Nitroblue Tetrazolium Reaction Research Articles

Активність системи антиоксидантного захисту в організмі молоді коропа за згодовування олії розторопші плямистої

Purpose. Determination of the biological effect of milk thistle oil at different amounts of its introduction into the diet (0.5, 1.0 and 1.5%) on the antioxidant status of the body of juvenile carp in the conditions of recirculating aquaculture systems. Methodology. For the experimental studies, 4 groups of juvenile carps were formed, 150 individuals in each, with an average weight of 0.012 g, which were kept in 150 dm3 tanks with artificial water aeration. The period of their adaptation to holding conditions lasted 7 days. Over the next 21 days, the fish of the control and experimental groups were fed with compound feed with a protein content of 23%. The daily feed of fish of the experimental groups were additionally supplemented with milk thistle oil at amounts of 0.5 (Experiment I); 1.0 (Experiment II) and 1.5% (Experiment III). The juveniles were fed with the addition of oil three times a day. At the end of the experiment, skeletal muscle samples of juveniles were taken for the biochemical study. The concentration of diene conjugates was studied by the method based on the reaction of the optical density of the heptanisopropanol extract of lipids. Determination of the concentration of TBC-active products was carried out spectrophotometrically by a color reaction with thiobarbituric acid. The activity of superoxidedismutase was determined by the percentage of inhibition of the reduction reaction of nitroblue tetrazolium in the presence of phenazine metasulfate. Catalase activity depended on the change in the concentration of hydrogen peroxide (H2O2). Protein content was determined by the Bradford method. Processing of experimental results was carried out by the method of variational statistics. A statistically significant difference in obtained values was assessed by the Student’s t-test. Findings. It was found that the main parameters of water quality were within the normal values and the environment during the cultivation of the young-of-the-years was optimal. A positive effect of the use of milk thistle oil on the average weight was noted, which exceeded the average weight of fish of the control group by 3.8; 11.5; 7.8%. The survival rate of the fish of the experimental groups was higher in Experiment II and Experiment III by 7.4; 4.9% relative to the control group. An increase in superoxidedismutase activity was observed in all experimental groups, namely in Experiment I by 20.0%, Experiment II and Experiment III by 55.0% and 57.0% (p<0.01), respectively. At the same time, a decrease in the intensity of lipid peroxidation processes was observed in all experimental groups of fish. It was found that the addition of 1.0 and 1.5% milk thistle oil to the diet of juvenile carps had a positive effect on productive and biochemical parameters of their body. Originality. For the first time, the optimal norms for the introduction of milk thistle oil into the daily diet of juvenile carps and the effect of feeding this phytosupplement on the activity of antioxidant enzymes and the content of products of lipid peroxidation were determined. Practical value. The obtained results indicate the advisability and effectiveness of using milk thistle oil as a phytoaddition in growing and feeding juvenile carps. Keywords: juvenile carp, milk thistle oil, antioxidant enzymes, content of lipid peroxidation products, weight gain, survival rate.

Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37oC showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O2− localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions.

Long-lived photoinduced charge-carriers in Er3 + doped CaTiO3 for photocatalytic H2 production under UV irradiation

The incorporation of Er3+ ions in the CaTiO3 structure provides an enhanced H2 photoproduction due to the formation of long-lived charge-carriers in the semiconductor. Nitroblue tetrazolium reaction, photoluminescence and time resolved microwave conductivity techniques were used to investigate the generation of the superoxide species and the lifetime of the charge-carriers. The improved photoactivity has been explained in terms of the dopant agent, finding that phenomena such as a higher concentration of the superoxide radicals, a more efficient separation of the photogenerated charge-carriers, and a slower recombination process take place in the Er3+ doped CaTiO3 compared to the undoped CaTiO3.

Immunological Effects of Acute Testicular Torsion on the Contralateral Testis in Rats

The mechanism by which unilateral testicular torsion leads to injury of the contralateral testis is still controversial. The present study aimed to evaluate immunohistopathological changes in the contralateral testis of rats after acute testicular torsion. 40 pubertal rats were randomly divided into 3 groups. Group 1 (n=10) functioned as controls. Group 2 rats (n=10) underwent a sham operation, and Group 3 (n=20) underwent 720° torsion of the left spermatic cord (3 h) followed by detorsion (24 h). Immunofluorescence tests were done to investigate contralateral lesions in half of the animals in Group 3. Nitro blue tetrazolium (NBT) was injected in both testes to identify the presence of oxygen-derived free radicals. 24 h after reperfusion, bilateral samples of the testes were obtained for histological analysis. Fisher's exact test was used for statistical analysis with p-values <0.05 considered significant.Immunofluorescence tests were positive for the contralateral (right) testis but with a lower intensity than that of the testis submitted to torsion. NBT reaction was intense all over the left testis while a minor reaction was visible in the right testis. In Group 3 the left distorted testicle showed diffuse edema with cell elimination and extensive necrosis of the seminiferous tubules. Histological lesions were moderate to mild in the contralateral testis. An autoimmune IgG-mediated mechanism deposited on the basement membrane of seminiferous tubules seems to be involved in the genesis of contralateral testis injury.

Application of an Oxygen Electrode to Evaluate Superoxide Anion-scavenging Ability

The ability to scavenge superoxide anion radicals ((*)O(2)(-)) was determined using an oxygen electrode. The method is based on the determination of (*)O(2)(-) generated by the reaction of nitrilotriacetatoiron(III) with hydrogen peroxide and a decrease in the concentration of (*)O(2)(-) by a scavenging reaction, converting into a change in the generation of oxygen molecules through an electron-transfer reaction from (*)O(2)(-) to nitrilotriacetatoiron(III). Oxygen generation, which enhanced proportionally with an increase in the concentration of hydrogen peroxide, was inhibited depending on the concentration of superoxide dismutase. Hence, we applied the present reaction system to evaluate the (*)O(2)(-)-scavenging abilities of an antioxidant, measuring the degree of inhibition of oxygen generation using an oxygen electrode. A good correlation was obtained between the present method and conventional colorimetry, monitoring the formation of blueformazan by the reaction of nitro blue tetrazolium with (*)O(2)(-), to estimate the (*)O(2)(-)-scavenging activities of antioxidants.

Moxifloxacin Alleviates Oleic Acid-provoked Neutrophilic Respiratory Burst in the Rat Lung through the Inhibition of Cytosolic Phospholipase A2

Background: According to the notion of the immunoregulatory functions of moxifloxacin (MFX), the effect of MFX on the neutrophilic respiratory burst in conjunction with the expression of cytosolic phospholipase A(2) (cPLA(2)) was investigated. Methods: The effects and possible mechanisms of MFX on neutrophilic respiratory burst in oleic acid (OA)-induced acutely injured rats lung and OA-stimulated, isolated murine neutrophils were probed, associated with the expression of cytosolic phospholipase A(2) in vivo and in vitro. Results: In the OA-induced acutely-injured lungs, neutrophils were accumulated, which was attenuated by MFX. The parameters denoting a neutrophilic respiratory burst, such as nitro blue tetrazolium reaction, cytochrome-c reduction, neutrophil aggregation, H(2)O(2) production in neutrophils revealed increased neutrophilic respiratory burst by OA, and MFX decreased all of these parameters. In addition, the enhanced expression of cPLA(2) in the lung and isolated murine neutrophils by OA were decreased by MFX. Conclusion: MFX suppresses the OA-induced neutrophilic respiratory burst by the suppression of cPLA(2) in neutrophils.

Effect of the association of reduced glutathione and ciprofloxacin on the antimicrobial activity inStaphylococcus aureus

We report the effect of glutathione and the role of reactive oxygen species (ROS), assayed by a nitro blue tetrazolium reaction, on the antibacterial action of ciprofloxacin, gentamicin and chloramphenicol in Staphylococcus aureus 22 resistant to ciprofloxacin and gentamicin, and in S. aureus ATCC 29213 sensitive to the above three antibiotics. The association of glutathione with ciprofloxacin or gentamicin significantly reduced the value of the minimum inhibitory concentration (MIC) in resistant S. aureus 22, measured using the macrodilution method, with a concomitant increase of intracellular ROS and a decrease of extracellular ROS. However, glutathione did not induce modifications in MIC or ROS generated by chloramphenicol. Furthermore, in the sensitive S. aureus ATCC 29213, the association of glutathione with ciprofloxacin, gentamicin or chloramphenicol did not induce any significant variations of MIC or ROS. There was a correlation between the stimulus of intracellular ROS and the decrease of MIC caused by exogenous glutathione. According to the results obtained, it is possible to modify the sensitivity of resistant strains of S. aureus by the addition of exogenous glutathione.

OXALATE TOXICITY IN CULTURED MOUSE INNER MEDULLARY COLLECTING DUCT CELLS

Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin. However, to our knowledge it is unknown whether inner medullary collecting duct (IMCD) cells, which are physiologically exposed to higher concentrations of oxalate, also behave in a similar manner. A line of IMCD cells was exposed to oxalate (0.2 to 10 mM) for various time points. Trypan blue, and hematoxylin and eosin stains were used to assess cell morphology and membrane integrity. The production of reactive oxidative species was determined using the nitro blue tetrazolium reaction and crystal violet staining was used to measure cell density. Exposure of IMCD cells to oxalate produced time and concentration dependent changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in alterations in cell viability with net cell loss following exposure to concentrations of 2 mM and greater. Free radical production was time and concentration dependent. Crystal formation occurred in less than 1 hour and cells in proximity to crystals lost membrane integrity. Compared to IMCD cells LLC-PK1 and HK2 cells showed significant toxicity starting at lower oxalate concentrations (0.4 mM and above). To our knowledge the results provide the first direct demonstration of toxic effects of oxalate in IMCD cells, a line of renal epithelial cells of the inner medullary collecting duct, and suggest that cells lining the collecting duct are relatively resistant to oxalate toxicity.

New aspect of the synergistic antibacterial action of ampicillin and gentamicin

A new aspect in the action of ampicillin and gentamicin was detected in Enterococcus faecalis. Reactive oxygen species (ROS) increased in sensitive strains during treatment with each antibiotic up to a certain concentration of antibiotic, above which ROS diminished as a consequence of oxidative stress. Tiron, a scavenger of the superoxide anion O 2 −, counteracted the effect of the generated ROS. The oxidative stress was a consequence of an increase in ROS in the cytoplasm of bacteria, as observed by the nitroblue tetrazolium reaction. The viability of sensitive strains was significantly reduced at concentrations of antibiotics that increased the ROS, and this increment was parallel to the bactericidal effect. Sensitive E. faecalis strains showed an immediate increase of ATP in the presence of both antibiotics, thus an energy-dependent process had been triggered, indicating a bacterial reaction against the stress. The combination of both antibiotics augmented the effect of ROS, which helps to explain the synergism between ampicillin and gentamicin.

Assembly of Human Immunodeficiency Virus Precursor Gag Proteins

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 A is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers.

Down-regulation of nitric oxide synthase in the diabetic rabbit kidney: potential relevance to the early pathogenesis of diabetic nephropathy

SUMMARYObjectives: Nephropathy is a well-recognised complication of diabetes mellitus (DM). The aim of this study was to investigate the effect of DM on the density and distribution of nitric oxide (NO) synthase (NOS) in the rabbit kidney. Quantification of the NOS radioligand on slide-mounted sections was compared with the nitroblue tetrazolium reaction, where the intensity of the reaction varies with the nicotinamide adenine dinucleotide diaphorase (NADPH-d) activity of NOS.Materials and methods: DM was induced with alloxan in six New Zealand White (NZW) rabbits. Plasma creatinine, urea and electrolytes were monitored at monthly intervals. The kidneys were removed following 6 months of DM. Transverse serial sections were cut and low-resolution autoradiography was performed using a radioligand for NOS ([3H]-NOARG). Histochemical localisation of NADPH-d activity was also performed. Densitometric analysis was performed on the autoradiographs and the results compared with those obtained from six age-matched control rabbits.Results: There was a significant (p < 0.01) rise in plasma creatinine levels in the diabetic rabbits, although the mean values remained within the reference range. There was a significant (p < 0.0001) down-regulation of NOS binding sites in both the cortex and medulla of the DM kidney when compared with the controls. A similar decrease in NADPH-d activity was seen in the diabetic renal cortex and medulla. In addition, NADPH-d activity also appeared to be reduced in the diabetic glomeruli when compared with controls.Conclusions: NOS binding sites and NADPH-d activity are significantly decreased in the DM renal cortex and medulla. These changes are associated with a mild deterioration in renal function and may be an early event that could subsequently play a role in the progression of DM nephropathy. Manipulating the NO pathway during the early stages of DM nephropathy may be beneficial.

A convenient assay of glycoserum by nitroblue tetrazolium with iodoacetamide

Background: To determine glycoserum, the nitroblue tetrazolium (NBT) assay is quick, economical, convenient and easily automated. This method, however, is vulnerable to interference by thiol group, which should not be ignored during the analysis. Methods: Thiol group in glycoserum was blocked with iodoacetamide (IAM) before NBT was added. The reaction was carried out in a thermal bath and terminated on ice. The absorbance was measured at either 570 or 530 nm. Results: IAM (3–10 mmol/l) did not give any detectable interference in the NBT assay. The absorbance at both 570 and 530 nm was linearly related to the concentration of either glycoserum or 1-deoxy-1-morpholino- D-fructose (DMF) that had been treated with IAM. The assay showed a good discrimination between diabetic and normal subjects ( t-test, P<0.001). Uric acid and lipemia under physiological conditions did not interfere with NBT reaction. The assay was affected by hyperlipemia and hematolysis. Conclusions: IAM-modified method prevented NBT assay from the interference by thiol group and uric acid.

Oxidative stress induced by ciprofloxacin in Staphylococcus aureus

Staphylococcus aureus with multiple sensitivity to ciprofloxacin, was investigated to detect alterations in the production of superoxide anion (O 2 −), other reactive oxidant species (ROS), and superoxide dismutase (SOD), and to relate them with ciprofloxacin accumulation and sensitivity. Oxidative stress was studied by means of Nitroblue Tetrazolium reaction (NBT) and chemiluminescence (CL); lucigenin was employed to detect O 2 −, and luminol was used to measure other ROS. Sensitive strains exhibited higher intracellular O 2 − increase than resistant ones when incubated with ciprofloxacin. SOD was determined in normal conditions and induction was investigated in the presence of ciprofloxacin. These assays demonstrated that resistant and sensitive strains exported a great amount of SOD and that the induction of SOD intracellular was insufficient to counteract the augment of O 2 − in the cytoplasm of sensitive strains. Accumulation of ciprofloxacin, researched by spectrofluorometry, showed high levels of antibiotic in sensitive strains which increased the O 2 − causing more oxidative stress than in resistant S. aureus.

In situ histochemical monitoring of ozone- and TMV-induced reactive oxygen species in tobacco leaves

The pattern and time course of reactive oxygen species (ROS) production were monitored histochemically in vivo in tobacco ( Nicotiana tabacum L.) cv. Bel W3 and cv. Bel B leaves, comparing the hypersensitive (HR)-like reaction induced by the abiotic stressor ozone and the response to tobacco mosaic virus (TMV). The applied assays (nitro blue tetrazolium, NBT; 2’,7’-dichlorofluorescin diacetate, DCFH-DA; 3,3’-diaminobenzidine, DAB) gave positive results, evidencing scattered ROS production in a spotted pattern. DCFH-DA and DAB predominantly evidenced localised, long-lasting hydrogen peroxide production in cells prone to die, producing very similar pictures in TMV infected leaves of both tobacco cultivars and in ozone-sensitive Bel W3. Nitro blue tetrazolium reaction pointed out that a transient oxidative burst involving superoxide anion is induced early by ozone also in the tolerant cv. Bel B, despite the low malondialdehyde level.

The reduction of nitroblue tetrazolium by red blood cells: a measure of red cell membrane antioxidant capacity and hemoglobin-membrane binding sites

The reduction of nitroblue tetrazolium (NBT) with intact Red Blood Cells (RBCs) is biphasic with an initial rapid reduction followed by a slower second phase. This biphasic kinetics has been explained with the initial rapid phase attributed to antioxidants in the red cell which reduce membrane bound NBT and the slower phase associated with the reaction of NBT with membrane bound hemoglobin. This model has been confirmed by a utilization of a number of red cell modifications which either increase the red cell antioxidants (vitamin C and vitamin E) or damage the red cell membrane (cumene hydroperoxide and N-ethylmaleimide). The utilization of this assay for human blood samples was investigated by studying a series of 20 human subjects ranging between 34 and 87 years of age. It was possible to fit all of these samples with two adjustable parameters which reflect the red cell membrane antioxidant capacity (x) and the hemoglobin membrane interactions (m). The antioxidant capacity shows a significant (p < 002; R = -.67) decrease with age. This finding is consistent with a decrease in the level of antioxidants in aged subjects. In addition, the number of hemoglobin membrane sites are negatively correlated with the antioxidant capacity (p < .02; R = -.52) suggesting that the oxidative stress associated with reduced antioxidants results in increased hemoglobin-membrane interactions.

Specific association of increased cyclin-dependent kinase 5 expression with monocytic lineage of differentiation of human leukemia HL60 cells.

Hematopoietic cell differentiation takes place in phenotypically recognizable stages characterized by morphology as well as by the expression of enzymes and surface markers. It is recognized that differentiation results from an interaction of environmental cues, such as cytokines and hormones, with internal cellular programs, but the precise mechanisms are not entirely clear. HL60 cells, a human acute myeloid leukemia (AML) cell line with promyelocytic features, provide a model for such studies because they behave like stem cells, which can differentiate into two different lineages, granulocytic or monocytic/macrophage, depending on the inducer. Protein levels and kinase activity of cyclin-dependent kinase 5 (Cdk5) were reported [F. Chen and G. P. Studzinski (1999) Exp. Cell Res. 249, 422, 1999] to increase in HL60 cells induced to monocytic differentiation by 1alpha,25-dihydroxyvitamin D3 (1,25D3), but the specificity of the association of Cdk5 with the monocytic phenotype has not been established. We show here that up-regulation of Cdk5 does not occur in granulocytic differentiation, whereas inhibition of Cdk5 activity by olomoucine, or its expression by a plasmid construct expressing antisense Cdk5, switches the 1,25D3-induced monocytic phenotype (a combination of positive nonspecific esterase reaction, expression of the CD14 marker, and morphology) to general myeloid phenotype (positive nitro-blue tetrazolium reaction, CD11b marker and morphology). The transcriptional up-regulation of Cdk5 by 1,25D3 was not inhibited by olomoucine. These findings show that in human myeloid cells up-regulation of Cdk5 is specifically associated with the monocytic phenotype.

Macrophages are essential for lymphocyte infiltration in formyl peptide-induced cholangitis in rat liver.

Cholangitis in rats induced by N-formyl L-methionine L-leucine L-tyrosine (fMLT) is characterized by infiltration of mononuclear cells around bile ducts in portal tracts. We investigated the initial process in fMLT-induced cholangitis histochemically. Administration of fMLT into the colons of adult male Wistar rats with acetate-induced colitis resulted in an infiltration of mostly macrophages and granulocytes into the portal tracts on day 1. Abnormal peroxidation as demonstrated by the nitro blue tetrazolium (NBT) reaction occurred in bile duct cells as well, although no apparent necrosis of the bile duct cells was observed. On day 4, the majority of the inflammatory cells in the portal tracts were CD4+ or CD8+ T lymphocytes. The oxidative products of the NBT reaction also disappeared from the bile duct cells. Administration of carrageenan, a potent inhibitor of macrophage function, resulted in a significant decrease in lymphocyte infiltration into the portal tracts. On day 8, portal inflammation subsided. In formyl peptide-induced cholangitis, macrophages and granulocytes may injure bile ducts transiently. Further, macrophages are necessary for the subsequent migration of T lymphocytes around the bile ducts.

Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system

Background & Aims: Superoxide anion (O 2 −) plays an important role in gastric pathophysiology. The aims of this study were to identify O 2 −-producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. Methods: The amount of O 2 − was measured by the reduction of cytochrome c, and O 2 −-producing cells were visualized by nitroblue tetrazolium reaction. Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox. Results: Gastric pit cells, but not parietal cells, spontaneously released O 2 − at 50 nmol · mg protein −1 · h −1. NADPH or guanosine 5'- O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity. p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O 2 − release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O 2 −-dependent activation of nuclear factor κB occurred in these primed cells. Conclusions: Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase–like activity. GASTROENTEROLOGY 1998;115:1186-1196

Hemagglutinating virus of Japan-liposome–mediated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine vein grafts under conditions of poor runoff

Purpose: Late graft failure is a critical problem, particularly in the presence of poor runoff vessels. Intimal hyperplasia is considered to be the main cause of graft failure. We have already reported that intimal thickening of experimental vein grafts in dogs with poor runoff vessels is more pronounced than that in dogs with normal vessels. We and others also have reported that production of nitric oxide (NO) in the endothelium of canine vein grafts is impaired. In the present study, we asked whether in vivo gene transfer of endothelial cell NO synthase (ecNOS) would inhibit intimal hyperplasia of autogenous vein grafts implanted in limbs with poor distal runoff in dogs. Methods: After exposing femoral veins, the nuclear-targeted lac Z gene, bovine ecNOS cDNA, or control vector plasmid encapsulated in the hemagglutinating virus of Japan-liposomes was infused intraluminally, followed by incubation for 10 minutes at room temperature under a distending pressure of 100 mm Hg. Twenty reversed vein grafts were implanted under normal runoff conditions, and 4 days later these were used to confirm gene transfer to the vein grafts. Twelve reversed vein grafts were implanted under conditions of poor runoff, and 4 weeks after the operation intimal thickening was evident. Results: In vein grafts under normal runoff conditions, lac Z gene transfer exhibited diffuse and frequent X-Gal-positive signals in both medial and adventitial layers 4 days after implantation (n = 3). In case of the ecNOS gene–transferred vein grafts, bovine ecNOS protein was mainly detected in medial smooth muscle cells and adventitial cells 4 days after implantation, determined using immunohistochemical techniques and bovine ecNOS specific antibody (n = 3). In addition, ecNOS-transferred vessels showed intense purple signals by reduced nicotinamide adenine dinucleotide phosphate diaphorase and nitroblue tetrazolium reaction, in both medial and adventitial layers, whereas weak NOS activity was recognized at the adventitial vasa vasorum of the untreated veins or control vector transferred veins (n = 3, respectively). In vein grafts under poor runoff conditions, the intimal thickness at 4 weeks after implantation was significantly reduced by ecNOS gene transfer (n = 4; 90.0 ± 7.6 μm and 1.18 ± 0.07 mm 2) in comparison with buffer-treated vessels (n = 4; 195.8 ± 25.7 μm and 2.62 ± 0.48 mm 2) or vector vehicle–treated vessels (n = 4; 193.0 ± 15.8 μm and 2.65 ± 0.22 mm 2). Conclusions: Our findings show that gene transfer of ecNOS inhibited intimal hyperplasia of canine vein grafts caused by poor runoff conditions, as a result of an increased local production of NO. Thus ecNOS gene transfer warrants further study as a possible approach to prevent late graft failure. (J Vasc Surg 1998;27:135-44.)

A simple method to identify NBT-positive cells in isolated glomeruli.

Reactive oxygen radicals are probably involved in the pathogenesis of human and experimental models of renal disease, yet current methods are inadequate to quantify and identify the cells producing reactive oxygen radicals. We used the nitroblue tetrazolium reaction to determine superoxide anion production in glomerular cells in phorbol myristate-stimulated glomerular suspensions and in isolated glomeruli from rats with nephrotoxic nephritis, ureteral obstruction, and puromycin aminonucleoside nephrosis. We were also able to identify these nitroblue tetrazolium + cells using specific appropriate antibodies. When the technique was tested in conditions known to increase reactive oxygen radicals, as phorbol myristate-stimulated glomeruli and glomeruli from animals with nephrotoxic nephritis and ureteral obstruction, increased number of nitroblue tetrazolium + cells were found. These cells were identified as glomerular intrinsic cells (Thy-1 +) or infiltrating leukocytes (leukocyte common antigen + or antineutrophil +). This method may be useful to determine cells participating in glomerular damage induced by reactive oxygen radicals.