the ligand inducing a 50% inhibition in neutral red uptake compared to control cells, was determined. In several metal/ligand combination experiments, the effects for L2 were difficult to interpret, whereas for L3 in combination with copper ions, a severe increase -- and for zinc ions, a significant decrease of cell toxicity -- relative to the metal control was observed. To further examine the different effects observed with L3 in combination with, respectively, Cu2+ and Zn2+, the glutathione (GSH) content was measured. The relative GSH content decreased as the concentration of L3 increased. It was proposed that the increased toxicity of the combination Cu(2+)/L3 could be caused by the depletion of GSH and a subsequent inability to scavenge the produced reactive oxygen species (ROS). This hypothesis was supported by experiments during which vitamin E or C was added to the Cu(2+)/L3 system.

Alkanes↗
PubMed2006

Evaluation of mammalian cell adhesion on surface-modified porous silicon.

Porous silicon is a promising biomaterial that is non-toxic and biodegradable. Surface modification can offer control over the degradation rate and can also impart properties that promote cell adhesion. In this study, we modified the surface of porous silicon surface by ozone oxidation, silanisation or coating with collagen or serum. For each surface, topography was characterised using atomic force microscopy, wettability by water contact angle measurements, degradation in aqueous buffer by interferometric reflectance spectroscopy and surface chemistry by Fourier-transform infrared spectroscopy. The adhesion of rat pheochromocytoma (PC12) and human lens epithelial cells to these surfaces was investigated. Cells were incubated on the surfaces for 4 and 24 h, and adhesion characteristics were determined by using a fluorescent vital stain and cell counts. Collagen coated and amino silanised porous silicon promoted cell attachment for both cell lines whereas cells attached poorly to ozone oxidised and polyethylene glycol silanised surfaces. We showed that the two cell lines had different adhesion characteristics on the various surfaces at different time points. The use of the vitality assays Alamar Blue (redox based assay) and neutral red (active cellular uptake assay) with porous silicon was also investigated. We reveal incompatibilities between certain resazurin (Alamar Blue), lysosomal incorporation assays (neutral red) and porous silicon.

Animals↗
PubMed1995

Possible role of mononuclear phagocytes in clearance of immune complexes in pulmonary tuberculosis.

Phagocytic activity of peripheral blood mononuclear cells (PBMC) was investigated in 30 patients with pulmonary tuberculosis. The test was done using neutral red. Higher percentages of phagocytic cells were detected with PBMC of tuberculosis patients as compared with controls. An inverse relationship was observed between neutral red positive cell counts vs. relative percentages of several proteins (IgG, C3 and C4) in PEG precipitates in tuberculosis patients. It was interpreted that phagocytes might have mopped up the CICs via their surface Fc receptors.

Adolescent↗
PubMed1979

Characterization of two quenchers of chlorophyll fluorescence with different midpoint oxidation-reduction potentials in chloroplasts.

The properties of two redox quenchers of chlorophyll fluorescence in chloroplasts at room temperature have been investigated. (1) Redox titration of the fluorescence yield reveals two n = 1 components with Em7.8 at--45 and --247 mV, accounting for approx. 70 and 30% of the total yield, respectively. (2) Neutral red, a redox mediator often used at redox potentials below --300 mV, preferentially quenches the fluorescence controlled by the --247 mV component. Titrations using neutral red artifactually create an n = 2 quenching component with Em7.8 = --375 mV. (3) Analysis of fluorescence induction curves recorded at different redox potentials indicates that both the --45 and --247 mV components can be photochemically reduced. The reduction of the --247 mV component corresponds to a fast phase of the induction curve whilst the slower reduction of the 45 mV component accounts for the tail phase. (4) The excitation spectra for the fluorescence controlled by the two quenchers show small differences in the ratio of chlorophyll a and b. (5) Whereas the --247 mV component readily shows a 60 mV per pH unit dependency on solution pH, the ability of the --45 mV component to respond to pH change is restricted. (6) Triton Photosystem II particles contain both quenchers but the --247 mV component accounts for approx. 70% of the fluorescence and the high component has an Em7.8 of +48 mV. The relative merits of sequential and parallel models in explaining the presence of the two quenchers are considered.

Chlorophyll↗
PubMed1963

THE PARTICULATE HYDROLASES OF MACROPHAGES. II. BIOCHEMICAL AND MORPHOLOGICAL RESPONSE TO PARTICLE INGESTION.

The influence of phagocytosis on the morphological and biochemical properties of macrophage hydrolase-containing granules has been studied in vitro. Following the uptake of large numbers of heat-killed bacteria, an intracellular rearrangement of hydrolytic enzymes occurred. This was associated with the solubilization of 50 to 60 per cent of the total cell content of acid phosphatase, cathepsin, lysozyme, beta glucuronidase, acid ribonuclease, and acid desoxyribonuclease and with a corresponding decrease in granule-bound enzyme. With more prolonged incubation the majority of the soluble intracellular pool of acid ribonuclease and lysozyme was lost to the extracellular medium. No change in the total content of any of the hydrolases was noted during 180 minutes of incubation in vitro. The morphological fate of the granules was studied by a histochemical method for acid phosphatase. After the phagocytosis of yeast cell walls there was a disappearance of acid phosphatase-positive granules and an accumulation of reaction product about the ingested particle. Experiments employing macrophages which were supravitally stained with neutral red also demonstrated the loss of neutral red-positive granules and the accumulation of the dye about the yeast cell walls. These results strongly suggest that lysis of macrophage granules occurs following phagocytosis and that a portion of the granule contents are then resegregated within the newly formed phagocytic vacuole.

Acid Phosphatase↗
PubMed1994

New in vitro fluorimetric microtitration assays for toxicological screening of drugs.

Flow cytometry has been widely used to quantify fluorescent probes in cell culture. However, FCM is not adapted to toxicological screenings due to the cost, the length and the poor reproducibility of this technique. Moreover, several multicenter studies have preferred microtitration methodologies for drug screening. A new fluorimetric technology has been designed that is sensitive and adapted to direct screening in 96-well microplates. This fluorimeter uses cold light technology (CLF) with chemical and physical modifications of the lighting system (Rat et al., 1995). CLF allows reading of UV, visible and near infrared fluorescence by increasing light energy (from 1000 to 2300 lumens) and reducing the calorific part of light (IR > 900 nm, Joule effect). It induces a decrease in background and a 500- to 1000-fold improvement of detection limit of probes in comparison with classical fluorimeters and permits detection of pg/ml to fg/ml. CLF allows easy evaluation of cell injury induced by physical agents (UVA) or chemical toxins (CCl4). Four biological endpoints for cytotoxicity evaluation have been tested with several probes: proliferation (H33258); viability (fluorescent Neutral Red); cell-cell adhesion (calcein-AM); and mitochondrial metabolic effects (Rhodamine 123). Rh123 assay appeared more sensitive than fluorimetric or photometric detection of Neutral Red assay. Cold light fluorimetry (CLF) permits direct detection of low concentrations of probes (pg/ml to fg/ml). CLF is shown to improve classical cytotoxicity assays and, owing to its adaptability to microtitration (in 6-, 12- or 96-well plates and in Petri dishes), it is thus a promising alternative to flow cytometry for drug cytotoxicity screening.

Carbon Tetrachloride↗
PubMed2000

Effect of light-harvesting complex II on ion transport across model lipid membranes.

The effect of the incorporation of the major light-harvesting complex of photosystem II (LHCII) to planar bilayer lipid membranes (BLMs) formed from soybean asolectin and unilamellar small liposomes formed from egg-yolk phosphatidylcholine on ion transport across the lipid bilayer has been studied. The specific conductivity of the BLM rises from 5.2 +/- 0.8 x 10(-9) up to 510 x 10(-9) O(-1) cm(-2) upon the incorporation of LHCII. The conductivity of the membrane with LHCII depends upon the ionic strength of the bathing solution and is higher by a factor of five when the KCl concentration increases from 0.02 to 0.22 M. Such a strong effect has not been observed in the same system without LHCII. The liposome model is also applied to analyse the effect of LHCII on the bilayer permeability to protons. Unilamellar liposomes with a diameter less than 50 nm have been prepared, containing (trapped inside) Neutral Red, a pigment sensitive to proton concentration. A gradient of protons on the membrane is generated by the acidification of the liposome suspension and spectral changes of Neutral Red are recorded in time, reflecting the penetration of protons into the internal space of liposomes. Two components of proton permeation across liposome membranes are observed: a fast one (proceeding within seconds) and a slow one (operating on the time scale of minutes). The rate of both components of proton transport across LHCII-containing membranes is higher than for liposomes alone. The enhancement effect of LHCII on the ion transport across the lipid membrane is discussed in terms of aggregation of the pigment-protein complexes. The possible physiological importance of such an effect in controlling ion permeability across the thylakoid membrane is discussed.

Electric Conductivity↗
PubMed1993

Evaluation of skin damage of cyclic monoterpenes, percutaneous absorption enhancers, by using culture human skin cells.

The cytotoxicity of monoterpenes, percutaneous absorption enhancers, to cultured human skin cells was investigated in order to quantitatively estimate their skin damage. A neutral red bioassay with epidermal keratinocytes and a contraction test of collagen gel in which dermal fibroblasts were cultured were employed for evaluating the cytotoxicity of terpenes. In the neutral red bioassay, keratinocyte proliferation was inhibited on the addition of terpenes, and cell survival remarkably decreased with an increase in the concentration of terpenes fed into the culture well. When the fibroblasts were cultured in a collagen gel matrix, the lattice of collagen contracted as the cells grew. Therefore, the application of cytotoxic agents brings about an inhibition of collagen gel contraction induced by the fibroblasts. Strong inhibition was observed in the cases of hydrocarbons in terpenes, and the inhibition was dependent on the concentration of these compounds added in the culture medium. The cytotoxicity of terpenes was compared with the skin damage evoked by the application of terpenes in rats in vivo. As a result, it was considered that the skin irritation caused by terpenes was predictable to a certain extent by means of the cytotoxic study of cultured human skin cells.

Animals↗
PubMed2001

Cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities.

A novel series of cyclopentane derivatives have been found to exhibit potent and selective inhibitory effects on influenza virus neuraminidase. These compounds, designated RWJ-270201, BCX-1827, BCX-1898, and BCX-1923, were tested in parallel with zanamivir and oseltamivir carboxylate against a spectrum of influenza A (H1N1, H3N2, and H5N1) and influenza B viruses in MDCK cells. Inhibition of viral cytopathic effect ascertained visually and by neutral red dye uptake was used, with 50% effective (virus-inhibitory) concentrations (EC(50)) determined. Against the H1N1 viruses A/Bayern/07/95, A/Beijing/262/95, A/PR/8/34, and A/Texas/36/91, EC(50)s (determined by neutral red assay) of the novel compounds were < or =1.5 microM. Twelve strains of H3N2 and two strains of avian H5N1 viruses were inhibited at <0.3 microM. Influenza B/Beijing/184/93 and B/Harbin/07/94 viruses were inhibited at <0.2 microM, with three other B virus strains inhibited at 0.8 to 8 microM. The novel inhibitors were comparable in potency to (or slightly more potent than) zanamivir and oseltamivir carboxylate. No cytotoxicity was seen with the compounds at concentrations of < or =1 mM in cell proliferation assays. The antiviral activity of RWJ-270201, chosen for clinical development, was studied in greater detail. Its potency and that of oseltamivir carboxylate decreased with increasing multiplicity of virus infection. Time-of-addition studies indicated that treatment with either compound needed to begin 0 to 12 h after virus exposure for optimal activity. Exposure of cells to RWJ-270201 caused most of the virus to remain cell associated, with extracellular virus decreasing in a concentration-dependent manner. This is consistent with its effect as a neuraminidase inhibitor. RWJ-270201 shows promise in the treatment of human influenza virus infections.

Acids, Carbocyclic↗
PubMed2004

Ecotoxicological evaluation of the additive butylated hydroxyanisole using a battery with six model systems and eighteen endpoints.

The occurrence and fate of additives in the aquatic environment is an emerging issue in environmental chemistry. This paper describes the ecotoxicological effects of the commonly used additive butylated hydroxyanisole (BHA) using a test battery, comprising of several different organisms and in vitro test systems, representing a proportion of the different trophic levels. The most sensitive system to BHA was the inhibition of bioluminescence in Vibrio fischeri bacteria, which resulted in an acute low observed adverse effect concentration (LOAEC) of 0.28 microM. The next most sensitive system was the immobilization of the cladoceran Daphnia magna followed by: the inhibition of the growth of the unicellular alga Chlorella vulgaris; the endpoints evaluated in Vero (mammalian) cells (total protein content, LDH activity, neutral red uptake and MTT metabolization), mitotic index and root growth inhibition in the terrestrial plant Allium cepa, and finally, the endpoints used on the RTG-2 salmonid fish cell line (neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity, and glucose-6-phosphate dehydrogenase activity). Morphological alterations in RTG-2 cells were also assessed and these included loss of cells, induction of cellular pleomorphism, hydropic degeneration and induction of apoptosis at high concentrations. The results from this study also indicated that micronuclei were not induced in A.cepa exposed to BHA. The differences in sensitivity for the diverse systems that were used (EC50 ranged from 1.2 to >500 microM) suggest the importance for a test battery approach in the evaluation of the ecological consequences of chemicals. According to the results, the levels of BHA reported in industrial wastewater would elicit adverse effects in the environment. This, coupled with its potential to bioaccumulate, makes BHA a pollutant of concern not only for acute exposures, but also for the long-term.

Aliivibrio fischeri↗