Anthony J. Sbarra

The role of the phagocyte in host-parasite interactions: XIII. The direct quantitative estimation of H2O2 in phagocytizing cells

Abstract 1. 1. A technique that allows the direct quantitative estimation of H2O2 in polymorphonuclear leukocytes has been developed. 2. 2. Lysis or homogenization followed by dialysis of polymorphonuclear leukocytes results in a dialysate containing fluorescence-producing material. 3. 3. This material is catalase sensitive. 4. 4. Catalase specificity cannot be demonstrated without preliminary dialysis, indicating the presence of a non-dialyzable catalase-limiting component(s) in the polymorphonuclear leukocytes. 5. 5. Phagocytizing cells contain 2–4 times as much measurable H2O2 as non-phagocytizing polymorphonuclear leukocytes.

The role of the phagocyte in host-parasite interactions: XIX. Leukocytic glutathione reductase and its involvement in phagocytosis

Abstract Glutathione reductase activity was found in guinea pig peritoneal polymorphonuclear leuckocytes. The cofactor requirement for the enzyme was found to be NADPH. This was determined by the requirement of GSSG for oxidation of the reduced cofactor and the stimulatory effect of KCN on GSH peroxidase which was also found in these cells. Enzyme activity was inhibited by the sulfhydryl antagonist N -ethyl maleimide. The addition of GSSG to the reaction mixture resulted in an increase in cellular GSH content. The activity of the enzyme was confined to the 20,000 g supernatant fraction. Phagocytosis caused a significant increase in GSSG reductase activity within 15 sec after particle addition. The NADPH oxidase stimulation which has been associated with the phagocytic process did not occur until some minutes after the addition of latex particles. Since NADP + is a limiting factor for the hexose monophosphate shunt and GSSG reductase can increase the NADP + content of the cell, one can now postulate that increased activity of this enzyme is an early event involved in the phagocytosis-associated increase in this metabolic pathway.

Management of multiple gestation.

The following perinatal intensive care management protocol is suggested to minimize morbidity and mortality in multiple pregnancy. 1. Early diagnosis is essential, with ultrasound examination regarding as invaluable for all pregnant patients. If ultrasound examination has not been done, multiple pregnancy should be suspected in all patients who have a family history of dizygotic births, are large for dates or anemic, or have a low-grade preeclampsia. 2. Maternal care should be provided at a tertiary care (level III) perinatatl center, which is more fully equipped to manage multiple gestation. 3. Bed rest, if instituted early enough (26-29 weeks) appears to be of value, especially in promoting increased birth weight. 4. A liberal approach to performing cesarean birth is suggested when any abnormal presentation exists. 5. Aggressive management, using tocolytic agents to delay premature labor and steroids to accelerate pulmonary maturity, should be strongly considered. 6. The personnel of a neonatal intensive care unit should employ a team approach to the preparation for and management of multiple preterm births.

Chlorination by the Myeloperoxidase-H2O2-CI-Antimicrobial System at Acid and Neutral pH

Summary The oxidation of Cl- ion by H2O2 yielding Cl+ is catalyzed by myelope-roxidase (MPO) prepared from human PMN. Both H2O2 and CI- in this reaction serve as substrates. Affinity (apparent) studies between MPO and CI- and MPO and H2O2 were carried out. Both (apparent) affinities vary according to pH. The higher the pH, the less the affinity for Cl-. In contrast, the affinity between MPO and H2O2 increases with pH. The dissociation constant of the MPO-CI- enzyme substrate complex is affected by the second substrate, H2O2. The effect of H2O2 on the MPO-C1- affinity is competitive. Likewise, the MPO-H2O2 affinity is decreased with increasing Cl- concentration. This influence is also competitive. From these interactions, it may be concluded that the optimal pH for chlori-nation is dependent on the [H2O2]/[C1-] ratio. The relationship of pH, H2O2, and CI- concentrations may be expressed by the following formula: pH = log [CI-] x560/ [H2O2]. Using the formula, it can be shown that optimal chlorination may occur between pH 4.0 and 7.4. We wish to thank Paul Wallace and Gerry Heatley for technical assistance, Roberta Lemon for preparing the manuscript, and George Daynes for photography. This investigation was supported by Grant CA-1567 awarded by the National Cancer Institute, DHEW, and USPHS Grant HD-1805, NIH.

The Respiratory burst and its physiological significance

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Infection and phagocytosis as possible mechanisms of rupture in premature rupture of the membranes.

The concept that premature rupture of the membranes is due to an infectious process is well accepted. However, no definitive data implicating a particular microorganism or a mechanism of action have been advanced. By the use of our recently developed experimental in vitro amnion-chorion reaction vessel model we have studied the effect of the peroxidase-hydrogen peroxide-halide antimicrobial system on these membranes. We have noted that amnion, chorion, decidua, and placental macrophages all possess peroxidase activity. Tissues collected from deliveries following labor (vaginal) are significantly higher in activity than those collected from deliveries with no labor (cesarean section). A mobilization of enzyme from macrophages to amnion appears to occur in the laboring patient. Increased protein hydrolysis is noted in membranes collected from patients without labor subjected to the peroxidase-hydrogen peroxide-halide cytotoxic system when compared with membranes from laboring patients. Bursting pressures of membranes collected from patients without labor are shown to be decreased when the membranes were incubated in the presence of lysolecithin or in the presence of amniotic fluid and phospholipase A2.

Peroxidase Mediated Antimicrobial Activities of Alveolar Macrophage Granules

The 20,000g pellet obtained by centrifugation of a homogenate of rabbit alveolar macrophages has antibacterial activity in the presence of a hydrogen peroxide-generating system and iodide. Peroxidase activity has been demonstrated in this fraction. Addition of 3-amino-1,2,4-triazole diminished the antibacterial activity of the pellet-hydrogen peroxide-iodide system.

Direct involvement of nadph oxidase with the stimulated respiratory and hexose monophosphate shunt activities in phagocytizing leukocytes

Abstract Guinea pig peritoneal exudate polymorphonuclear leukocytes (PMN) have KCN-sensitive and insensitive NADPH oxidation activity. These activities can be localized to different homogenate fractions. NADPH oxidation activity of the 19 000 g pellet is KCN-insensitive. Myeloperoxidase (MPO) activity which is also confined to this fraction is completely inhibited by 1 mM KCN. This observation indicates that KCN-insensitive NADPH oxidation is not due to MPO. KCN-sensitive NADPH oxidation activity is found in the 19 000 g supernatant fraction, as is catalase. Purified beef liver catalase does not oxidize NADPH, thus indicating that the NADPH oxidation by the supernatant is not due to the presence of catalase. NADPH oxidation activity of both the 19 000 g pellet and supernatant fractions collected from phagocytizing cells is significantly greater than that found in the corresponding fractions collected from resting cells. The increased oxidative burst associated with phagocytosis can be accounted for by the stimulated NADPH oxidase activity.

The role of the phagocyte in host-parasite interactions: VII. Di- and triphosphorydine nucleotide kinetics during phagocytosis

Abstract 1. 1. In order to add further conceptual information regarding the physiological functions of the different pathways of glucose metabolism in phagocytizing guineapig polymorphonuclear neutrophils, a study designed to establish the levels of oxidized and reduced di- and triphosphopyridine nucleotides was undertaken. 2. 2. The total DPN + and DPNH was found to be higher than the total TPN + and TPNH in both resting and phagocytizing cells. The total amounts of di- or triphosphorydine nucleotides did not change during the phagocytic period. 3. 3. The DPN + /DPNH ratio dropped significantly as metabolism was initiated. A further decrease was noted with phagocytizing cells. The TPN + /TPNH ratio does not change significantly in resting cells; in phagocytizing cells a significant increase was noted. 4. 4. Glycolytic inhibitors increased the DPN + /DPNH ratios of both resting and phagocytizing cells. Iodocetate dit not affect the TPN + /TPNH ratio in any case; sodium fluoride increased the TPN + /TPNH ratio of resting cells to level found in phagocytizing cells. 5. 5. The physiological significance of the higher TPN + /TPNH ratio foun in phagocytizing cells and its cyclic relationship to hexose monophosphate shunt stimulation and TPNH oxidation is presented.

The role of the phagocyte in host-parasite interactions: IV. The phagocytic activity of leukocytes in pregnancy and its relationship to urinary tract infections

Abstract 1. 1. Using Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus albus as test organisms, the phagocytic and bactericidal rates for leukocyte populations from pregnant women at various stages of pregnancy have been determined and compared with results obtained with leukocytes from nonpregnant women. 2. 2. Leukocytes from pregnant women usually were found to demonstrate increased phagocytic and bactericidal activities: the greatest frequency of enhanced leukocyte activity was observed when E. coli was the test organism. 3. 3. Leukocytes from pregnant women with significant bacteriuria generally showed only normal or reduced activity against the test organism which was of the same genus as the predominant organism in the urine. 4. 4. Antibody levels to E. coli and P. aeruginosa were determined in the sera of pregnant and nonpregnant women by the indirect hemagglutination technique. Sera from pregnant patients showed a higher hemagglutinin titer to E. coli when compared to sera obtained from nonpregnant women. No such differences were noted with P. aeruginosa hemagglutinins.