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PubMed2003

Bacterial contamination of a cornea tissue bank: implications for the safety of graft engineering.

PURPOSE: To analyze the difficulties involved in managing an episode of bacterial contamination in a cornea bank. We describe (1) the circumstances of bacterial contamination discovery, (2) the methods used to investigate the outbreak, (3) the corrective measures adopted, and (4) the method introduced to improve the reaction capacity in case of bacterial contamination. METHODS: All the samples collected were cultured in an attempt to identify the environmental reservoir of the contaminated epidemic clone. Bacteria were identified by Gram stain, oxidase test, and biochemical characteristics. The clonality of the strains was assessed by pulsed-field gel electrophoresis. RESULTS: The bacterial contamination was confirmed for 28 corneas, and 70 additional corneas were discarded. The source of the contamination was identified 17 days after the beginning of the episode. It consisted of a clonal bacterial strain that was found in trypan blue, the dye, used to examine all the tissues. The contaminating bacterium was Burkholderia cepacia, a well-known nosocomial pathogen. A total of 169 grafted corneas had been checked with the contaminated reagent. No cases of post-graft infection were recorded. CONCLUSION: Trypan blue played a major role in this outbreak. The mode and chronology of contamination remain unresolved. This exceptional event emphasizes the risk of bacterial contamination in tissue/cell banks, the necessity to improve methods for its prevention, and procedures to limit its consequences.

Burkholderia Infections↗
PubMed1982

Nonhuman primate in vitro fertilization: seasonality, cumulus cells, cyclic nucleotides, ribonucleic acid, and viability assays.

Since establishing our squirrel monkey (Saimiri sciureus) in vitro fertilization (IVF) system in 1972, 1788 oocytes have been laparoscopically recovered from follicles with 669 matured and 332 fertilized. Oocyte maturation was higher (P less than 0.05) from March to June. Among the oocytes, 50% showed first polar body extrusion by 21 hours after recovery. Maturation was significantly higher (P less than 0.05) in oocytes with corona radiata cells, compared with denuded oocytes. Sperm motility studies indicated that 2.6 and 5.2 mM caffeine enhanced motility by 10% to 30% over controls for the first 5 hours. Addition of 1 and 10 microM dibutyryl cyclic adenosine 3':5'-monophosphate (dbcAMP) enhanced fertilization by 50.3% and 50.0%, respectively. Preliminary studies of 3H-urindine incorporation and uptake by unfertilized oocytes showed levels of 3.43 +/- 0.21 and 55.01 +/- 7.16 fmole/embryo/3 hours, respectively. Exclusion of the vital dye trypan blue by oocytes and embryos gave a high correlation with morphology and survival. Autoradiographic analysis indicated nuclear grain densities in oocytes cultured with 3H-uridine. The results of the studies indicated that oocyte maturation was dependent on season and corona radiata cells, and that addition of dbcAMP enhanced IVF. Trypan blue was found be a useful embryo viability test.

Animals↗
PubMed2005

Reflux-free cannula for convection-enhanced high-speed delivery of therapeutic agents.

OBJECT: Clinical application of the convection-enhanced delivery (CED) technique is currently limited by low infusion speed and reflux of the delivered agent. The authors developed and evaluated a new step-design cannula to overcome present limitations and to introduce a rapid, reflux-free CED method for future clinical trials. METHODS: The CED of 0.4% trypan blue dye was performed in agarose gel to test cannula needles for distribution and reflux. Infusion rates ranging from 0.5 to 50 microl/minute were used. Agarose gel findings were translated into a study in rats and then in cynomolgus monkeys (Macacafascicularis) by using trypan blue and liposomes to confirm the efficacy of the reflux-free step-design cannula in vivo. Results of agarose gel studies showed reflux-free infusion with high flow rates using the step-design cannula. Data from the study in rats confirmed the agarose gel findings and also revealed increasing tissue damage at a flow rate above 5-microl/minute. Robust reflux-free delivery and distribution of liposomes was achieved using the step-design cannula in brains in both rats and nonhuman primates. CONCLUSIONS: The authors developed a new step-design cannula for CED that effectively prevents reflux in vivo and maximizes the distribution of agents delivered in the brain. Data in the present study show reflux-free infusion with a constant volume of distribution in the rat brain over a broad range of flow rates. Reflux-free delivery of liposomes into nonhuman primate brain was also established using the cannula. This step-design cannula may allow reflux-free distribution and shorten the duration of infusion in future clinical applications of CED in humans.

Animals↗
PubMed1990

Biological effects of shock waves: cell disruption, viability, and proliferation of L1210 cells exposed to shock waves in vitro.

L1210 cells were exposed in suspension to shock waves generated with a Dornier XL1 lithotripter. After 1000 discharges at 25 kV, the number of nondisrupted cells was 15% and the number of trypan blue excluding cells was 7% as compared to 100% in sham treated controls; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Overall proliferation of cells which were trypan blue negative after exposure exceeded 70% of the proliferation of sham treated controls, except after 1000 shocks at 25 kV, where proliferation was reduced to 42%. The latter reduction in proliferation was found to be due to a reduced growth for 24 h after exposure, with a return to normal proliferation during the following days. Limiting dilution analysis revealed that the reduced growth was mainly due to a transitory increase of the doubling time and not to a reduction of the number of proliferating cells. Cell disruption by shock waves was completely inhibited by exposing the cells at an elevated pressure of 101 atmospheres, pointing to the possible involvement of cavitation in the shock wave effect.

Animals↗
PubMed1997

Production of lipid-derived free radicals in L1210 murine leukemia cells is an early oxidative event in the photodynamic action of Photofrin.

Photofrin photosensitization initiates a sequence of oxidative events that begins with singlet oxygen formation and ultimately leads to cell death. We hypothesize that membrane lipid-derived free radical formation is an early event in this process. In the presence of iron and ascorbate, lipid free radicals are generated during cellular photosensitization of L1210 cells as detected by electron paramagnetic resonance spin-trapping techniques. Tocopherol levels decline in an inverse manner to lipid radical formation. Trypan blue dye exclusion by membranes also decreases inversely to lipid radical formation but at an initially slower rate than alpha-tocopherol depletion. Propidium iodide nuclear staining as an alternative measure of cell integrity was a later event, occurring when alpha-tocopherol levels had fallen by 90%, trypan blue survival had decreased to below 10%, and lipid radical formation was nearing plateau levels. Likewise, the formation of cellular debris did not occur substantially until alpha-tocopherol was virtually exhausted and radical intensity had nearly reached a maximum. These temporal observations suggest the following sequence of events that leads to Photofrin photosensitization-induced cytotoxicity in the presence of iron and ascorbate: (1) singlet oxygen-derived lipid hydroperoxide formation and subsequent radical production; (2) cellular alpha-tocopherol depletion; (3) trypan blue-detectable membrane leakage; (4) nuclear exposure to propidium; (5) cell disintegration. These observations are consistent with membrane lipid-derived free radical formation being an early and perhaps seminal event in photosensitization by Photofrin, which leads to a concatenated series of events terminating in cell destruction.

Animals↗
PubMedDate not supplied

Hypothermic storage of periportal and perivenous rat hepatocytes.

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96 h-4 degrees C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37 degrees C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.

Animals↗
PubMed1997

Reducing cellular autofluorescence in flow cytometry: an in situ method.

Cellular autofluorescence affects the sensitivity of flow cytometric assays by interfering with detection of low level specific fluorescence. These detection limits increase with use of protocols, such as thermocycling and fluorescent in-situ hybridization (FISH), that can increase intrinsic cellular fluorescence to 5,000-20,000 fluorescein isothiocyanate (FITC) equivalents. In order to improve signal to noise ratios when using FITC labeled probes in these procedures, we employed a method using the polyanionic azo dye, trypan blue, to reduce intracellular autofluorescence. Dyes such as these are commonly used in immunofluorescent microscopy to reduce background fluorescence. By using this method, we realized an approximately 5-fold increase in signal to noise ratio (S/N) in the direct detection of RNA target probes using flow cytometry. Trypan blue aided in the resolution of dim surface antibodies, internal markers and probes, and functions to reduce background autofluorescence after thermocycling and hybridization. This technique is rapid and easily applicable for reducing intracellular autofluorescence, and can be used in single and dual color applications.

Coloring Agents↗
PubMed1994

Protein accumulation in cerebrospinal fluid during -90 degrees head-down tilt in rabbit.

Plasma proteins are only somewhat larger than the intercellular spaces of the cerebral microvessels that constitute the blood-brain barrier or of the choroid plexus villi that elaborate cerebrospinal fluid (CSF). We hypothesized that the integrity of these barriers in anesthetized rabbits might be compromised during head-down tilt (HDT). Plasma protein and osmolality, hematocrit, and CSF protein concentration were compared in rabbits exposed to 1 h of HDT (n = 20) and prone rabbits (n = 10). In addition, the concentration of trypan blue dye, injected intravenously at the end of HDT or the prone position, was measured in brain homogenate. Finally, arterial blood pressure was measured via a catheterized carotid artery. HDT disrupted the barrier between blood and CSF, as indicated by a significantly (P < 0.01) greater brain trypan blue concentration in the HDT rabbits [172.2 +/- 14.4 (SD) micrograms/g dry wt] than in the prone rabbits (29.8 +/- 4.4 micrograms/g dry wt). Moreover CSF protein 5 min after HDT onset was significantly increased compared with control in HDT rabbits (54.6 +/- 1.9 vs. 81.4 +/- 5.2 mg/dl; n = 8) but not in prone rabbits (55.6 +/- 2.7 vs. 57.2 +/- 5.0 mg/dl; n = 6). Changes in the plasma protein-to-hematocrit ratio in the HDT animals, but not in the prone animals, were also compatible with a loss of fluid from the vascular compartment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Cytotoxic effects on the plasma membrane of human diploid fibroblasts--a comparative study of leakage tests.

Confluent monolayers of human diploid lung fibroblasts were treated with cytolytic agents. The induced membrane damage was investigated by different test systems. Changes of membrane permeability were compared with morphological alterations. Four tests employed leakage of cytoplasmic markers of different sizes as criteria of membrane damage. Radioactive markers of the following decreasing size order were used: [3H]RNA (MW greater than 200,000) greater than 51Cr greater than [3H]nucleotides greater than [1-14C]alpha-amino-isobutyric acid (AIB, MW 103). Uptake of trypan blue was employed as a fifth criterior of changed membrane permeability. A comparison between the tests indicated the following order of sensitivity for detection of membrane damage: leakage of AIB-label greater than leakage of nucleotide label greater than leakage of 51Cr-label=uptake of trypan blue= morphological changes greater than leakage of RNA-label. Two distinct types of leakage patterns were evident: 1. Upon incubation with Triton X-100 all four release curves coincided. This was considered as representing large functional "holes". 2. With the polyene amphotericin B the smallest marker (AIB) was released to a strikingly greater extent than other markers. This was regarded as representing very small functional "holes". Melittin from bee venom and theta-toxin from Clostridium perfringens induced leakage patterns of two intermediate types. The results indicate that a combination of several different size markers may be useful for characterizing induced membrane lesions.

Amphotericin B↗
PubMed1975

[Detection of neoplastic cells in the bloodstream].

A study was made of the efficacy of trypan blue, acridine orange, tetracycline and oxytetracycline for detection of tumour cells injected into the blood stream of rats. The cells were identified in the mesenteric microvessels by intravital microscopy. Fluorescence of fluorochromized cells was observed in the blue-violet (lambda max = 400 nm) and ultra-violet (lambda max = 365 nm) irradiation of the fluorescent lamp and in the laser irradiation (lambda = 337 nm). The cells stained with acridine orange had a higher fluorescence intensity and a more distinct structure than those labelled with tetracyclines. Identification of cells with trypan blue was more difficult. The fluorescent method of determination is rather simple and permits to indentify tumour cells directly in the blood stream.

Acridines↗
PubMed1992

Effects of high glucose concentration on survival and respiration of human endothelial cells.

To determine whether or not endothelial cell survival was decreased after incubation with high glucose concentrations in culture media, we studied the influence of D-glucose or L-glucose (a non-metabolizable stereoisomer of D-glucose) on cell survival using the trypan-blue exclusion test. Simultaneously, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay was used to measure both the mitochondrial respiratory chain activity and cell viability. Respiratory chain activity per cell increased when D-glucose concentrations rose but at the same time trypan-blue excluded cells were decreased. Comparison with data in the literature showed that the MTT assay was not reliable for studies involving endothelial cell survival when glucose reduction was affected on these cells. It seems important to check MTT assay reliability carefully when it is used for drugs affecting glucose metabolism, or with other cell types.

Cell Survival↗
PubMed1995

Active pancreatic digestive enzymes show striking differences in their potential to damage isolated rat pancreatic acinar cells.

Active digestive enzymes are involved in the pathophysiology of acute pancreatitis. Previous studies have mainly focused on the role of trypsin in the autodigestive process. The present study compares the noxious potential of different pancreatic enzymes to damage acinar cells. Acinar cells were isolated from rat pancreas by collagenase digestion. Cell viability was studied by (1) exclusion of trypan blue, (2) release of lactate dehydrogenase, and (3) release of newly synthesized proteins identified with methionine labeled with sulfur 35. Cells were then incubated in oxygenated N-2-hydroxyethylpiperazine-N-'-2-ethanesulfonic acid-Ringer solution containing different concentrations of various active digestive enzymes. Uptake of trypan blue was the most sensitive and reliable test of cell damage when compared with release of lactate dehydrogenase or radiolabeled newly synthesized proteins. All active digestive enzymes studied caused dose-dependent cell damage. The noxious potential, however, was strikingly different for the various enzymes. Pancreatic elastase in nanomolar concentrations caused marked cell damage after 45 to 90 minutes of incubation. Lipase and chymotrypsin caused a similar damage only at micromolar concentrations, whereas even millimolar concentrations of trypsin failed to cause significant damage. The present results confirmed recent work showing that lipase and phospholipase A2 probably cause cell damage through release of free fatty acids and lysolecithin. Although activation of trypsin might be the trigger to start the activation cascade in acute pancreatitis, trypsin itself is markedly less noxious to acinar cells when compared with other digestive enzymes. Elastase by far had the greatest noxious potential of all enzymes evaluated. Studies analyzing therapeutic effects of protease inhibitors should evaluate not only the inhibitory potential against trypsin but also that against other digestive enzymes, particularly elastase.

Acute Disease↗
PubMed2001

Adaptation of an [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish.

AIMS: Adaptation of a colorimetric assay using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) to evaluate the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. METHODS AND RESULTS: The optimal conditions for the colorimetric assay were determined and this method was compared with the trypan blue exclusion assay. The protein concentration of extracellular products causing the death of 50% of the cell population (CI50) was determined. CONCLUSIONS: This assay enables quantitative and objective comparison of the cytotoxicity of the extracellular products of micro-organisms pathogenic to fish. It was shown to be more accurate than conventional counting with the trypan blue exclusion assay. SIGNIFICANCE AND IMPACT OF THE STUDY: This method may also be useful for characterizing the cytotoxicity of specific components of extracellular products.

Animals↗
PubMed1996

Interlaboratory validation of a new assay for DNA-protein crosslinks.

In 1992, a simple and sensitive assay for detecting DNA-protein crosslinks was developed [1]. In an effort to facilitate the greater use of the assay, a number of studies were conducted to evaluate its reliability and reproducibility. During this work, the assay was used to assess whether various metals and other compounds could induce crosslinks in cultured human lymphocytes (Epstein-Barr virus-transformed Burkitt's Lymphoma cell line). Potassium permanganate, mercury chloride, lead nitrate, magnesium perchlorate, aluminum chloride, and cadmium chloride did not induce DNA-protein crosslinks at either cytotoxic or non-cytotoxic levels. Copper sulfate, arsenic trioxide, and potassium chromate induced DNA-protein crosslinks only at cytotoxic concentrations. Acute lethality of the cells was assessed immediately after exposure to metals by trypan blue exclusion while long-term lethality was assessed by cell proliferation and trypan blue exclusion following an incubation period of 5 days after exposure to the metal compound. All metals exhibited more toxicity in the long-term lethality assay compared to the short-term assay. The cultured human lymphocytes treated with various doses of lead acetate, cadmium chloride, arsenic trioxide and copper sulfate, as well as cis-platinum and chromate, were sent to four different laboratories to compare the reliability and reproducibility of the DNA-protein crosslink assay. Depending on the chemical studied, there were quantitative differences in the results observed among the various laboratories using the assay. However, all laboratories generally showed that cis-platinum, chromate, arsenic trioxide and copper sulfate induced DNA-protein crosslinks at levels that produced acute cytotoxicity, whereas cadmium chloride and lead acetate did not.

Arsenic Poisoning↗
PubMed1997

Intermittent anoxia reduces oxygen free radicals formation during reoxygenation in rat hepatocytes.

The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.

Animals↗
PubMed2000

Contribution of adenosine A(2) receptors and cyclic adenosine monophosphate to protective ischemic preconditioning of sinusoidal endothelial cells against Storage/Reperfusion injury in rat livers.

A brief period of liver ischemia decreases sinusoidal endothelial cell killing after cold liver storage and improves graft survival after liver transplantation, a phenomenon called ischemic preconditioning. In this study, we investigated the mechanism of sinusoidal endothelial cell protection after ischemic preconditioning. Livers were preconditioned by 5 minutes of ischemia and 5 minutes of reperfusion. Subsequently, livers were stored for 30 hours in cold University of Wisconsin (UW) solution and reperfused briefly with physiological buffer containing Trypan blue. Ischemic preconditioning decreased sinusoidal endothelial cell killing after storage/reperfusion, as assessed by Trypan blue staining of nonparenchymal cells. Adenosine A(2) receptor blockade prevented the protective effect of ischemic preconditioning. By contrast, adenosine A(1) receptor blockade did not prevent protective ischemic preconditioning. Other rat livers were treated with adenosine A(1) and A(2) receptor agonists or dibutyryl-cyclic adenosine monophosphate (DB-cAMP) before storage. The adenosine A(2) receptor agonist, CGS-21680, and DB-cAMP decreased sinusoidal endothelial cell killing to the same extent as ischemic preconditioning, but the adenosine A(1) receptor agonist, 2-chloro-N(6)-cyclopentyladenosine (CCPA), had no effect. The adenosine A(2) agonist and prostaglandin E(2), another agent that preconditions sinusoidal endothelial cells against storage/reperfusion injury, but not the adenosine A(1) agonist, increased cAMP levels in cultured sinusoidal endothelial cells. In conclusion, an adenosine A(2) receptor pathway coupled to increased cAMP mediates sinusoidal endothelial cell protection by ischemic preconditioning.

Adenosine↗
PubMed2001

Ischemic preconditioning of rat livers against cold storage-reperfusion injury: role of nonparenchymal cells and the phenomenon of heterologous preconditioning.

Brief periods of ischemia followed by reperfusion render tissues resistant against subsequent prolonged ischemia, a phenomenon called ischemic preconditioning. The effect of ischemic preconditioning on liver transplantation was investigated in relation to sinusoidal endothelial cell injury and Kupffer-cell activation, which are prominent features of storage and reperfusion injury leading to liver graft failure. Rat livers were preconditioned by 5 or 10 minutes of ischemia and 5 minutes of reperfusion and stored in University of Wisconsin (UW) solution for 30 hours. Livers were then reperfused for 15 minutes with physiological buffer containing trypan blue. Under these conditions, injury occurs predominantly to sinusoidal endothelial cells, reflected by trypan blue staining of nonparenchymal cells in histological sections. Ischemic preconditioning decreased nonparenchymal cell killing by more than 50%. When half the liver was preconditioned, sinusoidal endothelial cells were also protected in the contralateral half. Other stored livers were reperfused with nitroblue tetrazolium, which is converted to insoluble formazan by superoxide radicals. Ischemic preconditioning decreased the intensity of formazan deposition over Kupffer cells. Finally, stored livers were transplanted into nontreated rats. Ischemic preconditioning improved recipient long-term survival after 30 hours of cold ischemic storage in UW solution from 30% to 80% and decreased serum tumor necrosis factor-alpha levels in posthepatic blood 4 hours postoperatively from 98 to 54 pg/mL. In conclusion, ischemic preconditioning protects sinusoidal endothelial cells and suppresses Kupffer-cell activation after storage and reperfusion. As a result, graft survival improves after liver transplantation. Moreover, ischemia to half the liver confers protection to the other half. Such heterologous preconditioning provides a new means to protect liver tissue against ischemia-reperfusion injury without imposing ischemia on the target tissue.

Animals↗
PubMed1984

Na and K changes in animal virus-infected HeLa cells.

Na-K changes and cytotoxic effects were monitored in HeLa cells infected with encephalomyocarditis (EMC) virus, human rhinovirus or vesicular stomatitis virus (VSV). Whereas in all three cases Na-K changes followed viral inhibition of cellular protein synthesis, such changes preceded trypan blue staining of rhinovirus-infected or VSV-infected cells and paralleled trypan blue staining of EMC virus-infected cells. In each case a progressive reduction in cellular electrical volume was associated with Na-K changes. K leakage from EMC virus- or rhinovirus-infected cells clearly exceeded Na gain by these cells. These findings are basically similar to those previously reported for poliovirus-infected HeLa cells. It is concluded that Na-K changes induced by picornaviruses or VSV do not trigger shut-off of HeLa cell protein synthesis, but may contribute to late manifestations of cytotoxicity.

Encephalomyocarditis virus↗