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PubMed2002

Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro.

BACKGROUND: Previous work has shown that cutaneous microbial species associated with skin conditions of microbial aetiology are susceptible to killing by photodynamic therapy (PDT) using visible light and methylene blue. Antimicrobial PDT (APDT) in vivo would require a therapeutic regimen where bacteria could be killed without damaging adjacent tissue. OBJECTIVES: To study keratinocyte killing in vitro using APDT. METHODS: We used a combination of methylene blue (100 microg mL(-1)) and visible light (42 mW cm(-2)), previously used for microbial killing, to study cytotoxic effects on keratinocytes. Kill rates and subsequent D-values were determined against a human keratinocyte cell line (H103) using trypan blue and neutral red dye viability tests. RESULTS: The kill rates for keratinocytes were exponential over the 90- and 180-min period of the experiment for neutral red and trypan blue, respectively. The corresponding D-values were shown to be 198 and 205 min using trypan blue exclusion and neutral red uptake viability tests, respectively. CONCLUSIONS: The kill rates for keratinocytes were 18-200-fold slower than those previously determined for cutaneous microbial species, suggesting that in vivo, APDT sufficient to reduce microbes by seven log cycles would have little cytotoxic effect on keratinocytes. This approach may offer a safe alternative to conventional antimicrobial treatment.

Bacteria↗
PubMed1982

Complement does not play a role in promoting Babesia rodhaini infections in Balb/C mice.

A critical role of C3 and the C3b receptor for the erythrocyte invasion and the development of the parasitemia of B. rodhaini in rats has been described recently (Jack and Ward 1980a). In the present study the influence of the C system on B. rodhaini infection in Balb/C mice is documented. Depletion of serum C3 to less than 5% of the normal level by treatment of mice with CoF, the C3 inactivator isolated from cobra venom, did not affect the course of B. rodhaini parasitaemia. Treatment of mice with trypan blue, a reagent that inactivates the C3b receptor on human polymorphonuclear leukocytes, inhibited the development of parasitemia. However, when B. rodhaini parasitized erythrocytes were incubated in vitro with trypan blue and subsequently tested for the in vivo and in vitro replication of the parasites, this old-fashioned therapy for babesiosis in cattle showed its babesiacidal activity. This indicates that the inhibition of parasite development by trypan blue is caused by its parasitotoxicity. These data suggest that the C system does not play an essential role in the development of B. rodhaini infection of the Balb/C mouse.

Animals↗
PubMedDate not supplied

Successful preservation of human skin by vitrification.

The vitrification technique was applied to the preservation of human skin. This technique was simple, and no expensive equipment was needed. Split-thickness human skins from 8 patients were immersed in vitrification solution for 10 minutes at room temperature, immediately plunged into a liquid nitrogen tank, and cryopreserved for 3 weeks. The vitrification solution consisted of 40% ethylene glycol (vol/vol) and phosphate buffered saline solution that contained 30% Ficoll 70 (vol/vol; Wako Junyaku, Co, Tokyo, Japan) and 0.5 mol/L sucrose. The viability of vitrified and cryopreserved skin was evaluated with the trypan blue dye exclusion test, the methyl-thiazoldiphenyl-tetrazolium (MTT) colorimetric assay, and a culture test of the keratinocytes obtained from vitrified skin. The results of the trypan blue dye exclusion test showed 87.4% of viable cells, and MTT developed an average 0.817 absorbance. When vitrified skin was compared with 4 degrees C refrigerated skins after 3 weeks of storage, the difference of viability was significant both on the trypan blue dye exclusion test (P < .05) and on the MTT assay (P < .01). However, there was no significant difference in the viability of vitrified skins compared with fresh skin. Furthermore, keratinocytes from vitrified skin grew uneventfully in culture test. We used these vitrified skin allografts for patients with flame burns and electric burns. These allografts took well in both cases and promoted wound healing. We concluded that the vitrification method for skin preservation is simple and reliable, and this method could contribute to skin banking.

Burns↗
PubMed1988

Binding and biological actions of prostaglandin E2 and I2 in cells isolated from rabbit gastric mucosa.

1. We have investigated the binding of the tritiated forms of prostaglandin E2 (PGE2) and a stable analogue of prostacyclin (Iloprost) to isolated cells of rabbit oxyntic mucosa. 2. The highest degree of specific [3H]PGE2 binding occurred in a cellular fraction enriched in parietal cells. [3H]Iloprost binding occurred predominantly in cells identified as mucous cells. 3. PGE2 binding to the parietal cell fraction was associated with its ability to inhibit histamine-stimulated aminopyrine accumulation by these cells. Iloprost binding did not correlate with a biological action on the parietal cells. 4. PGE2 and Iloprost reduced Trypan Blue staining in cells exposed to 10% (w/v) ethanol. Iloprost (10(-8) to 10(-6) M) reduced Trypan Blue staining in cells identified as mucous and parietal cells. PGE2 (10(-8) M) significantly reduced Trypan Blue staining in parietal cell-enriched fractions. 5. Cyclic AMP stimulation in response to either prostanoid occurred most potently on non-parietal cell fractions. However PGE2 or Iloprost binding affinities did not correlate with cyclic AMP formation. 6. These data provide evidence for true PGE2 receptors on oxyntic mucosal cells. The receptors appear to mediate inhibition of acid secretion. Iloprost binds to sites which might mediate cellular protection.

Aminopyrine↗
PubMed1989

Iron-induced lipid peroxidation and inhibition of proliferation in Ehrlich ascites tumor cells.

The purpose of this study was to find further experimental evidence for the postulated negative association between the extent of lipid peroxidation in tumor cells and their proliferative behavior. After incubation of Ehrlich ascites tumor cells at 37 degrees C for 30 min with increasing concentrations of Fe(II) histidinate (Fe/His) the following parameters were determined: the formation of lipid hydroperoxides was measured fluorimetrically after reaction with dichlorofluorescein; 4-hydroxynonenal was determined by reversed-phase high-pressure chromatography after derivatization with dinitrophenylhydrazine; as a third parameter of lipid peroxidation the formation of 2-thiobarbituric-acid-reactive substances was determined. The proliferative activity was determined by measuring the growth rate in vivo after reimplantation i.p. of the tumor cells into mice. Trypan-blue exclusion tests for viability were performed before reimplantation. The reliability of the trypan-blue exclusion tests was checked by comparing the results with another parameter of viability, the release of the cytosolic enzyme lactate dehydrogenase. The concentration both of lipid hydroperoxides and of 2-thiobarbituric-acid-reactive substances showed a biphasic dependence on the concentration of Fe/His with maximal increase at iron concentrations of 0.25 mM and 0.1 mM respectively. 4-Hydroxynonenal, in contrast, showed a continuous increase up to 41.1 nM (corresponding to 0.58 pmol/10(9) cells) with increasing iron concentration in the range from 0.1 mM to 0.6 mM. The total number of tumor cells, when determined 5 days after reimplantation, continuously decreased with increasing iron concentration, showing half-maximal inhibition at about 0.22 mM Fe. The exclusion of the trypan-blue dye was unaffected by the presence of iron at any concentration used. Similarly, iron had no influence on the release of lactate dehydrogenase. The results support the hypothesis that 4-hydroxynonenal may act as an inhibiting messenger between endogenic lipid peroxidation and proliferation.

Animals↗
PubMed1984

[The effect of antitumor vaccine on the recurrence of solid tumors in mice subjected to surgery].

The frequency of tumour recurrences in A/Sn mice with operated Ehrlich carcinomas and treated with lyophylized antitumour vaccine and trypan blue or both of them was investigated. The tumours in vaccinated mice relapsed by the 47th day after the operation in 10% of cases, in 50% of control animals, the injection of trypan blue increased the tumour recurrences by 20%. The antitumour effect of vaccination is associated with the enhancement of the activity of the cellular and humoral immune systems. The trypan blue inhibition of the macrophage function suppresses the antitumor activity of the immunity cellular components.

Animals↗
PubMed1990

Merocyanine 540-sensitized photoinactivation of leukemia cells: role of oxygen and effects on plasma membrane integrity and mitochondrial respiration.

Merocyanine 540 (MC 540) is a photosensitizing dye that is used clinically for the purging of autologous bone marrow grafts and preclinically for the inactivation of enveloped viruses in blood products. In this paper we present evidence that the MC 540-sensitized photoinactivation of leukemia cells is an oxygen-dependent process and that unsaturated plasma membrane lipids are substrates for singlet oxygen and/or other activated oxygen species generated by photoirradiated MC 540. A comparison of the inhibition of clonal growth, the inhibition of mitochondrial respiration, and the exclusion of trypan blue by the plasma membrane after exposure to MC 540 and graded doses of light showed that mitochondrial respiration is compromised relatively early in the course of the dye-mediated photoinactivation of cells, well before the plasma membrane loses its capacity to exclude trypan blue. It also showed that trypan blue exclusion assays can greatly underestimate the cytotoxic effects of MC 540-sensitized photoirradiation.

Animals↗
PubMedDate not supplied

Vital staining of corneal endothelium.

The efficacy and applicability of dual vital staining of human and porcine corneal endothelium with trypan blue and alizarin red S stains was investigated. Exposure of endothelial cells to different concentrations of these two dyes for varying periods of time revealed that 0.3% solution of trypan blue for 1 minute followed by 0.2% solution of alizarin red S (pH 4.2) for 1/2 to 1 minute was the most useful technique to assess the cell viability, micro to macro damage, cell division and cell counts, etc. Alizarin red S dye should not be used in in vivo evaluation, while trypan blue was very effective to evaluate the endothelial viability of donor cornea just prior to keratoplasty.

Animals↗
PubMed1996

A new method for isolation of cardiac myocytes by percutaneous endomyocardial biopsy.

The objective of this study was to demonstrate the feasibility of isolating viable canine cardiac myocytes from percutaneous right ventricular endomyocardial biopsy specimens. Although histologic data can be obtained from percutaneous endomyocardial biopsies, this approach has not been used as a source of viable cells for evaluating pathological conditions. Study of isolated viable myocytes may provide insight into the electrical, biochemical, and physiologic functions of the heart. Using a standard 8F sheath, a 5F bioptome was introduced via the right femoral vein and advanced to the right ventricle, where 85 biopsies were obtained from 8 mongrel dogs. An average of six biopsy specimens were pooled for processing to provide adequate tissue substrate. This resulted in 14 groups of specimens which were then processed to isolate individual myocytes. Viable myocytes were striated, rod-shaped, and excluded trypan blue dye. Nonviable myocytes were rounded, had no cross-striations, and did stain with trypan blue. Partially-injured myocytes contracted spontaneously and had a region of loss of cross-striations. The average number of viable cells recovered per group of pooled specimens was 1.8 x 10(4) (1.8 x 10(3) cells/mg of tissue). The greatest yield of viable myocytes recovered was 8.0 x 10(4), which represented a viability of 90% by trypan blue dye exclusion and morphological criteria. Percutaneous right ventricular endomyocardial biopsy is a novel method for obtaining viable cardiac myocytes. Its feasibility and utility in humans warrant further investigation.

Animals↗
PubMed2005

Manual small incision cataract surgery in eyes with white cataracts.

PURPOSE: To assess the safety and efficacy of Manual Small Incision Cataract Surgery (MSICS) in cases of white cataract with the use of trypan blue as an adjunct for performing continuous curvilinear capsulorthexis (CCC). MATERIALS AND METHODS: Prospective observational study on 100 consecutive eyes of 100 patients with white cataract who had undergone MSICS with trypan blue assisted CCC. The nucleus was prolapsed into anterior chamber by using a sinskey hook and extracted out of the eye using irrigating vectis. Intraoperative and postoperative findings (according to OCTET classification) as well as postoperative visual outcomes were used as main measures to report the safety and efficacy of the surgery. RESULTS: Of the 100 eyes, 16 had intumescent, 67 had mature and 17 had hypermature cataract. Intraoperatively CCC was incomplete in 4 eyes (4%) and had to be converted to canopener capsulotomy. None of the eyes had posterior capsular rupture or zonular dialysis and no eyes were converted to conventional Extra Capsular Cataract Extraction (ECCE). Postoperatively, 6 eyes (6%) developed corneal oedema with >10 Descemets folds and 7 eyes (7%) had corneal oedema with CONCLUSION: In developing countries like India where phacoemulsification may not be affordable to a majority of those requiring cataract surgery, MSICS proves to be a safe and efficacious alternative for white cataracts especially with the adjunctive use of trypan blue dye.

Adult↗
PubMed1986

Cytoskeletal changes as an early event in hydrogen peroxide-induced cell injury: a study in A549 cells.

Hydrogen peroxide (H2O2) and other oxygen metabolites have been implicated in the pathogenesis of cell and tissue injury. The nature of the injury occurring in cells exposed to oxygen metabolites is unknown. A549 cells, derived from human lung carcinoma, were exposed to glucose-glucose oxidase or hydrogen peroxide in vitro. The distribution of actin and cytokeratin filaments, as well as 51chromium (51Cr) release and trypan blue dye exclusion were assessed. Both glucose-glucose oxidase and H2O2 resulted in changes which were time- and dose-dependent. Alterations in the cytoskeleton were detected by immunofluorescence microscopy at two hours, at which time the cells excluded trypan blue dye, while 51Cr release and trypan blue uptake first occurred at 8 h and required a five-fold greater concentration of glucose oxidase. The addition of catalase to glucose-glucose oxidase or H2O2, or inactivation of glucose oxidase by boiling, abrogated the injury. Therefore, one of the early targets of H2O2-induced cell injury may be the cytoskeleton.

Actins↗
PubMed2004

Cytotoxicity of fungal metabolites to lepidopteran (Spodoptera frugiperda) cell line (SF-9).

The toxicity of sixteen fungal metabolites produced by some entomopathogenic fungi or biological control fungi agents was evaluated on lepidopteran Spodoptera frugiperda (SF-9) cell line by Trypan blue dye exclusion and MTT-colorimetric assay, after 48 h of incubation. No statistical difference was found between IC50values (50% Inhibiting Concentration) and CC50 values (50% Cytotoxicity Concentration) obtained by MTT test and Trypan blue dye exclusion for each fungal metabolite. By MTT assay, the cytotoxicity ranking was fusarenon X (IC50 0.3 microM) = diacetoxyscirpenol (IC50 0.5 microM) = beauvericin (IC50 2.5 microM) = nivalenol (IC50 5.3 microM) = enniatin (IC50 6.6 microM) > or = gliotoxin (IC50 7.5 microM) > zearalenone (IC50 17.5 microM) > deoxynivalenol (IC50 47.6 microM). By Trypan blue dye exclusion the cytotoxicity ranking was fusarenon X (CC50 0.4 microM) = diacetoxyscirpenol (CC50 1.1 microM) beauvericin = (CC50 3.0 microM)=gliotoxin (CC50 4.0 microM) = enniatin (CC50 6.7 microM) > or = nivalenol (CC50 9.5 microM) > zearalenone (CC50 18.3 microM) > deoxynivalenol (CC50 45.0 microM). The comparison with other bioassays showed that the SF-9 insect cell line could represent a further tool to screen for the toxic effects of fungal metabolites especially for beauvericin, gliotoxin, and zearalenone.

Animals↗
PubMed1983

Survival of cultured cells after functional and structural disorganization of plasma membrane by bacterial haemolysins and phospholipases.

Lesions were induced in the plasma membranes of cultured human fibroblasts by membrane damaging toxins of bacterial origin (haemolysins). Structural disorganization of the membrane was measured as leakage of a radiolabelled small cytoplasmic marker and functional membrane damage was measured as decreased uptake of aminoisobutyrate. Cell survival was scored 24 and 48 hr later by measuring uptake of Trypan Blue and by light microscopical evaluation of cell morphology and proliferation. The membrane damage induced by most bacterial toxins was reversible upon removal of the toxin, since toxin-treated cells recovered and excluded Trypan Blue although they had been permeable to the dye immediately after the toxin treatment. Among ten bacterial toxins tested, the only exception of this general behavior was the Aeromonas hydrophila beta-haemolysin, which irreversibly damaged human fibroblasts. Thus, the action of bacterial haemolysins on cultured cells generally seems restricted to a plasma membrane permeabilization, which is reversible regardless of the mechanism of membrane damaging action of the toxin or of the relative size of the structural lesions induced. Furthermore, the use of Trypan Blue uptake as a measure of cell death caused by membrane damaging agents appears to be of limited value.

Aeromonas↗
PubMed1998

Toxicity of Xylocaine to rabbit corneal endothelium.

PURPOSE: To assess the toxicity of lidocaine hydrochloride (Xylocaine) to the corneal endothelium. SETTING: Department of Ophthalmology, Hôtel-Dieu Hospital, Paris, France. METHODS: Rabbit corneas were excised and the endothelium was exposed to balanced salt solution (BSS), Xylocaine 1%, or Xylocaine 5% (5 corneas/group) for 20 minutes. The endothelium was then stained with trypan blue and alizarin red, and 5 photomicrographs were taken of each cornea at a standard magnification and analyzed with a digital imaging system (Biocom 200). RESULTS: Xylocaine solutions produced changes in endothelial cell morphology, but there was no cell staining with trypan blue. Corneas exposed to Xylocaine 5% had more marked cell alterations. Small areas of cells were lost from all 15 corneas, mainly at the periphery, but the differences among the 3 groups of corneas were not significant. CONCLUSION: Exposure of rabbit corneal endothelium to Xylocaine solutions in vitro was not associated with trypan blue staining of endothelial cells.

Anesthetics, Local↗
PubMed1979

Amphetamines reduce embryonic size and produce caudal hematomas during early chick morphogenesis.

Experiments were designed to study some of the similarities and differences in the effects of amphetamines and trypan blue on early chick morphogenesis. Both dextroamphetamine sulfate (0.5 mg/egg) and methamphetamine hydrochloride (1.0 mg/egg) were capable of inducing, in 3-day chick embryos, caudal hematomas which were similar in appearance and location to those routinely observed following treatment with trypan blue. It was found, too, that both dextroamphetamine and methamphetamine treated embryos frequently exhibited a significant decrease in crown rump length and cross-sectional area of the notochord, neural tube, dorsal aortae and whole body section, when compared with unopened or saline injected controls. Trypan blue treated embryos had only a rare decrease or increase in the size of structures when compared to either control group. These findings suggest that the amphetamines have an ability to decrease or retard embryonic growth in the chick.

Abnormalities, Drug-Induced↗
PubMed2003

A chicken model to study the embryology of cloacal exstrophy.

BACKGROUND/PURPOSE: The embryology of bladder and cloacal exstrophy is a mystery. Reasons for this are the lack of human embryos showing these malformations as well as the scarcity of appropriate animal models. Here, the authors present cases of cloacal exstrophies found in chick embryos subsequent to the application of suramin and trypan blue. This animal model might facilitate insight into the embryology of cloacal exstrophy. METHODS: Fertilized chicken eggs were incubated at 38 degrees C and 75% humidity. Embryos were treated in ovo on incubation day 3. The egg shell was windowed, and solutions of suramin (stage 13, 2 x 40 microL/0.2%) or trypan blue (stage 14, 2 x 80 microL/0.03%) were injected into the coelomic cavity. The window was closed, and the embryos were reincubated until examination on incubation day 8. Fifty embryos were treated in each group. RESULTS: Among the surviving embryos, cloacal exstrophy was found in 2 cases in the suramin-treated group (2 of 29, 6.9%) and in 4 cases in the trypan blue-treated group (4 of 20, 20%). CONCLUSIONS: Suramin and trypan blue can induce cloacal exstrophy in chick embryos. The authors now are modifying their experimental protocols to increase the incidence of this malformation. This model might facilitate studies on the morphogenesis of cloacal exstrophy.

Animals↗