Increased respiratory burst response and alkaline phosphatase activity in human cord blood neutrophils

The priming effect of a bacterial lipopolysaccharide (LPS) on subsequent respiratory burst activity induced by either the chemoattractant formylmethionyl-leucyi-phenylalaninc (fMLP) or phorbol myristate acetate (PMA; a protein kinase C activator) was studied in human ncutrophils isolated from cord blood and adult peripheral blood. Cells from adults, but not from newborn babies, were primed by LPS pretreatment. The content and release of β-glucuronidase and vitamin-B12 binding protein, or marker for the azurophilic and specific granules, respectively, was similar for cells from infants and adult controls. In contrast, alkaline phosphatase activity was significantly increased in the cord blood neutrophils compared to neutrophils from adult peripheral blood. The latency of the alkaline phosphatase activity was, however, similar. Thus, the primed response of cord blood neutrophils could not be explained by an increased release of azurophilic or specific granule content. If the increased alkaline phosphatase activity of curd cells represents an increased number of secretory vesicles, however, then this would indicate a rapid turnover leading to delivery of new receptors and oxidase components to the cell membrane.     

Membrane NADPH oxidase activity and cell size in bovine neonatal and adult neutrophils

Neutrophils (PMN) from newborn calves generate significantly less superoxide anion (O2-) than do their adult counterparts after stimulation with direct protein kinase C agonists. To better understand this observation, we compared the activity and kinetics of NADPH oxidase in membrane fractions from phorbol 12-myristate 13-acetate-stimulated adult and newborn PMN. After phorbol 12-myristate 13-acetate stimulation, PMN were sonicated and the membranes assayed for O2- production with increasing concentrations of NADPH. O2- production was calculated 1 and 2 min after the beginning of the reaction. At all concentrations of NADPH used, there was no difference (p greater than 0.05) in O2- production between adult (n = 8) and newborn (n = 9) PMN membrane preparations. Enzyme kinetics calculations revealed no differences (p greater than 0.05) between age groups in Km and Vmax or in the velocity of the reactions. Determination of the protein content in the membrane pellet, however, showed that adult PMN yielded significantly (p less than 0.01) higher amounts of protein (2.82 +/- 0.14 mg/mL) than did newborn PMN (1.78 +/- 0.07 mg/mL). This difference could be partly attributed to cell size; flow cytometric analysis showed that newborn PMN had a significantly (p less than 0.01) smaller diameter (10.94 +/- 0.07 microns) than did adult PMN (11.65 +/- 0.06 microns), and calculated cell volume and surface area were also both significantly less (p less than 0.01) in newborn PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

NADPH oxidase activity in evaluating the status of newborn infants

The authors presented the results of studying NADPH-oxidase activity in the blood of neonates born to mothers with central endocrine disorders. The activity of the enzyme was detected in the blood and amniotic fluid withdrawn from the woman in birth. The relationship has been shown between the activity of NADPH-oxidase in the children's blood and their health status at birth together with a possibility to forecast the degree of the hypoxia-induced damage to the child in accordance with detecting the enzyme in the amniotic fluid.     

Low burst promoting activity production by phytohemagglutinin-stimulated mononuclear cells of cord blood

Burst-promoting activity (BPA) produced by phytohemagglutinin(PHA)-stimulated cord blood mononuclear cells (MNC) was examined using the two-stage cell culture assays. Burst-promoting activity was measured as the increase in the number of early erythroid progenitor cells in 2-day incubation of peripheral blood MNC with or without the conditioned medium of PHA-stimulated MNC (PHA-LCM). Burst-promoting activity in PHA-LCM of cord blood was significantly lower than that of adult blood (37 +/- 13 versus 105 +/- 19%, mean +/- SD, p less than 0.01). No elevation of inhibitors to the erythroid colony growth was noted in PHA-LCM of cord blood. In contrary, the response of cord blood MNC to PHA was similar to that of adult blood MNC, as determined by colony-stimulating activity production and cell proliferation. These results showed that burst-promoting activity production by PHA-stimulated MNC of cord blood was lower than that of adult blood.     

Increased activation of NADPH oxidase 4 in the pulmonary vasculature in experimental diaphragmatic hernia

Aim

Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). NADPH oxidases (Nox) are the main source of superoxide production in vasculature. Nox4 is highly expressed in the smooth muscle and endothelial cells of the vascular wall and increased activity has been reported in the pulmonary vasculature of both experimental and human pulmonary hypertension. Peroxisome proliferator-activated receptor (PPARγ) is a key regulator of Nox4 expression. Targeted depletion of PPARγ results in pulmonary hypertension phenotype whereas activation of PPARγ attenuates pulmonary hypertension and reduces Nox4 production. The nitrofen-induced CDH model is an established model to study the pathogenesis of pulmonary hypertension in CDH. It has been previously reported that PPARγ-signaling is disrupted during late gestation and H₂O₂ production is increased in nitrofen-induced CDH. We designed this study to investigate the hypothesis that Nox4 expression and activation is increased and vascular PPARγ is decreased in nitrofen-induced CDH.

Methods

Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into control and CDH. RT-PCR, western blotting and confocal-immunofluorescence-double-staining were performed to determine pulmonary expression levels of PPARγ, Nox4 and Nox4-activation (p22^phox).

Results

There was a marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary Nox4 levels were significantly increased whereas PPARγ levels were decreased in nitrofen-induced CDH compared to controls. Western blotting revealed increased pulmonary protein expression of the Nox4-activating subunit p22^phox and decreased protein expression of PPARγ in CDH compared to controls. Confocal-microscopy confirmed markedly increased pulmonary expression of the Nox4 activating subunit p22^phox accompanied by decreased perivascular PPARγ expression in lungs of nitrofen-exposed fetuses compared to controls.

Conclusion

To our knowledge, the present study is the first to report increased Nox4 production in the pulmonary vasculature of nitrofen-induced CDH. Down-regulation of the PPARγ-signaling pathway may lead to increased superoxide production, resulting in pulmonary vascular dysfunction and contributing to pulmonary hypertension in the nitrofen-induced CDH model. PPARγ-activation inhibiting Nox4 production may therefore represent a potential therapeutic approach for the treatment of pulmonary hypertension in CDH.     

Deficiency of NADPH oxidase activity in chronic granulomatous disease.

NADPH oxidase activity was examined in paired 27,000 x g granule fractions isolated from normal polymorphonuclear leukocytes from patients with chronic granulomatous disease. At 0.17 mM NADPH, the oxidase activity was not measurable in normal resting cells but was activated by phagocytosis.This activation was absent in CGD cells. At higher levels of NADPH, activity was present in cells from patients with CGD, although it was lower than normal, and no difference in activity was found between resting and phagocytizing cells. Granule fractions from phagocytizing normal cells exhibited higher than granule fractions from resting normal cells at all levels of NADPH. These results suggest that NADPH oxidase activity is defective in chronic granulomatous disease, and further that the defect is not the absence of the enzyme but rather a failure to activate it.     

Lack of correlation between NADPH-oxidase priming and elevated alkaline phosphatase activity in cord blood neutrophils

Human neutrophils were isolated from cord blood drawn after Caesarean section deliveries without labour and from peripheral blood from healthy adults. The alkaline phosphatase (ALP) activity in the cell populations was compared with the release of respiratory burst products after activation with the chemoattractant n-formyl-methionyl-leucyl-phenylalanine (fMLP). In contrast to cord blood neutrophils obtained from babies after normal vaginal deliveries, the Caesarean section neutrophils were not metabolically primed. However, like the neutrophils from vaginally delivered babies, the Caesarean section neutrophils showed an increased ALP activity compared to adult control neutrophils. These findings indicate that priming of cord blood neutrophils appears to be induced by labour and can be dissociated from increased ALP activity.     

Oxidase activity in cord blood neutrophils: a balance between increased membrane associated cytochrome b558 and deficient cytosolic components

INTRODUCTION: Newborn infants are prone to develop life-threatening pyogenic infections. Alterations in the function of neonatal phagocytes, including the activity of the neutrophil NADPH oxidase, have been suggested as one cause of increased susceptibility to such infections. METHODS: In the present study, comprehensive analysis of NADPH oxidase enzyme system was performed in cord blood neutrophils from vaginally and cesarean section (CS) delivered, healthy, full-term infants. RESULTS: Superoxide anion (O(2) (-)) production by intact neutrophils from cord blood in response to soluble stimuli was equal to or increased compared to levels generated by cells from adult controls. In the sodium dodecyl sulfate (SDS) cell-free system, cytosol and plasma membrane from cord blood neutrophils generated O(2) (-) at comparable rates to subcellular fractions from healthy adults. However, mixing experiments demonstrated higher O(2) (-) generation with combination of cytosol from adult controls and membrane from cord blood neutrophils and lower O(2) (-) production with combination of cytosol from cord blood neutrophils and membrane from adult controls. Kinetic parameters for cord blood specimens were no different from those obtained for fractions from adult controls. Quantitative analysis of cytosolic components showed moderately reduced amount of p40-phox, p47-phox, and p67-phox in neutrophils from cord blood. In contrast, cytochrome b(558) content of plasma membrane of cord blood neutrophils was approximately 2-fold higher compared to adult controls. CONCLUSION: The normal to increased respiratory burst of intact cord blood neutrophils is the result of alterations to oxidase components: increased content of cytochrome b(558) in the plasma membrane and decreased levels of cytosolic components p47-phox, p67-phox, and p40-phox.     

Prolactin in umbilical cord blood and the respiratory distress syndrome.

Prolactin was measured in umbilical cord serum obtained from 77 newborn infants of gestational age 28 to 40 weeks. A positive correlation with gestational age was demonstrated. Between 30 and 36 weeks of gestation the elevation of the regression line of the concentration of cord PRL versus gestation age was significantly lower (P less than 0.05) for those infants who developed respiratory distress syndrome compared to the regression line for infants who did not develop RDS. Between 32 and 33.5 weeks, the mean +/- SEM cord PRL concentration in infants who developed RDS (101.7 +/- 9.5 ng/ml) was significantly less (P less than 0.025) than the PRL concentration in those who did not develop RDS (161.8 +/- 18.9 ng/ml). Cord PRL did not correlate with cord cortisol or dehydroepiandrosterone sulfate concentrations. Cord growth hormone concentrations did not show any relationship to the occurrence of RDS. Serum PRL was not suppressed in a further 114 infants whose mothers were treated prenatally with betamethasone. These findings raise the possibility of a role of PRL in fetal lung maturation.     

Oxidative metabolism of cord blood neutrophils: relationship to content and degranulation of cytoplasmic granules

Generation of oxygen metabolites is an important component of the neutrophil's armamentarium against microbes. Production of superoxide anion (O2-) and generation of hydroxyl radical (OH) were measured in neutrophils from cord blood of 12 vaginally delivered, term newborn infants and 12 adults after stimulation with phorbol myristate acetate (PMA) and opsonized zymosan. With either stimulus, generation of OH was relatively less than production of O2- for all infants studied. This discrepancy might be related to abnormal release or diminished cell content of a cofactor necessary for production of OH from O2-. Since both lactoferrin (LF) found in specific granules and myeloperoxidase (MPO) found in azurophilic granules have been shown to enhance OH generation, we compared degranulation of both granule types in response to PMA and opsonized zymosan and total neutrophil content of MPO, LF, and lysozyme in cord blood and adult neutrophils. Degranulation, even after pretreatment with cytochalasin B, was the same for newborn and adult neutrophils. Content of MPO was identical (adult, 204 +/- 24 A units, mean +/- SEM, n = 9; newborn, 201 +/- 21, n = 9) but lysozyme was mildly diminished (adults, 111 +/- 10 A units; newborn, 89 +/- 6, n = 9, p less than 0.05), and lactoferrin was moderately decreased (adult, 89.0 +/- 7.3 micrograms/mg cell protein, n = 11; newborn, 43.2 +/- 7.0, n = 11, p less than 0.005). Generation of OH in response to PMA and LF content were measured in seven cord blood-adult control pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Superoxide dismutase activity in the maternal and cord blood.

Superoxide dismutase (SOD) activity was measured in the maternal and cord blood by the modified method of Beauchamp and Fridovich, using a carbonate-buffered (pH 10.2) xanthine-xanthine oxidase system. No great differences between maternal and cord blood in erythrocyte SOD levels were observed, with the exception of whole blood; namely, washed RBC showed a SOD activity of a fairly high level, which was comparable to the activities of crude SOD, but showed no difference between them. In contrast, the SOD activity in the maternal whole blood was significantly lower than that in the cord blood. In measuring SOD activity, the serum factor has a great effect, and serum contains a substance that inhibits NBT reduction. Only one band of SOD has been detected which shows identical Rf values both in maternal and cord blood by polyacrylamide-gel electrophoresis.     

细胞因子对脐血单个核细胞端粒酶活性的影响

目的研究集落刺激因子对脐血单个核细胞(CBMC)端粒酶活性的影响。方法体外培养3例足月正常新生儿的CBMC,并加入集落刺激因子IL?3、SCF、GM?CSF及其组合,培养5d后用TRAP?ELISA法测定每份标本的端粒酶活性。结果CBMC培养前有较弱的端粒酶活性,加入集落刺激因子体外培养后端粒酶活性均有不同程度的上调,以加入IL?3、SCF、GM?CSF组最为显著;CBMC与PBMC培养前端粒酶活性差异无显著性,培养后比较差异有显著性(P<0.01)。结论IL?3、SCF、GM?CSF的组合对CBMC端粒酶活性有明显影响;集落刺激因子对CBMC和PBMC均有扩增作用,但CBMC具有更高扩增潜能。     

Glutathione peroxidase activity in cord blood: effects of fetal sex and maternal smoking

Glutathione peroxidase activity was estimated in the cord blood of 47 newborn infants. In 23 cases corresponding maternal glutathione peroxidase activities were measured at time of delivery and compared with 23 non-pregnant women of the same age. Mean cord blood activity was 23.6 +/- 4.8 IU/g Hb at 37 degrees C. Maternal smoking and male fetal sex were associated with significantly lower fetal glutathione peroxidase activity. Maternal levels (33.0 +/- 5.0 IU/g Hb) were significantly higher than those in cord blood (p less than 0.001) but were the same as those for non-pregnant women. Maternal to cord blood level ratio was 1.5:1.