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PubMed1980

Suppression and acceleration of experimental amyloidosis in mouse model.

Leupeptins, protease inhibitors, suppress the appearance of experimental amyloidosis in CBA mice induced by the injections with complete Freund's adjuvant. This substance, however, should be administered continuously from 1 week prior to amyloid induction to the end of the experiment. On the other hand, Trypan blue, inhibitors of lysosomal enzymes, accelerates experimental amyloidosis in the mouse model above-mentioned. Trypan blue is effective when given either prior to or at the same time of the initiation of amyloid induction. Organic Germanium has not been confirmed to be a potent suppressor of experimental murine amyloidosis but the experimental group administered this substance continuously from 1 week prior to the induction shows a rather low incidence of amyloidosis, and the average number of amyloidotic organs per affected mouse is about half of that of the control group. The suppression and acceleration of experimental murine amyloidosis presented here are a useful tool for investigating the pathogenesis of amyloidosis.

Amyloidosis↗
PubMed1998

Sequential identification of apoptotic and necrotic cells on individual cytospin preparations.

Here, we describe a simple methodology which allows the consecutive differentiation of apoptotic and necrotic cells on the same cytospin preparation of fresh and cultured cells. In this methodology, necrotic cells are initially identified on the cytospin preparations using trypan blue followed by the identification of in situ DNA fragmentation using the TUNEL assay. Identification of trypan blue and TUNEL positive cells in the same section permits the simultaneous assessment of necrotic and apoptotic cells.

Apoptosis↗
PubMed2005

An experimental method for testing novel retinal vital stains.

There is uncertainty surrounding the safety of the vital stains currently used to assist macular surgery, and there may be other agents that are more suitable. This study aimed to validate a method of screening retinal vital stains for their potential surgical utility. Bovine retina was exposed to test agents at a range of concentrations. Masked observers determined the minimum dye concentration that reliably stained the retina, defined as the minimum visible concentration (MVC). Computer image analysis (CIE94 colour difference equation) was used to estimate the magnitude of the colour difference between stained and unstained retina. Agents that had favourable staining characteristics underwent safety testing using a retinal pigment epithelium and glial cell culture model. Cells were exposed to each agent and viability was assessed with a mitochondrial enzyme (MTT) assay, and fluorescent live-dead probe (ethidium homodimer-1/calcein-AM). Frozen sections were used to determine which retinal layers were stained. Techniques were tested on the following agents: alcian blue; diethyloxadicarbocyanine; Evan's blue; fast green; fluorescein; Janus green; methylene blue; naphthol green; neutral red; procian (reactive) yellow; rose bengal; and trypan blue. For most dyes, the results of image analysis showed that colour differences increased linearly with dye concentration, although some displayed a more exponential relationship. Five agents showed favourable staining characteristics: Evan's blue, rose bengal, naphthol green, neutral red, and trypan blue (MVC 0.02, 0.01, 0.1, 0.002, 0.01%, respectively). Safety testing of these five agents did not show toxicity, except in glial cells exposed to rose bengal. Relative to the negative control (saline), these showed a 48% reduction in viability using the MTT assay (p<0.001; t=4.71; CI 30-75%), and qualitative damage on fluorescence microscopy. Frozen sections showed that some agents produced diffuse staining of all retinal layers, others produced selective inner retinal staining. There are thousands of biological stains available and many of these may be more effective or safer than those currently used for retinal surgery. This study provides a means of screening potentially useful vital stains.

Animals↗
PubMed1985

Experimental examination of the direct damaging effects of Giardia lamblia on intestinal mucosal scrapings of mice.

Giardia lamblia is known to produce functional and structural derangement of the small intestine but the pathogenesis of this defect is not clear. To examine this, mucosal scrapings from the small intestine of mice were incubated with human G. lamblia trophozoites. The integrity of the mucosal cells was assessed by their ability to exclude trypan blue, and by the levels of brush border lactase, sucrase and maltase. As judged by the trypan blue test, more mucosal cells incubated with G. lamblia were found to be damaged than were in the control groups I (mucosal cells alone) and III (mucosal cells plus Giardia culture media). Similar results were obtained with disaccharidases where again the mucosal cells incubated with G. lamblia showed a statistically significant reduction in the activity of lactase, sucrase and maltase compared to that in the control groups. These findings suggest that G. lamblia causes direct damage to the small intestinal epithelial cells and that this effect is not mediated through factors such as bacterial proliferation, bile salt deconjugation and immunological reactions.

Animals↗
PubMed1996

Amelioration of pulmonary allograft injury by administering a second rinse solution.

OBJECTIVE: The use of rinse solutions before reperfusing liver allografts has been shown to reduce cell death in rats. Carolina rinse solution (an extracellular solution that contains antioxidants, vasodilators, and other substrates that help prevent ischemia-reperfusion injury) has also been shown to improve liver function clinically in liver transplant recipients. This pilot study evaluates the value of a second pulmonary artery flush before reperfusion of a lung graft. METHODS: Six groups of Sprague-Dawley rats (n = 6 each) were subjected to the following: Group 1 lungs were preserved with modified Euro-Collins solution followed by 24 hours of cold ischemia. Group 2 lungs were treated the same as group 1 but reperfused with blood. Group 3 lungs were preserved in Carolina rinse solution followed by 24 hours of cold ischemia. Group 4 lungs were treated the same as group 3 lungs and then reperfused with blood. Lungs in groups 5 and 6 were preserved with Euro-Collins solution, stored cold for 24 hours, and then rinsed with Euro-Collins or Carolina rinse solution, respectively, before reperfusion with blood. Lungs were subsequently stained with trypan blue solution for 5 minutes. Lung blocks were fixed and embedded in water-soluble methacrylate. Trypan blue--stained nuclei in nonviable endothelial cells and alveolar pneumocytes were counted in 10 different fields. RESULTS: Groups 1 and 3, preserved with Euro-Collins and Carolina rinse solutions for 24 hours but not reperfused with blood, had significantly more viable endothelial cells (groups 1 and 3 vs group 2, p < 0.0001; group 3 vs group 4, p < 0.02) and pneumocytes (group 1 vs groups 2 and 4, group 3 versus group 2, p < 0.0001; group 3 vs group 4; p < 0.035) than groups 2 and 4, which were subsequently reperfused with blood. Groups 5 and 6, which received a second rinse, also had significantly more viable endothelial cells (p < 0.0005) and pneumocytes (p < 0.0001) than control groups, which were not rinsed before reperfusion. CONCLUSIONS: We conclude that damage to pulmonary allografts resulting from prolonged ischemia is accentuated by reperfusion with blood. We also conclude that preservation with a single flush of Euro-Collins or Carolina rinse solution does not offer adequate protection, whereas a second rinse before reperfusion significantly decreases the number of damaged cells within the allograft.

Animals↗
PubMed2001

Cytolytic peptides induce biphasic permeability changes in mammalian cell membranes.

The cytolytic peptides melittin and gramicidin S are naturally occurring agents that provide a comparative model for studies of complement, immunotoxin and cell-mediated membrane permeability. Most attempts to characterize cytolytic peptides have used model membrane systems including phospholipid vesicles or erythrocytes. Membrane vesicles permit the use of self-quenching concentrations of fluorescent permeability markers, while erythrocytes release measurable hemoglobin. Attempts at measuring early membrane permeability changes in nucleated mammalian cells have been limited. To measure the kinetics of mammalian cell membrane permeability changes induced by cytolytic peptides, we developed a 96-well fluorescence cytolysis assay using the cytoplasmic fluorescent dye calcein as the membrane permeability marker. To facilitate rapid assessment of membrane permeability, trypan blue was added to the assay solution to quench (a) released fluorescence and (b) retained intracellular fluorescence. Trypan blue also provided a complementary visual assessment of cell viability. Using this assay, a detailed kinetic analysis demonstrated permeability of the cell membranes within seconds of exposure to the cytolytic peptides. The rapid permeabilization of the cell membranes was confirmed by flow cytometry using the calcium indicator dye fluo-3. The assay also demonstrated a second slower phase of marker release over the next several hours. The fluorescence cytolysis assay was able to reliably detect the biphasic permeability changes associated with the melittin and gramicidin S peptides suggesting the potential utility of this assay in the assessment of other cytolytic agents.

Animals↗
PubMed1983

A comparison of cell killing by heat and/or X rays in Chinese hamster V79 cells, Friend erythroleukemia mouse cells, and human thymocyte MOLT-4 cells.

The radiation and/or heat sensitivity of Chinese hamster V79 cells, Friend erythroleukemia (FELC) mouse cells, and MOLT-4 human transformed thymocytes were compared. MOLT-4 cells were more radiosensitive (D0 = 0.50 Gy) than FELC (D0 = 0.65 Gy) and V79 cells (D0 = 1.43 Gy). Arrhenius analysis showed that MOLT-4 cells were more heat sensitive than FELC or V79 cells below 42.0 degrees C, but more heat resistant at higher temperatures. In addition, the MOLT-4 cells showed a single-heat inactivation energy between 41.0 and 45.0 degrees C, while FELC and V79 cells both showed a transition in the inactivation energy at about 43.0 and 43.5 degrees C, respectively. These differences may be related to the fact that the upper temperature limit for the development of thermal tolerance during continuous heating was lower for MOLT-4 cells than for FELC or V79 cells. Killing of FELC and V79 cells was dependent on the sequence in which heat and X rays were applied, but the greatest effect was obtained when both treatments were given simultaneously. Recovery occurred when treatments were separated by incubation at 37.0 degrees C. The MOLT-4 cells did not show a sequence dependence for heating and irradiation. Survival of MOLT-4 cells after heating and/or irradiation was compared using trypan blue dye exclusion or colony formation. Both assays showed similar qualitative responses, but survival levels measured by the trypan blue assay were much higher than those determined from the colony-forming assay.

Animals↗
PubMed1986

The use of bioluminescence to evaluate the influence of chemotherapeutic drugs on ATP-levels of malignant cell lines.

Influence of various cytostatic agents on intracellular ATP concentrations of malignant cell lines was studied. The HL-60 promyelocytic cell line, the SW-480 cell line, derived from an adenocarcinoma of the colon and the SW-620 cell line, derived from a lymphonodal metastasis of the colon carcinoma, were investigated. Cell lines were incubated with cytostatic agents and changes in intracellular ATP concentrations were measured after various incubation times by means of bioluminescence. A marked fall in intracellular ATP concentrations was observed, when HL-60 cells were incubated with drugs used in clinical protocols for treatment of acute leukaemia (daunorubicin, vinblastine, vincristine), whereas only a slight decrease of ATP concentrations was measured after incubation with bleomycin, dacarbazin and prednisolone. The decrease in intracellular ATP concentrations of SW-480 and SW-620 cells was much less pronounced after incubation with cytostatic agents compared to the HL-60 cells. This is in accordance with the clinical experience of the known resistance of colon carcinoma against cytostatic agents. Dose-response curves were obtained for the single cytostatic agents. Comparison of intracellular ATP concentrations and cell viability as determined by the trypan blue and eosin dye exclusion test showed that the trypan blue dye exclusion test underestimated cell kill compared to the eosin dye exclusion test and the bioluminescence assay.

Adenosine Triphosphate↗
PubMedDate not supplied

The assessment of viability in isolated rat hepatocytes subjected to cold or subzero non-freezing preservation protocols using a propidium iodide modified test.

A rapid and simple assay (6 min, two steps) is described for determination of cell viability of hepatocytes subjected to cold preservation protocols. In this method, cells are incubated with the fluorescent marker propidium iodide (PI) and the fluorescence intensity is measured before (direct fluorescence--Fd) and after (total fluorescence--Ft) addition of digitonin, which allows the dye to enter the hepatocytes. The Fd originated from non-viable cells that have membrane damage and taken up PI. The Ft originated from all cells in the sample. The ratio between the two fluorescence values is used as an indicator of cell viability. The assay was challenged versus two classical viability tests: LDH retention and Trypan Blue exclusion. Our assay shows good correlation only with Trypan Blue test. In addition, a fluorescence confocal microscopy protocol was used to evaluate the possible toxicity of PI in hepatocyte suspensions.

Animals↗
PubMedDate not supplied

Enhancement of the cytolytic effect of anti-bacterial cecropin by the microvilli of cancer cells.

A comparison between IC50s of cecropin B on tumor cells such as KG-1 leukemia and Ags stomach carcinoma and non-tumor cells like fibroblasts and red blood cells was conducted. The IC50s of cecropin B for KG-1 leukemia and Ags carcinoma cells were 20.8 +/- 2.3 microM (MTT) and 18.9 +/- 3.3 microM (trypan blue) and 16.0 +/- 3.5 microM (MTT) & 15.3 +/- 3.7 microM (trypan blue), respectively. The IC50 of cecropin B for 3T6 fibroblast cells was 92.0 +/- 9.1 microM by MTT assay and the HE50 of cecropin B for human red blood cells was 180.0 +/- 20.1 microM at OD414nm. The cytolysis induced by cecropin peptides was more effective for the cancer cells than for the normal cells. Based on the observations from scanning electron microscopy, this may mainly due to the cancer cells having a high population of the irregular microvilli on the cell surface. Since peptides bound to the cell membrane are non-specific, the attraction of peptides by microvilli may be one of the main driving forces before the lysis in membrane bilayers can be efficiently initiated.

Animals↗
PubMed1993

The involvement of Kupffer cells in carbon tetrachloride toxicity.

Carbon tetrachloride (CCl4) is a classical pericentral hepatotoxicant; however, precise details of its mechanism of action remain unknown. One possibility is that Kupffer cells participant in this mechanism since CCl4 elevates calcium, and the release of toxic eicosanoids and cytokines by Kupffer cells is calcium-dependent. Therefore, these studies were designed to evaluate the role of Kupffer cells in CCl4 toxicity in the rat in vivo. Kupffer cells were destroyed selectively with gadolinium chloride treatment (10 mg/kg GdCl3 iv) 1 day prior to administration of CCl4 (4 g/kg ig). Twenty-four hours after CCl4 treatment, rats were anesthetized, blood samples were drawn for aspartate aminotransferase (AST) determination, which is indicative of parenchymal cell damage, and trypan blue was infused into the liver to stain the nuclei of dead hepatocytes. AST levels were in the normal range and trypan blue staining was negligible in livers from vehicle- or GdCl3-treated rats. As expected, CCl4 treatment alone elevated AST levels to values over 4000 U/liter and caused massive cell death (60-90 trypan blue-positive cells/pericentral field). In dramatic contrast, the elevation in AST and cell death due to CCl4 were almost completely prevented by GdCl3 treatment. In attempts to understand this phenomenon, metabolic and detoxification pathways were assessed. CCl4 is metabolized via cytochrome P450 II.E.1; however, GdCl3 treatment did not alter this pathway as assessed from p-nitrocatechol formation from the selective substrate, p-nitrophenol. GdCl3 treatment also had no effect on hepatic glutathione levels. On the other hand, GdCl3 treatment significantly reduced infiltration of neutrophils resulting from exposure to CCl4. These data clearly support the hypothesis that Kupffer cells participate in the mechanism of toxicity of CCl4 in vivo, possibly by release of chemoattractants for neutrophils.

Animals↗
PubMed2006

Nosocomial outbreak of Pseudomonas aeruginosa endophthalmitis.

We describe an outbreak of nosocomial endophthalmitis due to a common source, which was determined to be trypan blue solution prepared in the hospital's pharmacy service. We assume that viable bacteria probably gained access to the trypan blue stock solution during cooling after autoclaving. The temporal cluster of Pseudomonas aeruginosa endophthalmitis was readily perceived on the basis of clinical and microbiological findings, and an exogenous source of contamination was unequivocally identified by means of DNA fingerprinting.

Aged↗
PubMed1991

Reduction of glutamine synthetase activity in astroglia exposed in culture to low levels of inorganic lead.

Astroglia serve as a site of lead (Pb) deposition in Pb-exposed animals. Thus, the potential exists for astroglial function to be impaired by elevated intracellular Pb levels. We previously showed that the specific activity of glutamine synthetase (GS), an astroglial enzyme with a key role in glutamate and ammonia metabolism in the brain, is reduced in fetal guinea pigs exposed to low levels of lead. This observation indicates either a direct effect of Pb on astroglia or an indirect systemic effect. The purpose of the present study was to test the hypothesis that Pb directly reduces GS activity in astroglia. Cultured rat astroglia were fed three times per week with medium containing 0, 0.25, 0.5, or 1 microM Pb acetate for 7, 14, or 21 days. Trypan blue dye exclusion, cell number, and GS activity were measured. Lead treatment reduced the ability of cells to exclude trypan blue in a dose- and time-dependent manner, with the greatest reduction (28%) on day 21 in the group treated with 1 microM Pb. Cell numbers were reduced a maximum of 15% on day 15, but were unaffected on days 7 and 21. The effects of Pb exposure on GS activity were much more pronounced. For example, cultures exposed to 0.25 of 1 microM Pb for 7 days showed, respectively, 17 and 52% reduction in activity from the control value. Slightly greater reductions were measured on days 14 and 21. The much greater sensitivity in vitro of GS activity than dye exclusion or cell numbers to low level lead supports a specific enzymatic inhibition. In order to test the possibility that Pb might directly inhibit GS activity Pb-treated cells, we also measured the effect of Pb on GS activity in cytosolic extracts of the cells. Pb inhibited GS activity in a dose-dependent manner ranging from 27% inhibition at 0.1 nM to 67% at 0.1 microM and 100% at 10 microM Pb. These results indicate that astroglial function is vulnerable to Pb exposure, even at low lead levels.

Animals↗
PubMed2003

Cholic acid-carboplatin compounds (CarboChAPt) as models for specific drug delivery: synthesis of novel carboplatin analogous derivatives and comparison of the cytotoxic properties with corresponding cisplatin compounds.

This report continues our work on new compounds which consist of three functional parts--a transport fragment, a spacer and a biologically active 'drug' component. Here cholic acid functions as the transport fragment, linked via an alkyl spacer to a carboplatin analog, representing the drug (carbo-ChAPt-Fig. 1). We describe the synthesis and characterization of the series of complexes [Pt(Cyclobutane-1,1-dicarboxylato)(diamine)], [diamine=CholCOO(CH(2))(n)CH(CH(2)NH(2))(2) and THP(CH(2))(n)CH-(CH(2)NH(2))(2), n=4, 6, 8, 11]. The compounds were characterized by elemental analysis and NMR-measurements. Cytostatic activity data are given. In general, the cytostatic activity is similar to that of the parent compound and is strongly influenced by the length of the alkyl chain spacer separating the drug and transport fragments, the ones with long chain spacers being more toxic than the parent complexes. Preliminary investigations indicate the ability of the ChAPt to break resistance of tumor cells against common platinum tumor drugs, e.g. cisplatin. They are effective even on cell lines that have developed resistance to other drugs such as cis- and carboplatin. They are more cytotoxic so they are potentially effective at lower dose concentrations. The mode of cell death was examined by trypan-blue exclusion test and DNA gelelectrophoresis. Typical fragmentation of DNA was observed and the cells were still able to exclude trypan-blue.

Antineoplastic Agents↗
PubMed1979

Aged-related physiological studies comparing Candida albicans chlamydospores to yeasts.

From electron-microscopical observations, a decreased metabolic activity in 3-day-old Candida albicans chlamydospores was suggested, and progressive deterioration in chlamydospores aged 2-8 months was shown. Oxygen utilization by chlamydospore-pseudomycelium (CSP) preparations was less than that by yeast, while 3-day-old CSP preparations used significantly less O2 than 24-h CSP preparations. Amino acid incorporation was greater in yeast than in CSP preparations. Leucine incorporation by 20-h yeasts was twice that of 5-day yeasts and 5 times that of 20-h and 5-day CSP. Amino acid decarboxylation was similar in yeasts and CSP and was determined by end-product analyses to be via amino acid oxidase. Light microscopy autoradiography of [14C]leucine incorporation demonstrated that the metabolic activity in CSP preparations was due to the young growing tips of the pseudomycelium and not to mature chlamydospores. Yeasts did not take up trypan blue could be stained if first autoclaved or treated with 10% acid or 10% base. Young chlamydospores grown in the presence of trypan blue developed unstained and became permeable to the dye at 2 1/2 days. These data suggest that chlamydospores of C. albicans do not function in the classical role attributed to spores; i.e., mature chlamydospores cannot germinate, but rather age, deteriorate, and die.

Amino Acids↗
PubMed1999

Reperfusion injury in livers due to gentle in situ organ manipulation during harvest involves hypoxia and free radicals.

Kupffer cell-dependent injury in livers gently manipulated during harvest develops upon transplantation; however, underlying mechanisms remain unknown. Thus, the purpose of this study was to identify factors involved in mechanisms of injury. Livers from female Sprague-Dawley rats (200-230 g) were cold stored for 24 h in University of Wisconsin solution. Subsequently, livers were perfused at 37 degrees C with oxygen-saturated Krebs-Henseleit buffer containing fluorescein-dextran to assess microcirculation. Cell death was assessed by uptake of trypan blue, a vital dye. Minimal dissection during harvest had no effects on sinusoidal lining cells; however, gentle organ manipulation dramatically increased trypan blue uptake about 5-fold (p <.05). In contrast, perfusion with N2-saturated buffer after cold storage totally prevented cell death due to manipulation. At harvest, portal venous pressure was increased significantly by 70% due to manipulation. Furthermore, vascular space and microcirculation were decreased by more than 50% (p <.05), reflecting the rate of entry and exit of fluorescein-dextran. Pimonidazole, a 2-nitroimidazole marker, was given to rats before harvest to detect hypoxia in liver. Pimonidazole adduct binding was increased significantly about 2-fold by manipulation. To detect free radical adducts by electron spin resonance (ESR) spectroscopy in bile, C-phenyl-N-tert-butylnitrone was given as spin trapping reagent to the donor before operation. Free radical formation was increased about 3-fold by organ manipulation (p <.05). Donors given gadolinium chloride, a selective Kupffer cell toxicant, or dietary glycine, which prevents activation of Kupffer cells, significantly blunted microcirculatory disturbances, hypoxia, and death of endothelial lining cells. These data indicate for the first time that gentle organ manipulation during harvest causes oxygen-dependent reperfusion injury to endothelial lining cells via mechanisms involving hepatic microcirculation, hypoxia, and Kupffer cells.

Animals↗
PubMed1982

The viability of cells in the waste products of CO2-laser evaporation of Cloudman mouse melanomas.

The viability of cells present in waste products originating during CO2-laser evaporation of Cloudman S91 mouse melanomas was investigated. Cytologic smears showed mainly carbonized particles and thermally damaged cells but some morphologically intact cells as well. Viability was tested with the trypan blue test, by in vitro culture and intramuscular and intraperitoneal inoculations in syngeneic mice. No dye-excluding cells were found in the trypan blue test. Growth was noted neither in vitro nor in vivo in the 127 mice, intramuscularly or intraperitoneally injected with 10(5) x 10(5)/10 microliter smoke or debris particles, and killed four weeks after inoculation. Viable tumor cells, derived from a Cloudman melanoma cell line and added to the smoke and debris suspensions, remained viable. Thus, a toxic influence of smoke and debris particles in viable tumor cells, which would make the viability tests meaningless, was excluded. It is highly unlikely that viable tumor cells do occur in the waste products of CO2-laser tumor vaporization.

Animals↗
PubMed1985

Determination of isolated myocyte viability: staining methods and functional criteria.

Single ventricular cells tolerant to physiological levels of calcium have been successfully isolated from adult rat heart. These cells exhibit morphological as well as functional characteristics of the intact myocardium. Under anaerobic incubations either in the absence of extracellular calcium or in the presence of 6% PEG, myocytes excluded Trypan blue despite severe derangements in biochemical functions. We suggest that Trypan blue entry may be a rather insensitive criterion of cell injury. We recommend the combination of rod-shape morphology, ATP levels and the ability to contract under external pacing as alternative simple tests of cell viability.

Adenosine Triphosphate↗