Human newborns are more susceptible than adults to infection by gram-negative bacteria. We hypothesized that this susceptibility may be associated with a decreased response by leukocytes to lipopolysaccharide (LPS). In this study, we compared LPS-induced secretion of tumor necrosis factor alpha (TNF-␣) by mononuclear cells (MNC) from adult peripheral blood and newborn umbilical cord blood in vitro and attempted to determine the mechanisms involved in its regulation. At a high concentration of LPS (10 ng/ml) and in the presence of autologous plasma, MNC from adults and newborns secreted similar amounts of TNF-␣. However, in the absence of plasma, MNC from newborns secreted significantly less TNF-␣ compared to MNC from adults. Moreover, at a low concentration of LPS (0.1 ng/ml) and in the presence of plasma, TNF-␣ secretion was significantly lower for newborn MNC compared to adult MNC. Adults and newborns had similar numbers of CD14 and Toll-like receptor 4 (TLR-4)-positive cells as measured by flow cytometry. However, the intensity of the CD14 marker was greater for adult than for newborn cells. Incubation of cells with LPS led to an increase in CD14 and TLR-4 intensity for adult cells but not for newborn cells. The effect of LPS stimulation of adult or newborn cells was similar for ERK, p38, and IB␣ phosphorylation, as well as IB␣ degradation. Finally, we assessed levels of the TLR-4 adapter protein, the myeloid differentiation antigen 88 (MyD88). We found a direct relation between adult and newborn TNF-␣ secretion and MyD88, which was significantly decreased in newborn monocytes. Since TLR-4 signals intracellularly through the adapter protein, MyD88, we hypothesize that MyD88-dependent factors are responsible for delayed and decreased TNF-␣ secretion in newborn monocytes.

Previous research in our laboratory has shown that polymorphonuclear leukocytes (PMN) from neonates are not primed effectively in vitro with lipopolysaccharide (LPS) (from Escherichia coli O111:B4) compared with priming of adult PMN. This finding led us to speculate that differences between neonatal and adult LPS receptors may account for the lower response by neonatal PMN to LPS. In these experiments, we investigated if CD14 or other LPS receptors contribute to the priming activity of PMN by LPS. We found that unprimed neonatal and adult PMN expressed similar numbers of CD14 (11.6 ؎ 9.2 versus 18.6 ؎ 2.7 fluorescence units [FlU]; P > 0.05) and LPS-binding sites (2.94 ؎ 1.4 versus 4.94 ؎ 0.79 FlU; P > 0.05). Monoclonal antibody against CD14 (MY4) did not significantly change the binding of LPS to adult unprimed PMN, suggesting that LPS receptors other than CD14 receptors are predominant on PMN. However, when PMN were pretreated with LPS (10 ng/ml) for 45 min at 37؇C, expression of CD14 on adult PMN increased to 33.8 ؎ 4.9 FlU (P < 0.05 versus unprimed adult PMN) while that on neonatal PMN showed little change, increasing to 17.2 ؎ 10.3 FlU (P > 0.05 versus unprimed neonatal PMN; P < 0.05 versus primed adult PMN). Furthermore, MY4 specifically blocked oxidative-radical production from PMN primed with LPS (10 ng/ml) compared with that from control PMN (P < 0.01). These studies suggest that LPS primes PMN by activating CD14 expression. We conclude that lower expression of CD14 or failure to up-regulate CD14 after LPS pretreatment contributes to the inability of neonatal PMN to be primed by LPS.

We investigated the role of atrial natriuretic factor (ANF) and the renin-angiotensin system as well as the effects of losartan in rats with aortocaval (AC) shunts. Right atrial and left ventricular end-diastolic pressures (LVEDP) were higher and mean arterial blood pressure (MAP) was lower in AC shunt animals than in their controls. AC shunt rats presented marked cardiac hypertrophy, decreased right atrial ANF concentration, and increased ventricular ANF content and concentration. Plasma ANF levels were elevated, and hematocrit was lower in AC shunt animals than in controls. Captopril or losartan treatment decreased MAP and returned LVEDP to sham-operated control values. A clear regression of cardiac hypertrophy was evident in both treated AC shunt groups, with plasma ANF levels tending to follow those in sham-operated rats. Plasma COOH-terminal ANF levels were decreased and urinary volume and hematocrit were increased in losartan-treated AC shunt animals. We conclude that chronic angiotensin converting enzyme inhibition and angiotension II receptor antagonism improved hemodynamic conditions, diminished water retention, reversed cardiac hypertrophy, and restored plasma and tissue ANF to more "normal" levels in rats with moderate high-output heart failure.

We investigated the role of humoral factors in lipopolysaccharide (LPS) priming of polymorphonuclear leukocytes (PMN) using cells isolated from adults and from neonates. Plasma from newborn infants had decreased priming activity of adult plasma when mixed with LPS in studies measuring oxidative radical production of PMN after stimulation with a formyl bacterial oligopeptide (fMLP). This marked difference was not caused by LPS binding protein (LBP) because the LBP concentration in newborn and adult plasma were similar (138.4 ± 12.9 U for adults, and 126.9 ± 12.1 U for neonates, P = .53). Therefore, we attempted to identify other plasma factors that may contribute to LPS priming of PMN. We identified an LPS priming factor for PMN that is present in plasma, heat stable (56°C for 30 minutes), enhanced by heparin, and concentrated in cold precipitates of plasma. Because these properties resemble those of plasma fibronectin, we assessed the role of fibronectin in LPS priming of PMN. Although fibronectin in phosphate-buffered saline (PBS) had little effect on LPS priming of PMN, fibronectin in combination with other plasma factors appeared to play a role in LPS priming of PMN because (1) removing fibronectin from adult plasma dramatically decreased LPS priming activity from plasma (P < .005), (2) addition of fibronectin to fibronectin-depleted plasma restored its LPS plasma priming activity (P < .05), and (3) neutralizing fibronectin with antibody decreased the LPS priming activity of plasma (60.3 ± 1.3 v 30.2 ± 2.2, P < .01). Thus, plasma fibronectin plays a role in LPS priming of PMN in the presence of other factors in plasma.

(1) Chronically increased cardiac filling pressure stimulated not only ANF release but also ANF synthesis in each cardiac chamber, which in turn contributed to raised plasma ANF concentrations in aorto-caval shunt rats; (2) an attenuated renal response to endogenous ANF and sodium and water retention were apparent in A-C shunt rats. Activation of neurohormonal vasoconstrictor systems and gradually decreased plasma ANF concentrations may contribute to sodium and water retention at different stages of this experimental model of heart failure.