Cleavage of complement C3b to iC3b on the surface of Staphylococcus aureus is mediated by serum complement factor I.

Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 and 8 S. aureus strains after incubation in human serum. Using purified classical activation pathway complement proteins and the Western blot assay, we showed that when C3b was generated on the S. aureus surface no iC3b fragments were found, suggesting that other serum proteins may be required for cleaving C3b to iC3b. When C3b-coated S. aureus was incubated with serum factor I, a complement regulatory protein, iC3b was generated. Purified factor H, a serum protein cofactor for factor I, did not enhance factor I-mediated cleavage of C3b. These findings suggest that C3b cleavage to iC3b on S. aureus is mediated by serum factor I and does not require factor H.

Intracellular effect of ultrashort electrical pulses.

A simple electrical model for biological cells predicts an increasing probability for electric field interactions with cell substructures of prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses with electric field intensities of up to 5.3 MV/m to human eosinophils in vitro. When 3-5 pulses of 60 ns duration were applied to human eosinophils, intracellular granules were modified without permanent disruption of the plasma membrane. In spite of the extreme electrical power levels applied to the cells thermal effects could be neglected because of the ultrashort pulse duration. The intracellular effect extends conventional electroporation to cellular substructures and opens the potential for new applications in apoptosis induction, gene delivery to the nucleus, or altered cell functions, depending on the electrical pulse conditions.

Human milk anti-inflammatory component contents during acute mastitis.

Mastitis is a common complication of human lactation. We examined milk specimens from eight women with clinical mastitis to determine their content of anti-inflammatory components. Antioxidant activity (spontaneous cytochrome c reducing activity), selected pro-inflammatory cytokines (IL-6, IL-1beta), selected endogenous cytokine control molecules (sIL-6R, sIL-1RII, and sTNFRI), lactoferrin, Na(+):K(+) ratios, and milk bioactivities that cause shedding of sIL-1RII from human polymorphonuclear leukocytes (PMN), suppress PMN aggregation, and suppress PMN adherence responses were not increased compared to normal milks. Neither the bioactivities that deplete PMN intracellular Ca(2+) stores nor those that block Ca(2+) influx into fMLP-stimulated PMN were significantly increased in mastitis milks. In contrast, levels of TNFalpha, sTNFRII, and IL-1RA and bioactivities that cause shedding of sTNFRI from human PMN were significantly increased compared to normal milks. Mastitis milk has the same anti-inflammatory components and characteristics of normal milk, with elevations in selected components/activities that may help protect the nursing infant from developing clinical illness due to feeding on mastitis milk.

Anti-inflammatory characteristics of human milk: how, where, why.

When first proposed, the hypothesis that human milk was anti-inflammatory was supported by 2 observations: poor function of milk leukocytes and the presence in milk of components that could modify inflammatory processes. This hypothesis is now supported by studies documenting anti-inflammatory effects in animal models and suppression of humoral and cellular components of inflammation in vitro. To date, two mechanisms have been demonstrated: alteration of leukocyte function and modification of cytokine biology. It is not clear whether these mechanisms are only topical effects in the digestive tract, or whether absorption of milk components results in systemic effects. While inflammation benefits the host as a defense mechanism and precursor to immune responses, it also contributes to the clinical manifestations of illness and is an important early component of wound-healing responses that result in scar. The biological effects of milk's anti-inflammatory character may be to minimize clinical symptomatology without losing immunoresponsiveness for the breast-fed infant, and to minimize scar formation during healing responses.

Release of cytokines and soluble cytokine receptors after intravenous anti-D treatment in children with chronic thrombocytopenic purpura.

INTRODUCTION: Immunoglobulin anti-D administration is one of several methods used in treating children with chronic immune thrombocytopenic purpura. Fc receptor blockade of the reticuloendothelial cell system and of mononuclear phagocytes is an important mechanism of the action of anti-D in ITP. Recently other possible mechanisms by which anti-D works in ITP have been considered. METHODS: The aim of this study was to obtain a better understanding of the effect of anti-D administration on cytokine, soluble cytokine receptors and platelet count in children with chronic ITP and to determine the pre-treatment plasma cytokine profile in this group of patients. Eighteen children with chronic ITP were examined. In our study the impact of anti-D on the cytokine network was evaluated by analysing serum levels of IL-6, IL-8, tumor necrosis alpha and soluble TNF receptors I and II by the EASIA method before and 1, 3, 20 and 40 h then seven days and one month after anti-D infusion. RESULTS: Anti-D caused a significant increase in platelet count 20 h postinfusion in 10 out of 18 children, 96 h postinfusion in three children and 168 h postinfusion in one child. The mean duration of the response was four weeks. A significant and rapid increase in plasma levels of IL-6, IL-8 and TNF-alpha was seen within 1 to 20 h after anti-D infusion. This increase was accompanied by a prolonged elevation of soluble TNF receptors. There was a significant correlation between TNF-alpha and IL-8, IL-8 and IL-6, TNF-alpha and sTNFRI, and sTNF receptors I and II. CONCLUSION: These data demonstrate that anti-D infusion caused changes in the cytokine network and raises the question of whether the therapeutic effectiveness of anti-D is related to its immunomodulating properties.

Should central venous catheters be removed as soon as candidemia is detected in neonates?

BACKGROUND: Controversy exists regarding the most appropriate acute management of central venous catheters (CVCs) in neonates with candidemia, with up to two thirds of neonatologists preferring to attempt antifungal therapy without removing CVCs. OBJECTIVE: To determine whether CVCs should be removed as soon as candidemia is detected in neonates. Methods. A cohort study of candidemia and CVC was conducted in infants in a neonatal intensive care unit (NICU) over a 5-year period (1994-1998). RESULTS: Fifty infants had early-removal CVC (ER-CVC) within 3 days and 54 infants had late-removal CVC (LR-CVC) >3 days after the first positive blood culture for Candida species. All infants were treated with amphotericin B. There was no significant difference between infants in the ER-CVC and LR-CVC groups in terms of gender, ethnicity, birth weight, gestational age, age at candidemia, severity-of-illness scores, distribution of types of CVC, or in the distribution of Candida species causing candidemia. The ER-CVC group had significantly shorter duration of candidemia (median: 3 days; range: 1-14 days), compared with the LR-CVC group (median: 6 days; range: 1-24 days). The case fatality rate of Candida albicans candidemia was significantly affected by the timing of CVC removal: 0 of 21 (95% confidence interval [CI]: 0-14) infants died in the ER-CVC group in contrast to 9 of 23 (39%; 95% CI: 19-59) in the LR-CVC group. CONCLUSION: Failure to remove CVC as soon as candidemia was detected in neonates was associated with significantly increased mortality in C albicans candidemia and prolonged duration of candidemia regardless of Candida species.

Surfactant modulates calcium response of neutrophils to physiologic stimulation via cell membrane depolarization.

Pulmonary surfactant (PS) reduces inflammation in the lung by poorly understood mechanisms. We have observed that surfactant-associated proteins (SAP) insert monovalent cation channels in artificial membranes. Neutrophils are primary mediators of acute pulmonary inflammation, and their functions are activated by increases in cytosolic ionized calcium concentration ([Ca2+]) and by changes in membrane potential. We hypothesize that PS inserts SAP-dependent cation channels in neutrophils, causing membrane depolarization, altered [Ca2+] response, and depressed activation. Human neutrophils were isolated, exposed to PS+SAP (1% Survanta), PS-SAP (1% Exosurf), or buffer, and washed before activating with selected stimulants. PS+SAP reduced phorbol ester- and formyl peptide-stimulated adherence and aggregation by 38% (p < 0.05) and 54% (p < 0.02), respectively. PS+SAP also inhibited the formyl peptide-induced [Ca2+] response of neutrophils (p < 0.01), but only in the presence of external Ca2+. Further characterization of this inhibition demonstrated that PS+SAP blocked formyl peptide-induced influx of both Ca2+ and Mn2+, and that this inhibition was present during activation by other neutrophil stimulants (IL-8, immune complexes). Prior depolarization of neutrophils with gramicidin-D similarly inhibited the [Ca2+] response of neutrophils to formyl peptide, and analysis of neutrophil membrane potential by 3,3'-dipentyloxaearbocyanine iodide (diOC5(3)) fluorescence revealed that PS+SAP induced rapid neutrophil depolarization. In contrast, PS-SAP exhibited little effect on neutrophil function, [Ca2+], or membrane potential. We conclude that PS+SAP decreases neutrophil adherence and aggregation responses, blocks Ca2+ influx after physiologic stimulation, and decreases membrane potential. We speculate that these effects are caused by membrane depolarization via SAP-dependent cation channel insertion, and that all of these effects contribute to the antiinflammatory properties of PS+SAP.

Fulminant late-onset sepsis in a neonatal intensive care unit, 1988-1997, and the impact of avoiding empiric vancomycin therapy.

OBJECTIVE: To determine the pathogens associated with fulminant (lethal within 48 hours) late-onset sepsis (occurring after 3 days of age) in a neonatal intensive care unit (NICU) and the frequency of fulminant late-onset sepsis for the most common pathogens. METHODS: A retrospective study was conducted of sepsis in infants in a NICU over a 10-year period (1988-1997). RESULTS: There were 825 episodes of late-onset sepsis occurring in 536 infants. Thirty-four of 49 (69%; 95% confidence interval [CI]: 55%-82%) cases of fulminant late-onset sepsis were caused by Gram-negative organisms, including Pseudomonas sp., 20 (42%); Escherichia coli, 5 (10%); Enterobacter sp., 4 (8%); and Klebsiella sp., 4 (8%). The frequency of fulminant sepsis was highest for Pseudomonas sp., 20 of 36 (56%; 95% CI: 38%-72%) and lowest for coagulase-negative staphylococci, 4 of 277 (1%; 95%CI: 0%-4%). The very low frequency of fulminant sepsis caused by coagulase-negative staphylococci did not increase during the period when oxacillin was used instead of vancomycin as the empiric antibiotic for Gram-positive organisms. CONCLUSIONS: These data suggest that empiric antibiotics selected for treatment of suspected sepsis in infants >3 days old need to effectively treat Gram-negative pathogens, particularly Pseudomonas sp., because these organisms, although less frequent, are strongly associated with fulminant late-onset sepsis in the NICU. Avoiding empiric vancomycin therapy seemed to be a reasonable approach to late-onset sepsis, because of the very low frequency of fulminant sepsis caused by coagulase-negative staphylococci.

Human milk effects on neutrophil calcium metabolism: blockade of calcium influx after agonist stimulation.

Neutrophils are the predominant cellular mediators of acute inflammation, and human milk suppresses multiple neutrophil functions. We sought to determine whether these effects were mediated through disruption of normal intracellular Ca2+ homeostasis. Exposure of human neutrophils to human milk, followed by washing, resulted in altered Ca2+ transient responses to formyl-peptide stimulation in which the peak cytosolic free Ca2+ concentration ([free Ca]) was the same as in unexposed cells, but the postpeak decline in [free Ca] was more rapid. This effect was observed after human milk exposures as brief as 10 s, persisted for up to 4 h after human milk removal, and was concentration dependent. On the basis of experiments examining Ca2+-free conditions followed by Ca2+ supplementation, and experiments examining spontaneous and stimulated manganese and barium influx into neutrophils, the human milk effect was due to blockade of Ca2+ influx. Decreased Ca2+ transient responses to other physiologic stimuli (IL-8, opsonized Staphylococcus aureus, and immune complexes) were observed after human milk exposures. Rat intestinal epithelial cells and HL-60 cells failed to show these effects, suggesting a selective effect on mature inflammatory cells. Characterization of the Ca2+-blocking activity showed it was heat and acid stable in human milk with a molecular mass between 30-100 kD. Commercial human milk lactoferrin exhibited Ca2+ influx blockade activity, but recombinant human lactoferrin showed none. Separation of the activity by heparin affinity chromatography showed that it was distinct from lactoferrin. Human milk-induced blockade of Ca2+ influx provides a potential mechanism for broad suppression of neutrophil functions that may contribute to the antiinflammatory properties of human milk.

Membrane depolarization and depletion of intracellular calcium stores are associated with delay of apoptosis in human neutrophils.

Apoptosis occurs rapidly in human polymorphonuclear leukocytes (PMN) after exposure to 1 mM cycloheximide (CHX). We examined whether this form of stimulated apoptosis altered either resting cytosolic free Ca2+ concentrations ([free Ca]) or membrane potential (psi) in PMN and found no significant effects. However, manipulation of either PMN intracellular Ca2+ stores or psi was found to delay CHX-induced apoptosis. Depletion of PMN intracellular Ca2+ stores with thapsigargin caused membrane depolarization and significantly delayed CHX-induced apoptosis based on both morphological and annexin-V-fluorescein isothiocyanate binding criteria. Short-term suspension (4 h) of PMN in Ca2+-free buffer depleted internal Ca2+ stores, induced membrane depolarization at 2.5 h, and delayed spontaneous (24 h) apoptosis but had no effect on CHX-induced apoptosis. Rapid membrane depolarization with 150 mM KCl buffer significantly delayed CHX-induced apoptosis, suggesting that depolarization rather than Ca2+ stores depletion was the crucial event. Timing experiments revealed that depolarization within 12 min of CHX exposure significantly delayed apoptosis. Collectively, these observations suggest an early psi-sensitive step in the apoptosis pathway initiated by CHX. CHX exposure alone does not alter either resting PMN [free Ca] or psi; accompanying depolarization of plasma membrane (either electrochemically or via depletion of internal Ca2+ stores) delays CHX-induced apoptosis in a time-dependent manner.