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At least 469 records · Page 26Linked to original sources
PubMed1989

Enterocyte-lymphocyte interactions in the follicle-associated epithelium of the mouse Peyer's patch.

1. Measurements of membrane potential (Vm) have been carried out in the follicle-associated epithelium of the mouse Peyer's patch to investigate possible site-dependent anomalies in enterocyte development. Initial heterogeneity in Vm values obtained from carrying out random impalements in the upper third of the follicle-associated epithelium could be described as arising from the presence of three different cell populations. 2. The predominant cell type in this epithelium (T1) had a mean Vm of -35.3 mV which was partly depolarized by increasing concentrations of K+. These cells were not stained with the vital dye Neutral Red. Two other types of cell (T2L and T2H) were recorded together as consecutive negative jumps in Vm, but only in areas of the epithelium previously stained with Neutral Red. T2L behaved like T1 cells to K+ but with a low Vm of -23.3 mV. T2H cells had a high mean Vm of -47.5 mV showing transient hyperpolarization to increasing K+ concentration. 3. The electrophysiological and non-staining properties of T1 cells are similar to those expected from mature enterocytes. The anatomical size, site, staining properties and Vm of T2H cells correspond to those expected for intraepithelial lymphocytes. The low Vm, K+ depolarization and close association of T2L with T2H cells are typical of properties predicted for an immature-type of antigen-transporting enterocyte known to be present in this type of epithelium. 4. The possibility that intraepithelial lymphocytes either slow or reverse a more normal process of development of enterocytes in their immediate vicinity is discussed along with wider aspects of enterocyte-lymphocyte interactions leading to the transfer to enteric antigens across these modified cells.

Animals↗
PubMed2005

Photodynamic effect of argon and diode laser on cholesteatoma cell cultures after intravital staining with absorption enhancers.

Chronic epitympanic otitis media, or chronic suppurative osteitis, is a destructive form of chronic middle-ear inflammation. The therapy of choice is complete surgical removal of the squamous epithelium from the middle ear. It is often impossible to inspect all areas of the middle ear with the posterior canal wall intact. Not all recesses can be reliably monitored with the microscope, particularly in the area of the antrum and hypotympanum. Residual squamous epithelium here causes frequent recurrences following cholesteatoma surgery. This study examines the effect of argon and diode lasers on cholesteatoma tissue. The aim is to develop a laser treatment selectively directed against cholesteatoma cells that can be performed after cholesteatoma surgery to eliminate any residual squamous epithelium. Intraoperatively harvested monolayer-cultured cholesteatoma cells stained in vivo with various absorption enhancers served as the in vitro examination model. Argon (499 nm) and diode lasers (810 nm) were applied since their irradiation has an appropriate tissue penetration depth and is absorbed by various chromophores such as neutral red (475-500 nm), fluorescein (488 nm), and indocyanine green (790-810). Intracellular staining of cultured cells increased the optical density at the wavelength corresponding to the dye. Neutral red damaged 50-60% of cultured cells merely by intracellular accumulation at high concentrations. An additive cell destruction of about 30% was achieved by also applying argon laser irradiation. Fluorescein diacetate caused no appreciable stain-induced damage to cultured cholesteatoma cells. Argon laser irradiation destroyed up to 60% of the cultures. Indocyanine green resulted in only minor damage to cultured cells. The diode laser destroyed up to 60% of the irradiated cells. Selective staining of cholesteatoma cells was not achieved with any of the dyes examined. Thus, other stained tissue could be damaged. Staining and subsequent laser irradiation destroys up to 60% of cultured cholesteatoma cells. Unstained irradiated cells are not affected. Indocyanine green and fluorescein are nontoxic and may thus be used as absorption enhancers. The diode and argon lasers appear to be basically suitable. Cell staining is not selective, i.e., other tissues would also be stained and damaged. To avoid such unwanted damage, it would be desirable to couple the chromophore to a specific antibody that binds only to cholesteatoma cells.

Argon↗
PubMed1997

Validation of in vitro methods to single out photoirritants using mechanistically based tests.

In the recent OECD draft proposal for a new guideline on acute dermal photoirritation testing, in vitro screening tests have been included as part of the sequential test strategy. These screening tests were placed directly prior to animal tests proposed. In Europe some in vitro techniques - cell culture and mechanistic tests - are under validation in a joint project of the European Center for the Validation of Alternative Methods (ECVAM) and the European Cosmetic, Toiletry, and Perfumery Association (COLIPA). Two promising cellular in vitro tests are presented and discussed as tool for the screening of photoirritancy. The first one as a general core test performed in each participating laboratory is the 3T3 mouse fibroblast Neutral Red Uptake Phototoxicity test determining the cell viability by uptake of Neutral Red as end point, whereas the second performed only by three participating laboratories was the the Red Blood Cell phototoxicity test comprising a combination of two end points, the photohaemolysis and the oxyhaemoglobin oxidation. Besides this, other mechanistic tests can be used as additional support. Identification of photoirritation is generally considered to be one area for the successful research and validation of in vitro techniques.

3T3 Cells↗
PubMed1995

The effect of red and neutral density filters on the degree of eccentric fixation.

The reported shift of fixation to a more central position when a red filter is placed before an eye which fixates eccentrically, has never been satisfactorily explained. To further investigate this phenomenon, 14 subjects with eccentric fixation were recruited. A Wratten No. 92 filter was placed before the eccentrically fixating eye, and any change of fixation was assessed by the after-image transfer test. A neutral density (ND) filter was then used, to mimic the reduced retinal illumination caused by the red filter. The results suggest that there is a significant reduction in the eccentricity of fixation, when placing the red filter before an eccentrically fixating eye, and also when placing the ND filter. No significant difference in the effects produced by either filter was found. This suggests that it is the reduction in retinal illuminance caused by the red filter which contributes towards such improvements in fixation.

Afterimage↗
PubMed1996

Chromium- and nickel-induced cytotoxicity in normal and transformed human keratinocytes: an investigation of pharmacological approaches to the prevention of Cr(VI)-induced cytotoxicity.

Chromium and nickel compounds cause irritancy but can also induce allergic contact dermatitis. The aims of this study were to characterize the direct cytotoxic effects of Cr(VI), Cr(III) and Ni(II) salts on keratinocytes, and to investigate pharmacological strategies to protect cells against Cr(VI)-induced cytotoxicity. Normal human keratinocytes and the HaCaT keratinocyte cell line were used. Cell viability was assessed by neutral red dye uptake, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) eluted stain assay and measurement of lactate dehydrogenase (LDH) activity in the medium. The assays varied slightly in their sensitivities (neutral red > MTT > LDH) although all three gave similar results. In both cell types, the relative order of cytotoxicity of the salts was Cr(VI) >> Ni(II) > Cr(III). There were no major differences between chromium salts of a common valency. Normal human keratinocytes showed a much greater variability in their response to Cr(VI) and Ni(II) salts than HaCaT cells and were generally more resistant to Cr(VI)- and Ni(II)-induced cytotoxicity. Several drugs were screened for their potential to protect both cell types against the cytotoxic effects of Cr(VI), specifically the reducing agents ascorbic acid, cysteine and glutathione, and the Cr(VI) cellular uptake inhibitors 4,4'-diisothiocyanato-2,2'-stilbenedisulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanato-2,2'-stilbenedisulphonic acid (SITS). All five drugs provided concentration-dependent protection against Cr(VI)-induced cytotoxicity but only ascorbic acid offered complete protection. Several of these pharmacological approaches to the prevention of Cr(VI) cytotoxicity confirm previous clinical studies on the inactivation of Cr(VI), while the clinical potential of others has yet to be investigated.

Ascorbic Acid↗
PubMed1982

Improved yields and assay of simian varicella virus, and a comparison of certain biological properties of simian and human varicella viruses.

Studies were performed to define conditions under which propagation, assay and stabilization of the Delta herpesvirus (DHV) strain of simian varicella virus might be improved, and to compare biological properties of DHV with those of human varicella zoster virus (VZV). A mycoplasma contaminant was successfully eliminated from the DHV seed virus by treatment with a specific anti-serum. DHV was found to replicate more efficiently in the BS-C-1 line of African green monkey kidney cells than in Vero cells, and seed virus preparations in the form of virus-infected cells were produced which had infectivity titers greater than or equal to 1 X 10(6) p.f.u./ml. Greater yields of virus were produced in cultures infected as dispersed cells than as preformed monolayers. Infectious DHV could be released from host cells by sonic treatment of heavily infected cultures at 48 h post infection. Certain agents reported to enhance replication of herpes viruses (caffeine, carbaryl, the tumor promoter 12-0-tetra-decanoyl-phorbol-13-acetate, and DEAE-dextran) had no enhancing effect on replication of DHV. However, DEAE-dextran in the maintenance medium enhanced spontaneous release of DHV into culture fluids. Plaquing efficiency and plaque size of DHV were greater in BS-C-1 than in Vero cells, and plaque assays and plaque reduction neutralization tests were developed in this cell system using a solid overlay medium with neutral red vital stain. Neutralization of DHV was markedly enhanced by fresh guinea pig complement. The newly developed neutralization test demonstrated more vigorous antibody responses to DHV in active and latent VZV infections than were demonstrated with previous procedures. In addition to their preferential growth in monkey and human cells respectively, DHV and VZV were found to differ markedly in their rates of attachment to host cells, with DHV requiring over 6 h of adsorption, while VZV adsorption was essentially complete at 1 h. Also, cell-free DHV was much more resistant than cell-free VZV to repeated cycles of freezing and thawing.

Animals↗
PubMed2004

Comparison of trypsin treatment method and standard laboratory technique for diagnosis of dermatomycosis.

Dermatomycosis is prevalent worldwide. Discrepancy between microscopic examination and culture findings can create problems in the diagnosis of this common infection. In this study, samples from 60 patients were processed after trypsin treatment and examined by neutral red staining to distinguish viable and non-viable fungal elements. The trypsin treatment method was compared with standard laboratory techniques. A higher number of direct-microscopy-positive, culture-negative samples were obtained without trypsin treatment. Trypsin treatment increased the isolation of fungi from clinical samples, and neutral red staining was able to distinguish viable fungal elements.

Candida↗
PubMed2005

Genotoxic effects of selected biocides on RTG-2 fish cells by means of a modified Fast Micromethod Assay.

A sensitive in vitro assay for detecting DNA damage in RTG-2 cells culture is described. This assay employs a dye, PicoGreen double stranded DNA (dsDNA) quantitation reagent, which becomes intensely fluorescent upon binding nucleic acids. The assay includes a simple and rapid 50-min sample lysis in the presence of EDTA, SDS, and high urea concentration at pH 10, followed by time-dependent DNA denaturation at pH 11.6 after NaOH addition. The time course and the extent of DNA denaturation are followed in a microplate fluorescence reader at room temperature for less than 1h. Comparative studies between suspension and fixed RTG-2 cells indicated that it is possible to apply this methodology in both cases with good results. Neutral red assay was used for to determine the cellular viability when RTG-2 cultures were exposed to tetrakis(hydroxymethyl) phosphonium chloride (THPC) and benzalkonium chloride (BC), as biocides used in the disinfection of cooling towers. The results obtained by neutral red assay indicate IC(50(48)) values of 0.017 (0.011-0.028) and 2.71 (1.91-3.86) mg/L for tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. DNA damage has been evaluated for both disinfectants in RTG-2 culture, by exposure to 1/10-, 1/25-, 1/50-, and 1/100-IC(50(48)) value, and the results obtained indicate a strain scission factor (SSF) of 0.126+/-0.014, 0.181+/-0.014, 0.217+/-0.013, and 0.245+/-0.013 in cell suspensions, and 0.077+/-0.019, 0.107+/-0.014, 0.151+/-0.014, and 0.202+/-0.015 in attached cells for tetrakis(hydroxymethyl) phosphonium chloride; while the SSF values for benzalkonium chloride are 0.023+/-0.009, 0.033+/-0.017, 0.068+/-0.012, and 0.088+/-0.015 in cell suspensions, and 0.033+/-0.010, 0.044+/-0.011, 0.080+/-0.009, and 0.093+/-0.010 in attached cells. Thus, the assay proposed in this study has made it possible to show DNA damage in RTG-2 cells when exposed to 0.2(1/100 IC(50(48))) and 300(1/10 IC(50(48))) Hg/L of tetrakis(hydroxymethyl) phosphonium chloride and benzalkonium chloride, respectively. The results obtained indicate that the Fast Micromethod Assay, applied on RTG-2 cell line cultures, is a fast and sensitive method for the early DNA damage detection in the aquatic environment.

Analysis of Variance↗
PubMedDate not supplied

Genital herpes: Treatment with methylene blue and light exposure.

Fifty-six patients with genital herpes were treated by photodynamic inactivation of herpesvirus by methylene blue (36 patients), proflavine (101 or neutral red (10). The first 35 patients were randomly selected for treatment with one of the three dyes. All the others were initially treated with methylene blue. The disease was temporarily eradicated in about 70% of instances by exposure of the lesions to light plus topically applied methylene blue, neutral red or proflavine. Relapses, however, were not prevented or appreciably reduced.

Acridines↗
PubMed1990

Proliferation of human ocular fibroblasts. An assessment of in vitro colorimetric assays.

Growing interest surrounds the pharmacologic modulation of ocular wound healing and fibroblastic proliferation. Therefore many investigators have developed assays to screen potential antiproliferative drugs on cultured fibroblasts. Such assays are extremely labor intensive, especially when many compounds are tested. Using proliferating human ocular fibroblasts, the authors explored the feasibility of three in vitro colorimetric assays as rapid, simple alternatives to more tedious proliferation assays, such as Coulter counting. These were: (1) the mitochondrial metabolism of a tetrazolium dye, (2) the cytoplasmic activity of hexosaminidase, and (3) the lysosomal uptake of neutral red, a weak basic dye. Serial dilutions of fibroblasts were assayed with each colorimetric technique; Coulter counting was used as a standard. All three colorimetric methods showed strong linear relationships between optical density and Coulter-cell counts. The MTT and neutral-red techniques were relatively insensitive, unable to quantify reliably fewer than 20,000 or 50,000 cells, respectively. On the other hand, the hexosaminidase assay was far more sensitive, reliably detecting a few hundred cells. Despite their differences and limitations, these three colorimetric techniques are useful as inexpensive screens whenever multiple drugs are tested for antifibroblastic effects.

Cell Count↗
PubMed2004

In vitro cytotoxicity testing of three zinc metal salts using established fish cell lines.

The utilisation of fish cell lines has proven to be a valuable, rapid and cost-effective tool in the ecotoxicological assessment of chemicals and environmental samples. The main objective of this study was to investigate the value of multiple endpoint measurements in evaluating the cytotoxicity of three divalent zinc salts in three established fish cell lines (EPC, CHSE and RTG-2) and the potential for their employment as effective screening tools for zinc contaminated environmental samples. A significant stimulatory effect was detected with the neutral red assay in EPC and RTG-2 cells exposed to the lower doses of some zinc compounds. Significant (p < or = 0.01) lactate dehydrogenase release was detectable only with the highest exposure concentration of ZnCl2. Toxicity ranking based on IC50 values calculated from the neutral red and coomassie blue assay data found that in general, ZnC2 was the most cytotoxic metal compound to the cell lines employed. Differential cell sensitivities were observed to be dependant on the particular compound tested and the endpoint employed. It was found that the use of light microscopy in the identification of cell morphological changes was a valuable adjunct in verifying the results of colorimetric tests. In conclusion, careful consideration should be given to study design and statistics applied and use of a battery style approach is recommended for toxicological screening studies.

Animals↗
PubMed2005

Potential irritation of lysine derivative surfactants by hemolysis and HaCaT cell viability.

Surfactants represent one of the most common constituents in topical pharmaceutical and cosmetic applications or cleansers. Since adverse skin and ocular reactions can be caused by them, it is important to evaluate damaging effects. Amino acid-based surfactants deserve particular attention because of their low toxicity and environmental friendly properties. New lysine derivative surfactants associated with heavy and light counterions were tested. The ocular irritancy was assessed by hemolysis, and photohemolysis was employed to evaluate their phototoxicity. Cytotoxicity on HaCaT cells was determined by neutral red uptake and MTT assay to predict skin irritation. All lysine derivative surfactants were less hemolytic and thus less eye-irritating than the commercial surfactants used as model irritants. No phototoxic effects were found. All surfactants presented cytotoxic effects as demonstrated by decrease of neutral red uptake and reduction of MTT salt, with clear concentration-effect profiles. However, the rates of cytotoxicity on HaCaT for the new surfactants suggested that they were less cytotoxic and then, less skin-irritating than the reference ones; surfactants with heavy counterions were the less cytotoxic. The anionic surfactants investigated in the present work may constitute a promising class of surfactants given their low irritancy potential for pharmaceutical and cosmetic preparations.

Betaine↗
PubMed1981

Cellular differentiation and pattern formation in the absence of morphogenesis in the cellular slime mould Polysphondylium pallidum: evidence for a biochemical tip (organizer) in submerged aggregates.

When amoebae of Polysphondylium pallidum WS320 are placed in nonnutrient buffer in roller tube culture they form spherical or ellipsoidal aggregates. At first the aggregates demonstrate a "loose" morphology but by 12 h, with the formation a a cellulose-containing, peripheral sheath, they become "tight" aggregates. At this time stalk differentiation begins. Using various methods for the resolution of prespore (ultrastructure, spore antigen immunofluorescence, periodic acid - Schiff staining) and prestalk (ultrastructure, alkaline phosphatase histochemistry, neutral red staining, Calcofluor fluorescence) cell localization, the pattern of cell differentiation in submerged aggregates was shown to be essentially identical to that of normal pseudoplasmodia. Furthermore, using a cAMP bioassay it was revealed that the submerged aggregates, while devoid of a morphological tip, do possess a biochemical tip which is correlated with sites of neutral red staining and stalk cell differentiation. As a result of these studies, an earlier argument that the tip of the pseudoplasmodium is not essential for the establishment of pattern or in the "organization" of cellular differentiation during slime mould development is contradicted.

Alkaline Phosphatase↗
PubMed2005

[Study of trichloroethylene-induced apoptosis in normal human epidermis keratinocytes in vitro].

OBJECTIVE: To investigate the apoptosis-inducing effect of trichloroethylene (TCE) on cultured normal human epidermis keratinocytes (NHEK) in vitro. METHODS: NR(50) values (the concentration of neutral red absorbed is reduced to 50%) of TCE on NHEK were assayed by neutral red uptake (NRU), and the administered dose of TCE was determined. Lipid peroxidation (LPO) and oxidative stress were assessed by measurement of malondialdehyde (MDA) contens and superoxide dismutase (SOD) activity. Transmission electron microscope (TEM) were used to observe morphologic changes, flow cytometer (FCM) was used to measure DNA contents and calculate cell apoptosis rate and proliferation index (PI). RESULTS: NR(50) values of TCE on NHEK was found to be 4.53 mmol/L (95% CI: 3.92-5.13 mmol/L). The increase in MDA content and inhibition of SOD activity in a concentration-dependent manner were shown after NHEK was treated with a series of dose of TCE 4 h later, and typical morphologic changes of apoptosis were also observed by TEM examination. FCM analysis revealed a sub-G(1) peak in the apoptotic cells. The apoptotic rate in TCE 0.125, 0.500, 2.000 mmol/L exposed groups (31.83%, 38.63%, 44.35%, respectively) were significantly higher than that in blank control (18.42%), while PI in TCE 0.125, 0.500, 2.000 mmol/L group (3.26%, 2.48%, 2.07%, respectively) were significantly lower than that in blank control (4.99%). CONCLUSION: TCE may induce apoptosis of cultured NHEK in vitro, and inhibit cell proliferation through lipid peroxidation and oxidative stress.

Apoptosis↗
PubMed1994

Human bioassays to assess environmental genotoxicity: development of a DNA repair assay in HepG2 cells.

A direct assessment of the effects of environmental chemicals on human health has been hampered by the lack of suitable experimental systems. We have recently employed a human liver cell line (HepG2) to assess the biological effects of pollutants at both cellular and DNA levels. A Neutral Red dye uptake assay was used to assess potential cytotoxic effects of xenobiotics. DNA damage was quantified using an unscheduled DNA synthesis assay that measures repair that is induced following exposure to genotoxic compounds. HepG2 cells responded to the known mutagens, 4-nitroquinoline N-oxide and methylmethane sulfonate, both in the Neutral Red assay for cytotoxicity and two DNA repair assays for genotoxicity (monitored autoradiographically or by liquid scintillation counting). The HepG2 DNA repair and cytotoxicity assays also responded to an extract (containing polycyclic aromatic hydrocarbons) of sediment obtained from a polluted site in the Great Lakes. Results indicate that this system can be deployed further to assess potential cyto- and genotoxicity of pollutants. The development of human cell culture assays is a critical step towards a full assessment of the risk that such pollutants pose to human health.

4-Nitroquinoline-1-oxide↗
PubMed1977

Photodynamic inactivation of verrucae vulgares. I.

Verrucae vulgares (v.v.) stained in vivo and in vitro with 0.1% and 1% proflavine in 100% dimethylsulphoxide (DMSO) and 0.1% and 1% neutral red in 100% DMSO were examined grossly and by fluorescence microscopy. Light transmission studies were made using both whole v.v. embedded in methacrylate and 5 micrometer frozen sections. The dyes were seen to penetrate to the epidermal and dermal structures in the in vivo stained v.v. and accumulate in the cell nuclei. The average concentration of neutral red in the v.v. was estimated to be 2 X 10(-5) M. The concentration of proflavine was lower than that, but exceeded 10(-6)M. There was diffuse staining throughout the in vitro stained warts. From 400-600 nm the warts were penetrated by at least 1% of the light directed toward their surfaces.

Acridines↗
PubMed2005

Assays for the assessment of neutralizing antibody activities against Severe Acute Respiratory Syndrome (SARS) associated coronavirus (SCV).

Accurate assessment of neutralizing antibody activities is important either for patients infected with Severe Acute Respiratory Syndrome (SARS) or for animals and volunteers immunized with the experimental vaccines against the SARS associated coronavirus (SCV). However, the current assay based on the cytopathic effect (CPE) which has been frequently cited in literature has several limitations. The CPE assay relies on the visual observation on the damage of SCV infected target cells under a microscope. It is subjected to observer variations and it is difficult to generate a quantitative determination of neutralizing activities based on the level of CPE. In the current study, we established the utility of two additional assays to measure the neutralizing activities against SCV: the plaque reduction (PR) and the neutral red staining (NRS) assays. The PR assay described in this study was modified from the traditional viral plaque reduction assay by using an improved crystal staining method to achieve better plague formation in SCV infected Vero E6 cells. The NRS neutralization assay was adopted from a similar system used for detecting neutralizing antibody responses against human immunodeficiency virus type 1 (HIV-1). In this assay, the protective effect of neutralizing antibodies was determined by the cell viability which is measured by the uptake of neutral red dye at A540. The neutralizing antibody titers can be easily determined with either of the two new assays. In this report, we described the utility of these two new neutralization assays in measuring the neutralizing activities against SCV infection from rabbit sera immunized with various forms of spike protein of SCV.

Animals↗
PubMed2005

Symptomatic relief of herpetic skin lesions utilizing an energy-based approach to healing.

Herpes simplex virus induced oral and genital ulcerating lesions will fluoresce brightly yellow and yellow-orange, respectively, if treated with a chlorinated solution of neutral red and exposed to ultraviolet-A light. An orange to red fluorescence is seen with similarly treated and illuminated Herpes zoster virus induced shingles; while treated human papillomavirus induced genital warts display more of a purplish fluorescence. Pain and discomfort commonly disappear soon after the treatment and all lesions undergo expedited healing that is readily observable within 24 h. The mechanism of healing is thought to involve an interaction between neutral red and alternative cellular energy pigments (ACE pigments) present within the viral lesions that enhances responsiveness to ultraviolet light energy. The healing effects are not restricted to the treated lesions and may involve transmission of a biological energy throughout the body. Beyond its obvious clinical and diagnostic utility, this model system may help usher in a new era of energy-based medicine.

Coloring Agents↗