Indicators of spontaneous and stimulated nitroblue tetrazole test of polymorphous-nuclear leucocytes in acute dysentery patients

140 healthy individuals and 93 sick with acute dysentery were subjected to an examination by spontaneous and by bacterial preparations stimulated reaction with nitroblue tetrazole (NBT test). Indicators in healthy persons were normal in the spontaneous, and increased in the NBT test, stimulated by bacterial preparations. Indicators of the spontaneous NBT test in patients with acute dysentery were raised with a maximum in the period of early convalescence. Stimulation by a live shigella culture--the dysentery vaccine--revealed by means of Sonne diagnostic high, and when endotoxin from Serratia marcescens and dysenterin was used as an inductor, mild indicators of NBT test activity. When a polyvalent agglutinating dysentery serum was used as a stimulator, the activity increased considerably, and a simultaneous use of serum and vaccine had an inhibiting effect on the indicators of the stimulated NBT test. The obtained results testify the sufficient high reserve possibilities of leucocytes towards complete phagocytosis and the efficiency of the NBT test, stimulated by bacterial preparations for the study of functional and metabolic activity of leucocytes in the process of acute bacterial dysentery.     

Histochemical study of monoamine oxidase latency in the liver of the rat.

In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.     

Histochemical study of monoamine oxidase latency in the liver of the rat

Summary In a histochemical test system with adrenaline as substrate and nitroblue tetrazolium (NBT) as electron acceptor, an increase of NBT reduction in rat liver sections was found microspectrophotometrically following short hypotonic treatment. Investigations with iproniazide, a monoamine oxidase inhibitor, and non-enzymatic NBT reduction showed that the increased formazan formation was related to the presence of monoamine oxidase. It is suggested that the reason for the observed increase of formazan formation is due to increased permeability of the inner mitochondrial membrane to NBT. Consequently, the increase of monoamine oxidase observed in the histochemical test system does not represent mobilization of a latent activity, but rather complete assessment of activity that is normally present.     

The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L.

The phagocytosis of neutrophils and serum lysozyme activity were investigated in carp experimentally infected with Pseudomonas alcaligenes and Aeromonas punctata. The total leucocyte numbers, relative leucocyte counts, nitroblue tetrazolium (NBT) reduction, NBT test rate, lysozyme activity and lysozyme index were examined on days 7, 14 and 21 after injection. On days 7 and 14 there was a significant increase in total leucocyte numbers and serum lysozyme activity, but a decrease in the NBT test rate and lysozyme index. NBT reduction was unchanged compared with the control group. On day 21 the total leucocyte numbers and lysozyme activity had declined and were less than control values, but there was a significant increase of the NBT test rate, NBT reduction and lysozyme index.     

Direct spectrophotometric observation of intracellular nitro-blue tetrazolium and its formazan by multiple internal reflectance infrared spectroscopy.

A technique is described which permits the direct, infrared (IR) spectrophotometric observation of peripheral leukocytes by utilizing multiple internal reflectance (MIR) spectroscopy on zinc selenide prisms. The IR spectra of nitro-blue tetrazolium (NBT) and its derivative, nitro-blue diformazan (NBF), are described and examined and these spectra are compared with those of the intracellular NBT and NBF yielding several peaks which may prove to be analytically useful. Additionally, the presence of unreduced NBT in leukocytes is demonstrated, which microscopic and chemical "NBT tests" have not previously done.     

Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

Summary We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

Immunohistochemical detection of antibody in tissue sections of non-perfused and ex vivo-perfused organs using a tetrazolium alkaline phosphatase substrate

We have used nitroblue tetrazolium (NBT) as a color reagent to localize antibody-bound alkaline phosphatase in frozen tissue sections. In the method described, NBT is reduced to a stable black diformazan reaction product that contrasts well with nuclear counterstains such as hematoxylin and stands out strongly in black and white photographs. We have found NBT to be a suitable color reagent for the alkaline phosphatase: anti-alkaline immunohistochemical technique. The reaction product also contrasts well with fast red and can therefore be used as second reagent for two color immunoenzyme studies. In this report, we describe a novel two color immunoenzyme method to assess the ex vivo binding of antibodies against Class II histocompatibility antigens in whole organs connected to a perfusion circuit.     

植物冻害和氧化胁迫——甘蓝叶冻-融循环中超氧的产生

为检验植物冻害的发生和氧化胁迫这一假说,在冰冻前把氮蓝四唑（NBT）真空渗入到甘蓝叶圆片中,在叶圆片冻－融循环中NBT被还原为甲Zan,把其中的单甲Zan用乙醇提取出来,在分光光度计上比色,可作为冻融循环中产生的氧化胁迫的定量指标,NBT本身作为氧化剂,使冻害稍有增加,作为冰冻保护剂的二甲基亚砜真空渗入叶圆片使其抗冻性显著增加,而NBT还原则显著减少,表明二甲基亚砜在保护叶组织免受冻害上的作用和它减缓植物组织氧化胁迫的作用有关。实验结果支持植物冻害的发生和氧化胁迫有关这一假说。实验还表明还原NBT的还原剂很可能是超氧阴离子自由基。     

Metabolic activity of phagocytes in experimental sporotrichosis

Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to agressive microorganisms. In patients with sporotrichosis a diminished capability of killing engulfed yeast by their PMN has been described, but the origin of this deficiency remains unknown.In this work, partial aspects of the oxidative metabolism of PMN leukocytes and peritoneal macrophages of mongolian gerbils experimentally infected with sporotrichosis were studied. For this purpose the nitroblue tetrazolium (NBT) test as described by Baehner and Nathan (1) and myeloperoxidase activity measured according to Kaplow's method were utilized.The PMN and macrophages of mongolian gerbils infected with sporotrichosis showed increased reduction of NBT when compared with the phagocytic cells of normal ones, as is usually observed in most infections. Myeloperoxidase activity was diminished in both PMN and macrophages, but this diminution was statistically significant only in PMN leukocytes. These results show that part of the oxidative mechanisms of phagocytic cells can be impaired in experimental sporotrichosis, and could be correlated with the diminished fungicidal activity of PMN leukocytes obtained from patients infected with sporotrichosis.     

Influence of levamisole on the capacity of neutrophil granulocytes for ingestion and for the reduction of nitrobluetetrazolium

With the aid of a modification of the NBT test it has been shown in in vitro and in vivo studies that levamisole does not affect the NBT reduction capacity of neutrophil granulocytes. The increased NBT positivity of these cells under the influence of levamisol is due to stimulation of ingestion.     

Direct spectrophotometric observation of intracellular nitro-blue tetrazolium and its formazan by multiple internal reflectance infrared spectroscopy

Summary A technique is described which permits the direct, infrared (IR) spectrophotometric observation of peripheral leukocytes by utilizing multiple internal reflectance (MIR) spectroscopy on zinc selenide prisms. The IR spectra of nitro-blue tetrazolium (NBT) and its derivative, nitro-blue diformazan (NBF), are described and examined and these spectra are compared with those of the intracellular NBT and NBF yielding several peaks which may prove to be analytically useful. Additionally, the presence of unreduced NBT in leukocytes is demonstrated, which microscopic and chemical NBT tests have not previously done.     

Electrophoretic detection of ascorbate oxidase activity by photoreduction of nitroblue tetrazolium

A method for the detection of ascorbate oxidase in electrophoretic gels is described. This method relies on the ability of the enzyme to prevent the photoreduction of nitroblue tetrazolium (NBT). The method is based on that described by C. Beauchamp and I. Fridovich (1971, Anal. Biochem. 44, 276-287) for the superoxide dismutase and was made specific for ascorbate oxidase detection by treating the gel with 0.1 M hydrogen peroxide. Ascorbate (25 microM) or riboflavin (500 microM) was used as the electron donor. The possible reaction mechanism in the presence of ascorbate has been investigated. Western and Northern blot analyses confirmed the results obtained from the NBT staining procedure.     

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.     

Determination of the bactericidal strength of alveolar macrophages by using the NBT test

The NBT test on alveolar macrophages can be used for the indirect evaluation of their bactericidal potency. This test has made it possible to show that the development of the tuberculous process in the lungs is characterized by the increasing intensity of the reduction of NBT by alveolar macrophages, which indirectly indicates their considerable bactericidal potency. But 3-4 weeks after infection macrophages, though capable of considerable bactericidal action, do not react to stimulation by Mycobacterium tuberculosis.     

Characteristics of the functional activity of human blood neutrophils using the nitroblue tetrazolium reduction test]

A study was made of the test of restoration of the nitroblue tetrazolium (NBT) by the intact blood neutrophils and the neutrophils stimulated with various endotoxin doses of Gram-negative bacteria in 70 healthy persons. A high sensitivity of the test with the NBT for the quantitative assessment of the neutrophil response under conditions stimulating the phagocytic activity was demonstrated. A functional nonhomogeneity of the neutrophil population, the most distinct with the minimal stimulating action, was noted.     

Lithium carbonate effect on nitrotetrazolium blue (NBT) production in neutrophils of patients with granulocytopenia

In 30 persons with essential granulocytopenia, lithium carbonate in doses of 12.5 mg/kg per body weight was applied during 10 days. This resulted in an increased absolute neutrophil count and total leukocyte count as well as increased percentage of cells reducing NBT in the spontaneous NBT test according to Park. Prior to the beginning of treatment, patients with granulocytopenia exhibited an elevated percentage of NBT-positive cells in comparison to the control group of 28 healthy persons. The significance of this observation for treating granulocytopenia by means of the agent mentioned above is discussed.     

Flow cytometric evaluation of nitro blue tetrazolium (NBT) reduction in human polymorphonuclear leukocytes

Oxidative metabolic burst of activated human polymorphonuclear leukocytes (PMN) is most commonly investigated in clinical practice by evaluating nitroblue tetrazolium (NBT) reduction at the single cell level. Reduced NBT precipitates where the redox reaction has taken place and can be visualized as PMN-associated dark blue granules of formazan in light microscopy. Although widely used and not technically demanding, this method remains subjective and labor intensive, especially when large numbers of samples need to be investigated. We developed a new flow cytometry technique in which PMN membrane was rendered fluorescent by a short incubation with fluorescein-conjugated Concanavalin A. PMN were then incubated with NBT and increasing doses of a suitable stimulus, such as phorbol myristate acetate (PMA). Formazan has a distinct peak of absorption at 520 nm that represents the peak of emission of fluorescein. As a consequence, formazan quenches the PMN-associated fluorescence. Data show that a dose-dependent reduction of fluorescence can be obtained using graded amounts of PMA in normal PMN cultures. PMN-associated fluorescence remains unchanged in control patients with chronic granulomatous (CGD) disease, a disorder characterized by a selective impairment of PMN oxidative metabolism. Electronic cell size increases upon PMA incubation in normal PMN, irrespective of the presence of NBT. Conversely, forward light scatter intensity decreases in the presence, but not in the absence, of NBT indicating that the phenomenon is due to the capacity of formazan to absorb/scatter the incident light. The present method for easily detecting NBT reducing activity at single cell level by flow cytometry makes use of commonly available, inexpensive reagents and standard instrumentation. It could become a useful test for clinical purposes.     

Biological effects of cigarette smoking assessed with the NBT test

The NBT reduction spontaneous and stimulated test was carried out on 90 men; 30 of which had never smoked cigarettes, 28 subjects have smoked for up to 10 years and 32 subjects--since over 10 years. The investigations demonstrated show that in subjects smoking cigarettes for up to 10 years the spontaneous and stimulated ability of the neutrophils to reduce NBT was increased whereas in the subjects smoking cigarettes since over 10 years impairment of the stimulated NBT reduction was demonstrated. We've come to a conclusion that an exposure to tobacco smoke may activate oxidation-reduction processes of the neutrophils in the initial stage and impair them in longer stage of smoking.     

Neutrophil activation in vitro

Kinetics of spontaneous and induced reactions of Nitroblue tetrazolium (NBT) restoration by neutrophils was investigated in vitro. The index of activated neutrophils (IAN), commonly characterizing cell activation degree depending on the reaction time (t), was described by the equation: IAN = [NBT0](1-exp[-k1t]) + k0t; where (NBT0) is intracellular concentration of NBT, which reflects membrane permeability, k1 and k0--speed constants of the first and zero order reactions, characterizing, respectively, stimulation and spontaneous activities of enzymatic systems involved in NBT restoration. We detected the ability of heparin (2.0-7.5 mkg/ml) or chondrotin sulphate (0.25-2.5 mkg/ml) to activate neutrophils and presumably to maintain in vivo their spontaneous activity. The values of kinetics equation parameters enable us to speculate about relative hyper-, hypo- and normal function of neutrophils, related to the functional state of the whole organism. The value of [NBT0] changes from 8 to 103 conventional units, and a high correlation exists between this index and the reaction speed constants, differing in stimulated and spontaneously activated cells. The latter are represented by neutrophils only, which, as shown before, produce active forms of oxygen, used, probably, not for phagocytosis needs. The share of such cells makes approximately 30-40% of the neutrophil recirculating pool, and positively correlates with [NBT0], (r = 0.58; P < 0.05). This is determined by a very high membrane permeability of this neutrophil subpopulation. The detected different spontaneous and stimulated ability, various membrane permeability and enzyme system activity confirm the neutrophil functional unequality, which defines their activation peculiarity.     

Increased nitroblue tetrazolium dye reduction by human neutrophils stimulated by interferon in vitro

Human leukocyte interferon enhanced nitroblue tetrazolium dye (NBT) reduction by human neutrophils (PMNs). Increase in NBT reduction paralleled increase in interferon dose. When human leukocyte interferon was heated to 60 C or 80 C for 30 min, both the antiviral activity and the effect on NBT reduction decreased. Human leukocyte interferon neutralized with anti-human leukocyte interferon serum showed no effect on NBT reduction. A human fibroblast interferon preparation also enhanced NBT reduction. The species dependency of interferon was shown in NBT reduction as well as in antiviral activity.