Selvaraj Rj

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.

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.

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.

Positive correlation of optical density at 650 nm. with lecithin/ sphingomyelin ratios in amniotic fluid

In this study, we have attempted to correlate optical density measurements of amniotic fluids with L/S ratios. We may conclude, with over a 98 per cent accuracy, that fluids having optical density readings of 0.15 and above, at 650 nm. will have an L/S ratio over 2.0. Fluids having optical density readings up to 0.05 will have L/S ratios of about 1.3. Finally, amniotic fluids having optical densities greater than 0.05 and less than 0.15 will have L/S ratios of approximately 1.5.

Phagocytosis inhibition and reversal II. possible role of pyruvate as an alternative source of energy for particle uptake by guinea-pig leukocytes

Abstract The mechanism by which pyruvate reverses the phagocytosis blocked by glycolytic inhibitors (NaF and iodaecetic acid) has been studies. It has been shown that pyruvate reversal is not due to a restoration of glycolysis nor to a stimulation of the hexose monophosphate pathway. By using differentially-labeled pyruvate it has been demonstrated that pyruvate is rapidly oxidized to CpASAc. It is suggested that this high-energy compound, CoASAc, can substitute for the glycolytic energy required for particle uptake by synthesizing ATP at the substrate level.

Phagocytosis and onset of human labor

An in vitro experimental model has been developed which allows the study of amnion lysosomal enzyme release under controlled conditions. Briefly, a layer of human amnion membrane mounted on a specially designed reaction vessel serves as the reaction surface. We have noted that the addition of particulate material to these membranes incubating in pseudoamniotic fluid results in an increased release of the lysosomal marker enzyme N-acetylglucosaminidase when compared to the release in the absence of particles. This release is completely inhibited by iodoacetate and slightly by azide. A similar increased release is also noted with the use of term amniotic fluid as incubation medium when compared to centrifuged (30,000 g/20 min) amniotic fluid. Lecithin and lysolecithin were effective in releasing increasing amounts of enzyme. This increased release was noted only from membranes of placentas collected from subjects who had undergone cesarean section prior to labor. Membranes collected from vaginal deliveries after labor showed a baseline increased release but no further stimulation upon the addition of any of the substances. These results suggest that the release of lysosomal enzymes from amnion membranes is brought about by substance(s) present in amniotic fluid. Very probably, these are surfactants. The interaction of these substances with amnion cells would eventually result in an exponential burst of prostaglandin synthesis, which would result in labor.

Antimicrobial activity of amniotic fluid against Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum

Amniotic fluids obtained by amniocentesis at 16 weeks to term were examined for the presence of Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma urealyticum. Of 140 fluids tested, none harbored chlamydiae, and only one harbored mycoplasma, M. hominis. A number of amniotic fluids were subsequently tested for their ability to inhibit the growth of these microorganisms. Amniotic fluids and chlamydial suspensions in a 2:1 ratio were incubated 30 to 90 minutes before their inoculation in McCoy cells. Procedures were followed for chlamydial isolation. Genital mycoplasmas were incubated with amniotic fluid samples for 24 or 48 hours at 35 degrees C. Growth in amniotic fluid specimens was compared with growth in pseudoamniotic fluid and broth controls. Fourteen amniotic fluid specimens collected from gestations of 16 to 40 weeks, were found to be inhibitory to the formation of inclusions of C. trachomatis in McCoy cells. Ten amniotic fluid specimens (16 to 39 weeks, gestation) demonstrated various degrees of inhibition against M. hominis, and three fluids were inhibitory to the growth of Ureaplasma. The inhibitor was heat and protease resistant and activity was proportional to concentration. The molecular weight of the inhibitor was probably greater than 10,000 daltons, and pH, although perhaps a contributing factor, was not the cause of the inhibition.

The effect of cervical/vaginal secretions on measurements of lecithin/sphingomyelin ratio and optical density at 650 nm

Free-flowing amniotic fluid collected vaginally can be used in a reliable way for determination of fetal pulmonary maturity. Lavaging the vaginal/cervical area with sterile saline and examining the lavage fluid for lecithin/sphingomyelin (L/S) spots showed no detectable spots in the supernatants (one exception) and barely detectable L/S spots in the sediment. Vaginal-cervical saline-wash fluids did not affect fluid L/S ratios. Lavaging the vaginal-cervical area with abdominal amniotic fluid did not affect the L/S ratio of the original amniotic fluid.