Howard M Berger

Meconium Is a Source of Pro-Inflammatory Substances and Can Induce Cytokine Production in Cultured A549 Epithelial Cells

Inflammation plays an important role in the pathogenesis of meconium aspiration syndrome, and pneumonitis is one of the major characteristics. We have previously shown that meconium has chemotactic properties because of the presence of IL-8. We hypothesize that IL-8 and other proinflammatory substances in meconium may amplify inflammation in meconium aspiration syndrome, inducing endogenous cytokine production by lung epithelial cells. We measured proinflammatory substances in first-pass meconium from healthy newborns and evaluated the effect of sterile meconium on cytokine production in cultured A549 alveolar epithelial cells in vitro. IL-1β, IL-6, IL-8, and tumor necrosis factor-α were measured by ELISA, and heme was measured spectrophotometrically. After incubation of meconium samples with A549 cells, cytokine concentrations in the supernatant were measured. Meconium samples contained variable amounts of IL-1β, IL-6, IL-8, tumor necrosis factor-α, and heme. On stimulation of A549 cells with meconium, the IL-8 concentration in the culture supernatant significantly increased above baseline measurements, whereas tumor necrosis factor-α showed a variable pattern and IL-1β or IL-6 remained unchanged. There was no quantitative relationship between the concentration of the measured cytokines and heme in meconium and cytokine release by the A549 cells after meconium exposure. Meconium contains proinflammatory substances. All samples induced IL-8 release and some induced tumor necrosis factor-α release in cultured A549 epithelial cells. We speculate that proinflammatory substances in meconium can induce lung inflammation in meconium aspiration syndrome in two ways: directly via cytokines and heme present in meconium and indirectly by inducing cytokine release by the epithelial lung cells.

LIMITED PROTECTION AGAINST IRON-INDUCED LIPID PEROXIDATION BY CORD BLOOD PLASMA

The ability of plasma from newborn babies (cord blood) and adults to inhibit iron-induced lipid peroxidation was compared. The caeruloplasmin and transferrin concentrations, and latent iron-binding capacity were lower in the babies (p less than 0.001). The plasma of many of the babies had no latent iron-binding capacity and contained non-protein-bound iron (measured by the bleomycin assay). The in vitro ability of plasma to inhibit iron-induced liposome peroxidation by either ferroxidase antioxidant activity (caeruloplasmin) or iron-binding antioxidant activity (transferrin) was measured. The antioxidant activity in both assays was decreased in the babies (p less than 0.001). The percentage inhibition of peroxidation in the iron-binding antioxidant assay correlated positively with the latent iron-binding capacity (p less than 0.001) and negatively with the presence of bleomycin-detectable iron (p less than 0.02) in the babies. This assay produced stimulation of peroxidation in 42% of the babies but none of the adults. The diminished capacity of cord blood plasma to prevent iron-induced lipid peroxidation may predispose the newborn baby to the toxic effects of oxygen.

Recycling of Glutathione during Oxidative Stress in Erythrocytes of the Newborn

ABSTRACT: The ability of erythrocytes from newborn babies and adults to maintain reduced glutathione levels during oxidative stress was studied. In vitro incubation of erythrocytes with H2O2 with or without inactivation of catalase, caused a rapid depletion of reduced glutathione (GSH) and concomitant accumulation of oxidized glutathione followed by recovery of GSH and fall of oxidized glutathione to initial values in all subjects. Inactivation of catalase resulted in a 50% loss of intracellular glutathione (p < 0.005), a larger maximum GSH depletion (p < 0.05), and a longer GSH recovery time (p < 0.005). Erythrocytes from newborn babies showed a smaller maximum GSH depletion (p < 0.05) and a shorter GSH recovery time (p < 0.005) compared with those from adults. These differences between the newborn and adult groups persisted after inactivation of catalase. An increase in maximum GSH depletion and GSH recovery time (p < 0.005) was observed when a lower hematocrit was used for these GSH recovery studies. Effective glutathione recycling in erythrocytes may protect immature tissues of the newborn baby from peroxidative damage.

Preterm neonates with nephrocalcinosis: natural course and renal function.

The aim of the study was to evaluate the natural course of nephrocalcinosis (NC) in preterm neonates and the effect of NC on blood pressure and renal glomerular and tubular function. In a prospective observational study of 201 preterm neonates (gestational age <32xa0weeks) NC was present at term in 83 patients (41%), who were subsequently examined at 6, 12, and 24xa0months, and until August 2000 annually (with a maximum of 4xa0years) if NC persisted. Examination consisted of blood pressure measurement, renal ultrasonography, and glomerular and tubular function tests. The probability that NC, when present at term, would persist for 15 and 30xa0months was 34% [21–45, 95% confidence interval (CI)] and 15% (5–25, 95% CI) (Kaplan-Meier), respectively. Urinary tract infection did not occur more frequently in patients with NC (2.5%) than patients without NC at term (4.4%). Systolic and diastolic blood pressures above the 95th percentile were found in 39% and 48% of patients at 1xa0year and 30% and 34% at 2xa0years (P<0.001). Mean glomerular filtration rate (GFR) (inulin clearance) at 1 and 2xa0years was 92 and 102xa0ml/min per 1.73xa0m2, respectively. TP/GFR and excretion of α1-microglobulin were normal. The desmopressin test was impaired in 4 of 30 patients at 1xa0year and 2 of 25 at 2xa0years. It was concluded that while proximal tubular function is unaffected in children with neonatal NC, high blood pressure and impaired glomerular and distal tubular function might occur more frequently than in healthy children. Although no relationship can be proven between NC and hypertension or diminished renal function in this study, these results justify a large follow-up study with matched controlled study groups.

Cord blood Clara cell protein CC16 predicts the development of bronchopulmonary dysplasia

Clara cell protein (CC16) is an anti-inflammatory protein and a biomarker of pulmonary epithelial cells and alveolocapillary membrane injury in adults. We investigated whether low cord blood concentrations of CC16 are associated with the development of respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in preterm infants and the relationship between CC16 and its pro-inflammatory counterpart, the secretory phospholipase A2 (sPLA2) enzyme. CC16 concentration, sPLA2 activity and IL-6 concentration were measured in cord blood plasma from 79 preterm infants (25 controls, 37 infants who developed RDS and 17 infants who developed BPD). After adjustment for gestational age and Apgar score at 5 min, the CC16 concentration was lower in BPD infants than in preterm controls (p<0.01). sPLA2 activity was similar in all groups and the IL-6 concentrations were increased in both RDS and BPD infants (p<0.01 and p<0.05, respectively, vs. controls). We conclude that low cord blood CC16 concentrations in preterm infants independently predict the development of BPD. Low CC16 levels may reflect early lung injury, which contributes to the severity of RDS and progress towards BPD. Future studies are needed to assess whether the early administration of recombinant human CC16 in preterm infants with low cord blood CC16 prevents the development of BPD.

Non-protein bound iron release during chemotherapy in cancer patients

Non-protein bound iron (NPBI) is able to catalyse oxidative reactions, causing damage to vital structures. Adverse effects induced by cisplatin seem, in part, to be mediated by free radicals. In the present study, we have measured plasma NPBI, various other iron parameters and antioxidants in 28 cancer patients undergoing cisplatin-based chemotherapy at various time points before and during chemotherapy. No NPBI was present prior to therapy, but within 1-4 days following the first administration of chemotherapy, mean NPBI rose significantly to 10.6+/-6.6 micromol/l (range, 0.6-21.3 micromol/l) in 18 (64.3%) of the 28 patients measured. The rise in NPBI was accompanied by a significant rise in total plasma iron and ferritin and a marked decrease in the latent iron-binding capacity. Concomitantly, plasma vitamins C and E decreased significantly, indicating consumption of antioxidants. Similar observations were also made during the fourth chemotherapy cycle. The increase in NPBI preceded and correlated significantly with chemotherapy toxicity, such as a decrease in leucocyte count and haemoglobin, with a transient rise in various liver enzymes and with known cisplatin-related toxicity, i.e. the loss of renal and hearing function. In conclusion, cisplatin chemotherapy induces oxidative damage which rapidly leads to release of iron from intracellular proteins and the appearance of NPBI. Bone marrow, red blood cells, liver and kidney seem to be a likely source of NPBI. The observed high levels of NPBI may be a major causative determinant in chemotherapy-induced toxicity.

The capacity of different infusion fluids to lower the prooxidant activity of plasma iron: an important factor in resuscitation?

BACKGROUND: Prooxidant activity of non‐protein‐bound iron (NPBI) is an important contributor to reactive oxygen species‐induced injury after the resuscitation of critically ill patients. Plasma NPBI occurs in critically ill adults, children, and newborn babies, who often require resuscitation. The ability of the resuscitation fluids to bind iron and lower the patients′ NPBI levels in vitro has not previously been studied.

Pulmonary Secretory Phospholipase A2 in Infants with Respiratory Distress Syndrome Stimulates in vitro Neutrophil Migration

Background: The massive pulmonary neutrophil influx in respiratory distress syndrome (RDS) in preterm infants has been ascribed to the effect of leukotriene B₄ (LTB₄). Objectives: To investigate whether secretory phospholipase A₂ (sPLA₂), the rate-limiting enzyme in LTB₄ production, is present in lungs of RDS infants and stimulates neutrophil migration. Methods: sPLA₂ was measured in tracheal aspirates from 15 preterm infants with RDS. The effect of aspirates on cord blood neutrophil migration was first measured, and the contribution of sPLA₂ was assessed by addition of its endogenous inhibitor Clara cell protein (CC16) or absorption of sPLA₂ from the aspirates. The role of intracellular signal transduction activation and LTB₄ formation in sPLA₂-induced neutrophil migration was determined using purified sPLA₂, several inhibitors of signal transduction, a LTB₄ synthesis inhibitor and a LTB₄ receptor antagonist. Results: All aspirates contained sPLA₂, which significantly stimulated neutrophil migration. Addition of CC16 or absorption of sPLA₂ abolished the stimulatory effect. All inhibitors significantly reduced sPLA₂-induced neutrophil migration. Conclusions: sPLA₂ is present in tracheal aspirates of preterm infants with RDS. Human recombinant sPLA₂ and pancreatic type sPLA₂ stimulate in vitro cord blood neutrophil migration via activation of intracellular signal transduction pathways, LTB₄ production and receptor binding. We speculate that sPLA₂ contributes to pulmonary neutrophil influx in RDS. Further studies are needed to determine the potential of sPLA₂ inhibition as a treatment for RDS.

Antioxidant protection against free radicals is reduced in fetal plasma after intrauterine red blood cell transfusion.

After intrauterine transfusion for red cell alloimmunization, a 2- to 20-fold increase of plasma Hb, a strong pro-oxidant, was observed. The increase of fetal plasma Hb after transfusion leads to a highly significant reduction of plasma antioxidant capacity, measured as the peroxyl radical trapping capacity. Consequently, the posttransfusion reduced antioxidant protection may enhance the peroxidation of lipids in e.g. donor erythrocyte membranes, leading to the shortened life span of these cells in the fetus.

Iron Containing Feeding Formula and Oxidative Stress in Preterm Babies† 1580

Introduction: Plasma non-protein-iron (NPBI), a marker of iron overload and oxidative stress, is present at birth in preterm babies. Use of iron fortified formulas may prolong the presence of NPBI and increase oxidative stress. Methods:We compared iron status and indicators of oxidative stress in plasma of preterm babies (ga 27-34wks) fed iron fortified formula (0.8mg/dl iron, Fe+ group, n=17) or low iron formula (0.08mg/l, Fe- group, n=12). Measurements were started when babies reached 120ml/kg/d of formula feed and repeated at 2 and 4 wks thereafter. Results:The initial incidence of NPBI values did not differ (Fe+ group 18%, Fe-group 17%) but 4 wks later no NPBI was present in any infant. There were no differences in malondialdehyde, hemolysis test, reduced /oxidised glutathione and vitamin C ratios, antioxidant (transferrin, ceruloplasmin, vitamin E, A, and C) levels, and total antioxidant trapping capacity. Ferritin levels (mean (SD),μmol/l) fell in both the Fe+ and Fe- group from 206(83) and 162(60) resp. to 125(92) and 49(29) resp. at 4wks (difference between groups NS). However, 2 babies in the Fe-group compared to none in the Fe+ group developed ferritin levels of less than 20 μmol/l. Conclusion: Iron fortification did not increase iron overload and/or oxidative stress.