Cell damage and dye reduction in the quantitative nitroblue tetrazolium (NBT) test.

Nitroblue tetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles. Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction. This suggests that, in this test, dye reduction occurs largely as a result of contact between intracellular reducing compounds and NBT following damage of the outer cell membrane. The expression of dye reduction as a function of LDH release should enhance the sensitivity of the quantitative NBT test by correcting for the observed intersubject variation in cell damage. The relationship between cell damage and dye reduction is a measure of the reducing capacity of the cell. This was normal in immature, bone marrow neutrophils, but diminished in neutrophils of patients with chronic granulomatous disease.     

Serum and cellular factor involvement in nitroblue tetrazolium (NBT) reduction by human neutrophils

Summary The nitroblue tetrazolium (NBT) test has been modified in an attempt to separately assess leukocytic and serum factors in the reduction of NBT. For diagnostic purposes, NBT processing is assayed on leukocytes suspended in Hanks' solution in the absence and in the presence of serum, as well as with E. coli stimulation.The method has been tested for its reproducibility and for the suspceptibility of the leukocytes to stimulatory and inhibitory agents. Endotoxin and E. coli produce a dose-dependent stimulation, whereas prednisolone and cytochalasin B cause a dose-dependent inhibition of NBT processing.Applied to patients, different reaction patterns have been observed, revealing stimulatory and inhibitory serum factors, as well as increased and decreased cellular reactivity.Growth and Development Program of the University of Alabama, Birmingham, Alabama, USA.     

Glutathione reductase and nitroblue tetrazolium reduction deficiencies in neutrophils of patients with primary idiopathic myelofibrosis

Latex ingestion and nitroblue tetrazolium reduction were measured in neutrophils of 40 patients with primary idiopathic myelofibrosis. The percentage of neutrophils that ingested latex, in the presence of either autologous or control sera, was lower (P less than 0.001) than that of the controls. The percentage of the ingesting neutrophils that reduced nitroblue tetrazolium was also lower (P less than 0.001) in the patients than in the controls. Activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione peroxidase and NAD(P)H oxidases were not different from those of the controls. In contrast, glutathione reductase activity was significantly lower (P less than 0.001) in the patients than in the controls either with or without the addition of flavin adenine dinucleotide. Glutathione reductase and nitroblue tetrazolium reduction activities were correlated (r = 0.913). These results are discussed within the framework of the acquired enzymopathies and the increased susceptibility to infection observed in these patients.     

Assessment of neutrophil function in the elderly using antibody coated polyacrylamide gel (immunobeads) and nitroblue tetrazolium (NBT) reduction as a combined test

Neutrophil function was assessed in 35 elderly individuals (age greater than 75) and 20 normal young individuals (age 20-45) by combining ingestion of antibody coated polyacrylamide gel and nitroblue tetrazolium reduction in a single test. This test evaluates phagocytosis and metabolic integrity simultaneously and appears to be a sensitive and reliable test of neutrophil function. No significant difference was found (by using this test) between neutrophils from healthy elderly people and the neutrophils from young controls, or between the sexes in either age group.     

Effect of vitamin B12 and folic acid deficiency on small intestinal absorption

Three patients are described, and they provide further evidence that deficiency of folic acid and vitamin B(12) may sometimes affect small intestinal function. Malabsorption of both xylose and vitamin B(12) returned to normal in one patient after treatment of a megaloblastic anaemia due to dietary deficiency of folic acid. Impaired absorption of vitamin B(12) was corrected by vitamin B(12) therapy in the other two patients. The initial cause of the vitamin B(12) deficiency in one patient was not apparent, but she was taking Gynovlar 21, which may have been an aetiological factor. In the third patient the small intestinal defect was secondary to pernicious anaemia, and in a group of 98 other patients with pernicious anaemia intrinsic factor did not improve vitamin B(12) absorption in six, and only partially corrected absorption in 30. The significance of these observations is discussed.     

Activation of human monocytes for nitroblue tetrazolium reduction and the suppression of lymphocyte response to mitogens.

Human peripheral blood mononuclear cells freed from polymorphs reduce nitroblue tetrazolium (NBT). This reduction is due to monocytes, i.e. adherent, phagocytic, esterase-positive cells with Fc receptors. Monocytes allowed to phagocytose zymosan show increased NBT reduction which under optimal conditions is 12.2 +/- 2.4 x 10(-9) mol . hr-1 . 10(-6) monocytes. Monocytes which have phagocytosed zymosan depress the mitogen response of human lymphocytes to PHA. This effect of 'activated' monocytes is due to a soluble inhibitory mediator which appears in the supernatant after culture for 24 hr. Its appearance requires protein synthesis. It is suggested that NBT reduction of peripheral blood mononuclear cells can be used as a test for the state of monocyte activation in disease. The possibility that activated monocytes may depress blast transformation in vitro in disease states is discussed.     

Changes in activity of some mechanisms of specific and nonspecific immunity in vitamin B1 deficiency

The effect of thiamine deficiency on the immune response and activity of certain mechanisms of natural immunity was studied in adult rats. Thiamine deficiency was simulated experimentally by a single injection of hydroxythiamine, a vitamin B₁ antagonist. Administration of hydroxythiamine caused a marked decrease in complement activity, phagocytic activity of the peripheral blood leukocytes, bactericidal activity of the serum, and antibody production in response to immunization with sheep's red blood cells. Conversely, lysozyme activity increased. In vitamin B₁ deficiency the intensity of incorporation of [¹⁴C]leucine into liver protein synthesis was reduced.Department of Pathological Physiology, N. I. Sechenov First Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp. 60–62, July, 1979.     

Serum folic acid activity (L. casei) in Sephadex gel chromatography

The folic acid activity (FAA) of the protein zone in DEAE Sephadex A-50 chromatography of normal serum showed the following distribution: most (about 40%) was eluted with protein zone IV (alpha-2-macroglobulin), about 33% with protein zones II-III (transferrin), and the balance with zone V (albumin). In healthy human subjects the variations were relatively small. The repeatability of the chromatography in an individual was good.Towards the end of pregnancy the binding of FAA to protein zones II-III increased, while that to zone IV decreased. The return to normal started a few days after delivery.The binding to zones II-III was greatly increased in patients with pernicious anaemia but no neuropathy. It became normal during B(12) therapy. If the patient had neuropathy, his FAA chromatogram was close to normal. During therapy the amount of unbound FAA decreased rapidly.The use of diphenylhydantoin increased the binding of FAA to protein zone I and to zones II-III. In these patients the binding to protein zone IV was slighter than normal.     

Effects of nitroblue tetrazolium and vitamin E on platelet ultrastructure, aggregation, and secretion

All agents capable of triggering the platelet release reaction also stimulate prostaglandin biosynthesis in these cells. Information concerning the endoperoxides, thromboxanes, and more stable metabolites generated by the action of cyclooxygenase and lipoxygenase on arachidonic acid has accumulated rapidly, but little is known about the preliminary steps in the cleavage and preparation of arachidonic acid for insertion into the enzymatic pathways of prostaglandin synthesis. Studies in this laboratory have shown that the combination of nitroblue tetrazolium (NBT) and vitamin E which prevents oxygenation of arachidonic acid to a free radical also blocks platelet prostaglandin biosynthesis. The present study has evaluated the influence of NBT, vitamin E, and the combination of NBT and vitamin E on the fine structure and biochemistry of platelets during incubation, and the effects of these compounds on the aggregation and secretion of platelets stimulated by collagen, thrombin, epinephrine, and ADP. Results of the study demonstrate that NBT and vitamin E, rather than injuring platelets, appear to protect them during incubation. Together NBT and vitamin E blocked aggregation by epinephrine, collagen, and thrombin, but permitted a small first wave stimulated by ADP. Both ADP and thrombin induced shape change, pseudopod formation, and limited degrees of internal contraction in vitamin E-NBT-treated platelets, whereas epinephrine and collagen failed to significantly alter discoid form. This pattern of response to aggregating agents was identical to reactions observed in platelets pretreated with aspirin and indomethacin, both potent inhibitors of platelet prostaglandin synthesis. In addition, NBT-vitamin E virtually blocked the first wave of aggregation which is not affected by aspirin and indomethacin. The findings support the concept that conversion of arachidonic acid to an activated state is an important step in prostaglandin synthesis and that electron transfer or oxidation-reduction reactions are intimately involved in the development of platelet stickiness.     

Polymorphonuclear activation in leprosy. I. Spontaneous and endotoxin-stimulated reduction of nitroblue tetrazolium: effects of serum and plasma on endotoxin-induced activation.

Spontaneous nitroblue tetrazolium (NBT) reduction was evaluated in neutrophils from patients with the different types and forms of leprosy, and compared with reduction obtained form cells from normal controls. Leucocytes from the same subjects were stimulated in vitro by endotoxin, and the rise in percentage of cells reducing NBT was determined. Patients of all groups, with the exception of those with reactional lepromatous leprosy (RLL) had an essentially normal proportion of reducing cells. Neutrophils were normally activated by endotoxin. This indicates that while Mycobacterium leprae does not by itself stimulate leucocytes from leprosy patients, there is no overall anergy of neutrophils in lepromatous or other forms of leprosy. In RLL the proportion of reducing cells was significantly raised. Stimulation with endotoxin was able further to enhance this proportion, but not above levels reached by stimulation of normal cells. Neutrophil activation could not be reproduced by mixing serum from highly activated RLL patients with normal leucocytes. An inhibitory effect of serum and plasma over in vitro endotoxin activation of neutrophils was found.     

Effectiveness of the nitroblue tetrazolium test in demonstrating reduced bactericidal activity of polymorphonuclear neutrophils in schistosomal hepatosplenomegaly and ascites.

The nitroblue tetrazolium (NBT) test was carried out on blood from ten normal individuals and 20 schistosomal patients, ten of whom had hepatosplenomegaly and ten hepatosplenomegaly and ascites. The test revealed defective neutrophils in patients who had hepatosplenomegaly accompained by ascites. The phagocytic and bactericidal effects from five of each of the two groups of patients; results substantiated tose obtained by the NBT screening test. The NBT screening test thus proved useful in detecting acquired defective function of neutrophils in a helminthic parasitic disease that is accompanied by frequent bacterial infections.