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Oxidation of isonicotinic acid hydrazide (isoniazid) by horseradish peroxidase at the expense of H2O2 yielded reactive species which were able to reduce nitroblue tetrazolium and bleach p-nitrosodimethylaniline. Nicotinic acid hydrazide oxidation did not cause these effects. At slightly alkaline pH, oxidation of isonicotinic acid hydrazide by horseradish peroxidase proceeded at the expense of molecular O2, and the reaction was oxygen consuming. The addition of H2O2 abolished O2 consumption. Bovine liver catalase enhanced the rate of nitroblue tetrazolium reduction and decreased the maximal velocity of the reaction proportionately to catalase concentration. During oxidation of isonicotinic acid hydrazide by horseradish peroxidase-H2O2, splitting of the heme group of horseradish peroxidase took place as shown by the disappearance of the Soret and minor bands in the visible region of the spectrum.

We have developed a model of reperfusion injury in Krebs buffer-perfused rabbit lungs, characterized by pulmonary vasoconstriction, microvascular injury, and marked lung edema formation. During reperfusion there was a threefold increase in lung superoxide anion (O2-) production, as measured by in vivo reduction of nitroblue tetrazolium, and a twofold increase in the release of O2- into lung perfusate, as measured by reduction of succinylated ferricytochrome c. Injury could be prevented by the xanthine oxidase inhibitor allopurinol, the O2- scavenger SOD, the hydrogen peroxide scavenger catalase, the iron chelator deferoxamine, or the thiols dimethylthiourea or N-acetylcysteine. The protective effect of SOD could be abolished by the anion channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, indicating that SOD consumes O2- in the extracellular medium, thereby creating a concentration gradient favorable for rapid diffusion of O2- out of cells. Our results extend information about the mechanisms of reperfusion lung injury that have been assembled by studies in other organs, and offer potential strategies for improved organ preservation, for treatment of reperfusion injury after pulmonary thromboembolectomy, and for explanation and therapy of many complications of pulmonary embolism.

Functional capabilities of morphologically mature (segmented) and immature granulocytes (neutrophils and eosinophils) from bone marrow from cows were studied and compared with similar activities of segmented granulocytes from blood. Phagocytosis of Escherichia coli and postphagocytic oxidative metabolic stimulation, measured by nitroblue tetrazolium (NBT) reduction, were evaluated simultaneously. Phagocytosis was observed readily in segmented neutrophils, neutrophilic bands, and metamyelocytes and rarely in myelocytes. Phagocytosis was not seen in promyelocytes and myeloblasts. Neutrophilic bands and metamyelocytes were phagocytically less active than were segmented neutrophils. Washed segmented bone marrow neutrophils possessed phagocytic activity similar to that of blood neutrophils, whereas the activity of unwashed segmented bone marrow neutrophils was markedly less than that of blood neutrophils. Reduction of NBT was observed only in blood segmented neutrophils and bone marrow segmented neutrophils; the magnitude of NBT reduction was significantly (P = less than 0.005) less in bone marrow neutrophils than in blood neutrophils. Eosinophils were phagocytically less competent than were neutrophils. The NBT reduction was observed only in eosinophils from blood, but not in eosinophils from bone marrow.

We wished to measure the amount of reduced nitroblue tetrazolium (NBT) formed in corneal cells and to relate it to the viability of the cells in terms of their oxygen uptake. Two full-thickness corneal discs of identical size (3, 4, 5 or 6 mm in diameter) were cut from the eyes of guinea pigs. One disc was processed for the measurement of its reduced NBT and cellular nitrogen content; the other was processed for the measurement of its oxygen uptake and deoxyribonucleic acid (DNA) content. As the diameter of the corneal disc (that is, the number of cells) increased, the quantity of reduced NBT, nitrogen and DNA and the oxygen uptake increased proportionality. Since each of these measures correlated positively with each other (P less than 0.01) we were able to estimate the number of viable cells in a given cornea by measuring the amount of reduced NBT (formazan granules) formed in the cells.

Induction of luminol-enhanced chemiluminescence (CL) indicative of reactive oxygen formation was studied in glia cell cultures from newborn mice. A burst of CL could be induced by phorbol myristate acetate, zymosan, and antibody-coated bovine red blood cells, whereas Sendai virus and several other agents known to induce CL in myeloid cells were ineffective. Sodium azide failed to inhibit CL, indicating a myeloperoxidase-independent mechanism of light emission. In parallel experiments we identified the cells binding antibody-coated erythrocytes as macrophages characterized by the reduction of nitroblue tetrazolium and phagocytosis of zymosan and latex particles. Brain macrophages may use reactive oxygen intermediates (ROI) as a mechanism of antimicrobial defence; and, on the other hand, ROI formed by these cells may contribute to immuno-pathology in the brain.

In the original HL-60 cells (HL-60-S) and an HL-60 subline (HL-60-R) respectively susceptible and resistant to induction of differentiation by retinoic acid or dimethyl sulfoxide, 5-aza-2'-deoxycytidine inhibited growth equally but induced differentiation to a greater extent in HL-60-S. Flow cytometry showed that 5-aza-2'-deoxycytidine produced in both HL-60 lines an increased proportion of cells in G2+M rather than G0/G1 as with retinoic acid. 5-aza-2'-deoxycytidine may have a differentiation-inducing effect in HL-60 provided cells have the competence to differentiate, indicating the importance of an alternate mechanism of action.

Sporozoites of Plasmodium yoelii yoelii were incubated for 40 min with BALB/c peritoneal macrophages in the presence of nitro-blue tetrazolium (NBT). While immature oocyst sporozoites triggered the macrophage respiratory burst, as visualized microscopically by the localized reduction of NBT to insoluble formazan, 97.6% of mature salivary gland sporozoites did not induce such a response. The macrophage oxidative response was also induced by 82.7% of heat-inactivated and 95.7% of trypsin-treated salivary gland sporozoites. The relationship of these results to the infectivity and immunogenicity of malarial sporozoites is discussed.

During phagocytosis of opsonized lipopolysaccharide-coated paraffin oil droplets, rabbit alveolar macrophages reduced nitroblue tetrazolium, which effect was in part inhibitable with the use of superoxide dismutase. Exposure of cytochalasin-B-treated rabbit alveolar macrophages to opsonized zymosan led to the generation of superoxide, as quantitated by ferricytochrome C reduction. It was found that nitroblue tetrazolium in the presence of ferricytochrome C could in turn serve as scavenger of superoxide during stimulation of cytochalasin-B-treated rabbit alveolar macrophages. Following challenge with either opsonized zymosan or the membrane perturbant digitonin, rabbit alveolar macrophages released hydrogen peroxide into the extracellular medium. Employment of the surface membrane stimulant phorbol myristrate acetate led to activation of the hexose monophosphate shunt, which activity could be further enhanced in the presence of superoxide dismutase or attenuated in the presence of catalase. These studies demonstrate that rabbit alveolar macrophages release superoxide and hydrogen peroxide during surface membrane perturbation. In turn, hydrogen peroxide generation can stimulate the hexose monophosphate shunt.

A group of 2'-fluoro and 5-substituted arabinosyl pyrimidines and a group of base-substituted pseudoisocytidine analogs were evaluated for their capacity to induce differentiation in the human promyelocytic leukemia cell line, HL-60. These compounds were compared to 1-beta-D-arabinofuranosylcytosine (Ara-C) by monitoring: (1) inhibition of cell growth; (2) morphological maturation; (3) nitroblue tetrazolium (NBT) reduction; (4) expression of a myeloid differentiation antigen, Mo1; and (5) inhibition of colony formation. Exposure of logarithmically growing cells for 5 days to Ara-C, 2'-fluoro-Ara-C (FAC), 2'-fluoro-5-methyl-Ara-C (FMAC) and 2'-fluoro-5-ethyl-Ara-C (FEAC) resulted in cell growth inhibition at ED50 concentrations of 0.007, 0.11, 1.7 and 18 microM, and at cytostatic concentrations of 0.1, 0.5, 5.0 and 50 microM, respectively. These compounds induced granulocytic and monocytic maturation, reduction of NBT, increased expression of Mo1 antigen and a decrease or loss of both cell proliferation and colony formation in semisolid medium. There were few, if any, cell differentiation effects for the uracil nucleosides and pseudoisonucleosides tested. We found that Ara-C was the most cytotoxic of the compounds, and that when comparing absolute numbers of differentiated cells, i.e. percent of positive cells multiplied by the number of viable cells, FAC, FMAC and FEAC were superior to Ara-C inducing differentiation of HL-60 cells.

Bactericidal and metabolic activities were compared for polymorphonuclear leukocytes from normal controls, patients with sex-linked chronic granulomatous disease, five obligate carriers, and six potential carriers of CGD. Bacteria surviving at one hour were quantitated in a standardized assay which employed 1.25 Staphylococcus aureus per neutrophil. Heat-killed bacteria or a chemical agent, phorbol myristate acetate, were used to stimulate increases in utilization of oxygen, oxidation of [1--14C] glucose, and reduction of neotetrazolium chloride by PMN. The results demonstrate that PMN from the individual obligate carriers of CGD have a broad spectrum of functional capabilities. Neutrophils from one obligate carrier performed in the above in vitro tests and others on a par with normal control cells, whereas the PMN of others displayed deficiencies nearly as profound as those of the affected CGD patients. The observations parallel the broad range of phenotypic expression observed in heterozygotic carriers of other sex-linked recessive disorders as a result of random inactivation of the X chromosome. Although predictable from the current concept of random X inactivation, the spectrum has not been previously demonstrated for carriers of sex-linked recessive CGD and thus has important implications for the detection and counseling of carriers of CGD.

A procedure was developed for isolating large numbers of purified polymorphonuclear leukocytes (PMNs) from the peripheral blood of horses. Equine PMN function was evaluated by three procedures: 1) Staphylococcus aureus ingestion, 2) nitroblue tetrazolium reduction, and 3) iodination. Four preparations of R. equi were added to polymorphonuclear leukocytes (PMNs) in each test system. Live bacteria, heat-killed bacteria, the washed pellet from heat-killed bacteria, and the supernatant fluid from heat-killed bacteria were evaluated for effects on equine PMN function. None of the R. equi preparations had an effect on S. aureus ingestion by equine PMNs. Nitroblue tetrazolium reduction by PMNs, a measure of oxidative metabolism, was suppressed by pellet and supernatant fractions. Values for the iodination reaction were depressed by all R. equi preparations, indicating decreased activity of the myeloperoxidase-H2O2-halide system of the PMN. Further evaluation of the supernatant from heat-killed R. equi showed that it retained its inhibitory effect on iodination following autoclaving and/or passage through a 10,000 MW filter. R. equi fractions did not alter the enzymatic conversion of 125I to a protein-bound form in a PMN-free assay developed to evaluate this reaction. The presence of a surface component capable of inhibiting bactericidal mechanisms of the PMN may play an important role in intracellular survival of R. equi.

In thyroid gland, iodination takes place on the apical plasma membrane and requires the presence of the thyroid peroxidase and H2O2 generating system. H2O2 generation and NBT (nitro blue tetrazolium) reductase activity (both of which are NADPH-dependent) as well as peroxidase activity were compared for their respective orientations in membrane vesicles. The possible role of NADPH-NBT reductase activity in H2O2 generation was also examined. Results favor the conclusion that thyroid peroxidase is oriented towards the luminal side of the vesicles, whereas the NADPH site of NADPH oxidase-dependent H2O2 generation is located on the external side of the same or of different vesicles. Furthermore, it is shown that different NADPH-NBT reductase activities are present on both the outer and inner surfaces of the membrane vesicles, and that none of these activities is able to produce either H2O2 or O-2. The idea that a multi-component complex is involved in H2O2 generation is discussed, and a model is proposed which takes into account the possible spatial separation of the thyroid peroxidase site from the NADPH site of this H2O2 generation system on the apical membrane of the thyrocyte.

A modification of the NBT-reaction is described which allows a differentiation of the NBT uptake and NBT-reduction. By means of this method it has been shown that the capacity of NBT-reduction of neutrophil granulocytes taken from hematologically healthy persons is independent of the probands' age and, moreover, of the presence of acute infections and their etiology. It has been shown, that the increase in the NBT-reduction of neutrophilic granulocytes of newborns and persons with acute bacterial infections results from an increased NBT-ingestion. To grive an example of a strongly reduced capacity of NBT-reduction of the neutrophilic granulocytes 2 patients suffering from fatal granulomatous disease in childhood have been examined.

Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to aggressive 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.

The human promyelocytic leukemia cell line known as HL-60 can be triggered to mature to functional granulocytes and/or macrophages after exposure to a variety of compounds. The findings have generated enthusiasm for possible therapy of leukemia using compounds that induce leukemic cell differentiation. We investigated whether five compounds known to trigger HL-60 differentiation to granulocytes could trigger the maturation of blast cells from 12 patients with myelogenous leukemia. Maturation was judged by morphology, superoxide production, phagocytosis, expression of Fc receptors, and development of alpha-napthyl acetate esterase activity. The blast cells from most patients showed little morphological, histological or functional maturation after exposure to the various compounds as compared to the blast cells cultured without the compounds. Actinomycin was able to induce significant maturation of leukemic cells of some patients when maturation was analyzed by several statistical methods. Our study suggests that many compounds which trigger differentiation of promyelocytic leukemia cells may not trigger differentiation of less mature myeloid leukemic cells.

To determine whether normal aging interferes with the functional capability of polymorphonuclear leukocytes (PMNs), 6 tests of PMN function were performed in 285 healthy subjects whose ages ranged from 20 to 97 years. A second selection based upon blood measurement and a review of medical histories 6 months later, eliminated 68 subjects. The 217 remaining persons were sub-classed by age into 7 groups including equal numbers of males and females. The functional properties of PMNs in the aged, when compared to those of younger adults, were characterized by: (a) a decrease in the chemotactic response in the 80+ age group: (b) increased adherence, with onset after age 70, maximal after age 80; (c) a progressive decrease of NBT dye reduction capability, up to age 70-79, followed by an unexplained increase of the mean value after age 80; (d) diminished Candida-killing activity, appearing in the 60+ group and becoming lowest in the oldest group; and (e) lack of changes in spontaneous migration and endocytosis. The mechanisms by which this impairment occurs are hypothetical. It is proposed that normal PMNs, after leaving the bone marrow, are influenced by various humoral components such as metabolic byproducts or immune processes altered by aging. Thus the defective PMN may represent only another victim of the aging process.

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 non-stimulated and stimulated nitroblue tetrazolium (NBT) dye test was studied in five normal adults in sequence, viz. before and at intervals after prednisone was given as one large dose. A negative effect of prednisone on the NBT response was seen. This applied to both the non-stimulated and stimulated NBT test. For the latter five different toxins were used. At the same time a pronounced neutrophil granulocytosis was seen. The depression of the NBT test is present both in the percentage of NBT-stained neutrophils and when expressed in absolute counts of NBT-strained neutrophils.

It has been established that granulocytes generate superoxide (O-2) as well as hydrogen peroxide (H2O2) during phagocytosis. The chemiluminescence (CL) generated by phagocytes appears dependent on these oxygen radicals (or). however, recent studies suggest that oxygen molecules, including singlet oxygen (1O2) or hydroxyl radicals (OH-), may also be generated during phagocytosis and contribute to CL. We have tested this possibility by studying human granulocyte CL in the presence of 0.1 mM sodium azide, a known inhibitor of myeloperoxidase and catalase and a scavenger of 1O2. The effects of azide on CL were correlated with the effects of this compound on hexose monophosphate shunt (hmps) activity, nitroblue tetrazolium (NBT) dye reduction, formate oxidation, and cytochrome c reduction. CL generated by granulocytes during the phagocytosis of zymosan particles was markedly impaired by azide (24% to 47% of control values). On the other hand, phenomena dependent in part on the presence of O2 radicals, i.e., reduction of NBT dye and cytochrome c, were not impaired by the presence of azide. As would be expected, inhibition of catalase by azide virtually abolished the oxidation of formate, but the burst in HMPS activity associated with phagocytosis was augmented further. The latter observation indicated that azide did not impair generation of H2O2 but increased the relative amount detoxified via the HMPS. The experiment provides evidence that radicals other than O-2 and H2O2 are generated during phagocytosis and that these radicals are major contributors to the CL phenomenon.

The proto-oncogene c-myc, whose gene product has a role in replication, is overexpressed in the human promyelocytic leukemia HL-60 cell line. Treatment of HL-60 cells with an antisense oligodeoxyribonucleotide complementary to the start codon and the next four codons of c-myc mRNA has previously been observed to inhibit c-myc protein expression and cell proliferation in a sequence-specific, dose-dependent manner. Comparable effects are seen upon treatment of HL-60 cells with dimethylsulfoxide (Me2SO), which is also known to induce granulocytic differentiation of HL-60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examined and compared with Me2SO. Differentiation of HL-60 cells into forms with granulocytic characteristics was found to be enhanced in a sequence-specific manner by the anti-c-myc oligomer. No synergism was observed between the anti-c-myc oligomer and Me2SO in stimulating cellular differentiation. In contrast, synergism did appear in the inhibition of cell proliferation. Finally, the anti-c-myc oligomer uniformly inhibited colony formation in semisolid medium. It is possible that further reduction in the level of c-myc expression by antisense oligomer inhibition may be sufficient to allow terminal granulocytic differentiation and reverse transformation.

The effects of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and of hemin on the human erythroleukemia cell lines HEL and K-562 were studied using the parameters cellular morphology, cell proliferation, positivity in the nitro-blue tetrazolium (NBT)-reduction test, adherence to surface, development of pseudopodia, and isoenzyme profiles of carboxylic esterase, acid phosphatase, hexosaminidase and lactate dehydrogenase (LDH) separated by isoelectric focusing on horizontal polyacrylamide gels. TPA and hemin induced similar morphological changes in HEL and K-562, erythrocyte-like cells were seen to be the predominant population after exposure to hemin. Both TPA and hemin led to cessation of cell proliferation and to a dose-dependent decrease in the number of viable nucleated cells. Whereas HEL cells treated with TPA became positive in the NBT-reduction test, adhered to plastic surface and showed pseudopodia, HEL cells exposed to hemin and K-562 cells cultured with TPA or hemin did not show these markers indicative of the macrophage-cell lineage. TPA-treated HEL cells exhibited isoenzyme patterns which were characteristically seen in cells of the monocyte-macrophage complex. 'Myeloid isoenzyme profiles' were found in TPA-treated K-562 cells. As evidenced by the above described phenotypical changes, HEL cells appeared to differentiate along the macrophage and erythroid cell lineages. Induced K-562 cells displayed myeloid and erythrocytic markers.

The ability of blood and bone marrow neutrophils to reduce nitroblue-tetrazolium both before and after stimulation with Escherichia coli endotoxin was investigated. The polymorphonuclear cells of the bone marrow are less able to reduce the dye than blood cells. This difference is maintained after stimulation by endotoxin. Early forms of neutrophils (myelocytes and metamyelocytes) have some reducing ability but this is more marked after stimulation with endotoxin.

We have previously shown that cis-unsaturated fatty acids (c-UFAs) possess a selective tumoricidal action that can be blocked by antioxidants. This property of c-UFAs might be due to various factors, including increased uptake, unusual distribution, or an ability to alter free radical generation in tumor but not normal cells. 14C-labelled linoleic acid (LA) uptake was almost the same in normal and tumor cells, whereas that of 14C-labelled arachidonic acid (AA) and 14C-labelled eicosapentaenoic acid (EPA) in tumor cells was substantially less than in normal cells. Tumor cells incorporate major portions of the fatty acids in the ether lipid and phospholipid fractions, whereas normal cells incorporate the fatty acids primarily in the phospholipid fraction. LA, AA, and EPA augmented nitroblue tetrazolium reduction, an indication of free radical generation, selectively in the tumor cells. These results suggest that there are significant differences between normal and tumor cells in fatty acid uptake and distribution, and in the ability of fatty acids to generate free radicals.

GM3 ganglioside, added exogenously to a promyelocytic leukemia cell line (HL-60 cells) in serum-free synthetic medium, induced differentiation into macrophage-like cells. Macrophagic morphology and function of differentiation-induced cells were determined by cell growth behavior, May-Grünwald-Giemsa staining, activities of nonspecific esterase, phagocytosis and nitroblue tetrazolium (NBT) reduction. GM3 ganglioside may play a role in triggering differentiation of HL-60 cells into macrophage-like cells.

The present study shows that the dehydrogenation of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) can be coupled to tetrazolium nitro blue (TNB), a substance used as electron acceptor in many dehydrogenase reactions. The MPTP-TNB reductase system has an apparent Km value and inhibitor sensitivity similar to those of monoamine oxidase (MAO) type B. MPTP oxidation in the brain may very well be mediated by MAO-B.

The ingestion- and Nitro Blue Tetrazolium-(NBT)-reduction-capacity of neutrophil leukocytes in three newborn infants with lethal septicemia was studied using a modified NBT-test. In comparison to 25 controls in healthy newborns (umbilical vein-blood) the results indicate a failure in the functioning of leukocytes. Besides other disturbances the impaired ingestion- and NBT-reduction-capacity of the neutrophil granulocytes during an infection probably leads to septicemia.

Increases in cytochrome P-450 and cytochrome b5 and a decrease in NADPH-cytochrome c (P-450) reductase were generally brought about by feeding the riboflavin-deficient diet to young rats (50-120 g body weight) for 5 weeks, whereas no significant changes in these enzymes were observed with rats of 220 g body weight by feeding for 2 weeks. Amounts of lipid peroxides in the serum or the liver tissue and microsomes increased significantly after feeding the deficient diet for 5 weeks, in comparison with the respective control rats. On the other hand, NADPH-dependent lipid peroxidation in the presence of ferric ion and pyrophosphate, assayed as malondialdehyde, was decreased drastically in the liver microsomes of all groups of riboflavin-deficient rats irrespective of the period or body weight of animals. Lipid peroxidation could be detected by addition of EDTA-ferric ion or ferricyanide to the incubation medium, though the formation of malondialdehyde was less than that expected by the activity of NADPH-cytochrome c reductase. NADPH-reduction of nitroblue tetrazolium with liver microsomes was decreased in riboflavin deficiency, and was almost able to be correlated with the activity of NADPH-cytochrome c reductase. Following intraperitoneal injection of riboflavin into the deficient rats, NADPH-dependent lipid peroxidation in the presence of ferric ion and pyrophosphate recovered only to 10% of the control rates at 40 hr after the injection, when cytochrome P-450, cytochrome b5 and NADPH-cytochrome c reductase levels were restored to those of their respective controls. Activities of drug-metabolizing enzymes, aminopyrine demethylase and aniline hydroxylase were decreased by initiation of feeding from the weanling stage, but the activities changed only slightly by feeding from the 120 g of body weight stage.

The effects of human leukocyte, porcine spleen and bovine liver dialysate fractions on Listeria resistance were measured by survival studies and by assessing the capacity of peritoneal macrophages to produce superoxide anion (0-2) and prostaglandins. Leukocyte (DLE) and other tissue dialysates were fractionated on a Sephadex G-10 column. Thereafter the significant activities were found in fraction III of DLE, fraction II of porcine spleen and fraction II + III of bovine liver dialysate. The treatment with active porcine spleen dialysate fraction increased the capacity of peritoneal macrophages to generate superoxide anion. On the other hand, this fraction significantly decreased the production of prostaglandin PGE2 and thromboxan B2. These results may indicate that all dialysates can be a source of a non-specifically-acting immunomodulatory preparation and that the infection-resistance-increasing substances seem to operate via the monocyte/macrophage activation.

The superoxide production of BCG-infected and noninfected alveolar macrophages was measured by superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The cells were incubated with or without cell-free bronchial lavage fluid (pulmonary washings). When control alveolar macrophages were infected by BCG, superoxide production was decreased markedly, probably due to bacterial cytotoxic factors. In contrast, the production of superoxide in alveolar macrophages exposed to pulmonary washings was increased and not appreciably influenced by BCG infection. Superoxide production by alveolar macrophages was dependent on time and on the protein concentration in the pulmonary washings. In controls, it was inversely proportional to the infecting dose of BCG. We observed previously that alveolar macrophages activated by pulmonary washings inhibited intracellular growth of BCG. We now present evidence that enhanced production of superoxide contributes to such inhibition, especially in the presence of catalase at acid pH. These findings are pertinent to the defense of inflamed lungs, where serum and serum immunoglobulin G transuded from blood into alveolar spaces probably induce such activation on alveolar macrophages.

A method is described which permits the selection of mutants of Neurospora crassa that are deficient in succinic dehydrogenase activity. The method relies on the observation that succinic dehydrogenase-deficient strains fail to reduce the dye nitrotetrazolium blue when overlaid with the dye in the presence of succinate and phenazine methosulfate. Wild-type colonies reduced the dye and turned blue, whereas mutant colonies remained colorless. In this communication we present studies of a mutant, SDH-1, isolated by this method. The mutant had 18% of the succinic dehydrogenase activity of the parent strain used in the mutation experiments as determined from the ratio of Vmax activities obtained from Lineweaver-Burk plots. The SDH-1 mutant segregated in a Mendelian manner when back-crossed to its parent strain. Succinate oxidase activity in SDH-1 was low and was markedly inhibited by adenosine 5'-diphosphate. The succinate oxidase activity of the parent strain was high and was not affected by the presence of adenosine 5'-diphosphate.