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Complement-mediated opsonization of Staphylococcus aureus is a critical host defense in animal models. Specifically, C3b and CD35 play important roles in effective opsonophagocytosis of S. aureus. We have shown that complement control protein factor I mediates cleavage of the complement opsonin C3b bound to the S. aureus surface. In this study, we examined the physiologic relevance of this observation by determining whether factor I-mediated cleavage of S. aureus-bound C3b decreased phagocytosis of S. aureus by neutrophils. Compared with controls, anti-factor I antibody inhibited C3b-cleavage on the S. aureus surface by >83% (as measured by iC3b generation) and increased phagocytosis of S. aureus by >100%. Treatment of C3b-coated S. aureus with factor I increased generation of iC3b (75%), decreased the total amount of C3-fragments bound to the S. aureus surface (58%), and decreased the number of bacteria phagocytosed (40%). Testing specifically for C3-fragments shed from the S. aureus surface, we found that factor I increased shedding (43%). Notably, these factor I-mediated effects were of the same magnitude regardless of whether factor H, a known cofactor for factor I, was present. These findings indicate that S. aureus benefits from, and possibly manipulates, the normally host-protective activity of factor I cleavage of C3b, which results in bacterial escape from complement-mediated opsonophagocytosis. Because escaping opsonophagocytosis-mediated destruction is a necessary mechanism for bacterial survival resulting in human disease, preventing cleavage of C3b on the S. aureus surface, and thereby enhancing opsonophagocytosis, is a promising potential target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To determine how often neonates with Enterobacteriaceae (ENTB) bacteremia can be treated successfully without removing central venous catheters (CVCs). METHODS: A retrospective cohort study was conducted of ENTB bacteremia and CVCs in infants in a neonatal intensive care unit during a 7-year period (1994-2000). Cases of ENTB bacteremia were identified from a microbiology database and limited to late-onset cases occurring after 3 days of age. RESULTS: There were 53 cases of ENTB bacteremia in infants with CVCs. Blood cultures were positive for ENTB within a median of 10 hours (range: 5-43). Timing of CVC removal was at the discretion of attending neonatologists. Fifteen cases had early-removal CVC (ER-CVC) within 2 days, and 38 cases had late-removal CVC (LR-CVC) >2 days after the first positive blood culture for ENTB. There were no significant differences between infants in the ER-CVC and LR-CVC groups for case fatality, recurrence, or duration of ENTB bacteremia. Although 16 (42%) of 38 (95% confidence interval [CI]: 26%-59%) LR-CVC cases required CVC removal to resolve ENTB bacteremia, 17 (45%) of 38 (95% CI: 29%-62%) LR-CVC cases were treated successfully without removal of CVCs. ENTB bacteremia was successfully treated without CVC removal in 85% of 13 LR-CVC cases with 1 day of bacteremia in contrast to 24% of 25 LR-CVC cases with >1 day of bacteremia (relative risk: 3.5; 95% CI: 1.7-7.4). CVC removal was required to resolve ENTB bacteremia in 9 (82%) of 11 LR-CVC cases with severe thrombocytopenia compared with 7 (32%) of 22 LR-CVC cases without severe thrombocytopenia (relative risk: 2.6; 95% CI: 1.3-5.0). CONCLUSIONS: Retention of CVCs was successful in 45% of cases of ENTB bacteremia in which it was attempted, but success was unlikely when bacteremia lasted >1 day. ENTB bacteremia cases associated with severe thrombocytopenia rarely resolved unless CVCs were removed. [ABSTRACT FROM AUTHOR]

Purpose: An increase in postoperative infections after Nuss procedures led us to seek risks and review management. We report potential risk factors and make inferences for prevention of infections., Methods: An IRB-approved retrospective chart review was used to evaluate demographic, clinical, surgical, and postoperative variables of patients operated on between 10/1/2005 and 6/30/2013. Those with postoperative infection were evaluated for infection characteristics, management, and outcomes with univariate analyses., Results: Over this 8-year period (2005-2013), 3.5% (30) of 854 patients developed cellulitis or infection, significantly more than 1.5% (13) in our previous report of 863 patients, 1987-2005 (p=.007). The most frequent organism cultured was methicillin-sensitive Staphylococcus aureus. Patients who were given clindamycin preoperatively (5 of 26 patients) had higher infection rates than those who received cefazolin (25 of 828) (19% vs 3%, p<.001). Patients treated with a peri-incisional ON-Q (I-Flow, Kimberly-Clark, Irvine, CA) also had higher infection rates (8.3% vs 2.4%, p<.001). Of the 30 patients who developed an infection, eighteen (60%) with cellulitis or superficial infections did not require surgical treatment or early bar removal. The other twelve patients (40%) with deep hardware infections required an average of 2.2 operations (range 1-6), with 3 (25%) requiring removal of their stabilizer and 3 (25%) requiring early bar removal. None of these three patients experienced recurrence of pectus excavatum at 2 to 4 years of follow-up., Conclusion: Preoperative antibiotic selection and use of ON-Q's may influence infection rates after Nuss repair. Nuss bars could be preserved in 90% of all patients with an infection and even 75% of those with a deep hardware infection. Attempts to retain the bar when an infection occurs may help prevent pectus excavatum recurrence. Level of Evidence=III., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hospital breastfeeding initiation rates (75%) show that most mothers in the United States want to breastfeed and are trying to do so. Even from the very start, however, mothers may not be getting the breastfeeding support they need. Low breastfeeding rates at 3, 6, and 12 months illustrate that women face multiple additional barriers to maintaining breastfeeding. What can we do to help more mothers be more successful? As healthcare providers, we need to be believers that breastfeeding is worth the effort. Perhaps most important for us is to realize that human milk is not simply a food but rather a complex, human infant support system. We can then articulate to families the importance of breastfeeding as a clinical imperative, a preventer of acute and chronic illness and disease. It will take integration, normalization, and mainstreaming of breastfeeding into our culture for acceptance and growth of the practice. Once we assist families in making educated decisions about breastfeeding, we need to provide supportive environments in our hospitals, medical practices, workplaces, and communities that implement the best ways to support breastfeeding. Breast milk is worth the effort, and the time has come to be ardent supporters of mothers and infants and their breastfeeding intentions.

Proinflammatory cytokines have been variably linked to development of cerebral white matter injury (WMI) in preterm infants. Because soluble receptors tightly control cytokine bioactivity, we modeled cytokine-receptor interaction as a predictor of WMI. Plasma from 100 preterm infants was assayed for cytokines (tumor necrosis factor alpha, interleukin (IL-1beta, IL-6) and their soluble receptors (sTNF-RI), sTNF-RII, sIL-1RA, and sIL-6R). Cranial ultrasound (US) results were correlated with cytokine and receptor concentrations individually and with cytokine-receptor interaction models (PROC LOGISTIC; SAS Software). Receiver operating characteristic curves were constructed to determine the predictability of WMI. Fifty-two infants with normal US exams were compared with 21 infants with evidence of WMI. There was no association between individual cytokine or receptor concentrations and the development of WMI. However, modeling cytokines with their soluble receptors significantly improved the predictability of WMI. We concluded that consideration of cytokine-receptor interaction may be more important than individual cytokine concentrations alone in determining the role of inflammation in the pathogenesis of WMI in preterm infants.

West Nile Virus (WNV) infection is an important cause of encephalitis. Although the medical literature contains examples of WNV encephalitis in susceptible, mainly elderly, immunocompromised hosts, few case reports have described pediatric cases. The authors describe an adolescent with acute lymphocytic leukemia and WNV encephalitis. Surveillance studies indicate an increase in WNV activity. Physicians need to be aware of WNV activity in their community and consider WNV as a potential source of infection.

Complement-mediated opsonization of Staphylococcus aureus is a critical host defense in animal models. Specifically, C3b and CD35 play important roles in effective opsonophagocytosis of S. aureus. We have shown that complement control protein factor I mediates cleavage of the complement opsonin C3b bound to the S. aureus surface. In this study, we examined the physiologic relevance of this observation by determining whether factor I-mediated cleavage of S. aureus-bound C3b decreased phagocytosis of S. aureus by neutrophils. Compared with controls, anti-factor I antibody inhibited C3b-cleavage on the S. aureus surface by >83% (as measured by iC3b generation) and increased phagocytosis of S. aureus by >100%. Treatment of C3b-coated S. aureus with factor I increased generation of iC3b (75%), decreased the total amount of C3-fragments bound to the S. aureus surface (58%), and decreased the number of bacteria phagocytosed (40%). Testing specifically for C3-fragments shed from the S. aureus surface, we found that factor I increased shedding (43%). Notably, these factor I-mediated effects were of the same magnitude regardless of whether factor H, a known cofactor for factor I, was present. These findings indicate that S. aureus benefits from, and possibly manipulates, the normally host-protective activity of factor I cleavage of C3b, which results in bacterial escape from complement-mediated opsonophagocytosis. Because escaping opsonophagocytosis-mediated destruction is a necessary mechanism for bacterial survival resulting in human disease, preventing cleavage of C3b on the S. aureus surface, and thereby enhancing opsonophagocytosis, is a promising potential target for therapeutic intervention.

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.

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca(++) stores and that PS+SAP induces depletion through release of Ca(++) stores and through inhibition of Ca(++) influx. Our model predicts that PS+SAP releases Ca(++) stores through insertion of channels, depolarization of neutrophils, and activation of a G protein-dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin-a channel protein with properties similar to those of PS+SAP-is expected to mimic these effects. Human neutrophils were monitored for [Ca(++)] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca(++)] responses were reexamined following preexposure to inhibitors of internal Ca(++) release or the G protein pathway. We observed that (i) 1% PS+SAP-but not PS-SAP-causes transient increase of neutrophil [Ca(++)] within seconds of exposure; (ii) 1% PLG-but not gramicidin alone-closely mimics the effect of PS+SAP on Ca(++) response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca(++) stores releases or of G protein activation suppresses Ca(++) responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca(++) influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca(++) from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca(++) stores and by inhibiting Ca(++) influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.

Objectives: To elucidate the causes for treatment failure in children given extended-spectrum cephalosporins., Methods: During April 1998-March 2000, 18 isolates of members of the family Enterobacteriaceae, fulfilling microbiological criteria for carriage of extended-spectrum beta-lactamases (ESBLs) and carrying blaSHV, were isolated from paediatric inpatients. The collection was subjected to a retrospective molecular analysis., Results: Three species were represented in the collection: Citrobacter koseri (one isolate), Escherichia coli (one isolate) and Klebsiella pneumoniae (16 isolates). A common plasmid was found in these bacteria, as judged by restriction endonuclease digestion. This was able to transfer an ESBL phenotype from donors to a laboratory strain of E. coli. Nucleotide sequence analysis revealed that this phenotype was associated with a new variant in blaSHV encoding SHV-34., Conclusions: Analysis reveals the presence of an epidemic plasmid in this collection of bacteria. This carries a gene encoding the SHV-34 ESBL, described for the first time in this report. Nucleotide sequence analysis shows that there is a mutation from A-->G affecting the codon at amino acid position 64 (GAA-->GGA), changing the glutamic acid typically seen in this position to glycine.

Application of sub-microsecond duration (60-300 ns), intense (15-60 kV/cm) pulsed electric fields (sm/i-PEF) to six types of human cells was examined for its effects on individual cell surface membrane permeability and membrane potential. With short (60 ns) pulses, increasing percentages of Jurkat cells showed propidium iodide (PI) uptake at progressively shorter post-pulse times as the pulse train increased from 1 to 10 sequential pulses, while human blood polymorphonuclear leukocytes (PMN) were unresponsive to these short pulses regardless of train size. With 300 ns pulses, a similar pattern (increasing percentages of cells taking up PI, and progressively shorter times of onset after pulse applications as pulse train size increased) was seen with both Jurkat cells and PMN, but the patterns for both effects were different. Jurkat cell size did not appear to influence the responsiveness of this cell type. Comparisons of sm/i-PEF-induced PI uptake by human monocyte-derived macrophages vs. aged human mononuclear cells, human trunk skin (HTS) cells vs. fresh human mononuclear cells and human macrophages vs. HTS cells showed similar overall effects, but with differences between the patterns for each cell type compared (except the macrophages vs. HTS cells comparison). Application of sm/i-PEFs also caused different patterns of membrane potential loss in Jurkat cells vs. PMN. Jurkat cells developed significant decreases in t heir membrane potential only following the highest intensity pulse applications examined, i.e., 300 ns, 60 kV/cm x5, while PMN showed this effect over the entire range of pulse intensities (300 ns, 15-60 kV/cm, x5) applied. These data indicate that sm/i-PEF applications can have "specificity" (i.e., achieve different levels of effect in different cell types), that cell size does not appear to be the major factor determining sm/i-PEF effects in either Jurkat cells or PMN, that heterogeneous sm/i-PEF effects on cells tend to become homogeneous with increasing pulse train size, and that specificity of sm/i-PEF applications effects can occur at either end of the sm/i-PEF intensity spectrum examined.

Background: Nasal saline spray (NSS) used in the treatment of rhinitis and sinusitis often contains the preservative benzalkonium chloride (BKC). Previous studies have shown that corticosteroid nasal sprays and topical decongestants containing BKC damage respiratory mucosa, decrease mucociliary activity, and inhibit neutrophil functions in vitro., Objective: To evaluate the effects of NSS with BKC on human neutrophils., Design: Prospective, basic science observations., Methods: Human neutrophils were exposed to NSS with BKC or to phosphate-buffered saline (PBS) at varying times and concentrations. The cells were examined for morphologic changes by light microscopy and for viability as determined by trypan blue exclusion. Lactate dehydrogenase levels were measured to quantify neutrophil cell lysis. In vivo morphologic changes were studied in neutrophils obtained from the oral mucosa in human volunteers who rinsed their mouths with NSS or PBS., Results: Neutrophils exposed to NSS concentrations as low as 15% showed near-total cell lysis, and neutrophils exposed to 20% NSS demonstrated no cell viability by trypan blue staining. Phosphate-buffered saline-exposed cells were unaffected. The release of lactate dehydrogenase from lysed neutrophils increased sharply at NSS concentrations higher than 10% but remained stable in PBS-exposed cells. All neutrophils isolated from NSS oral rinses were lysed, while a mean of 78% of neutrophils from PBS rinses showed normal morphologic structure., Conclusions: Nasal saline spray with BKC is toxic to human neutrophils even at concentrations far lower than those found in commercially available preparations. Saline solutions without BKC appear to be safer alternatives, and additional studies are needed to determine the clinical significance of these findings.

A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both 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 (durations 100 micros-60 ns, electric field intensities 3-150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude.

Linezolid is an oxazolidinone that has activity against most gram-positive bacteria, including in vitro activity against all Nocardia species and strains. We describe 6 clinical cases of nocardiosis that were successfully treated with linezolid. Two patients had underlying X-linked chronic granulomatous disease, and 2 patients were receiving chronic corticosteroid therapy. Four of 6 patients had disseminated disease, and 2 of these 4 patients had multiple brain abscesses. Four patients primarily received monotherapy; for the fifth patient, linezolid was added to a failing multiple-drug regimen, and, for the sixth patient, it was used as part of combination therapy. All 6 patients were successfully treated, although 1 patient had a presumed relapse of central nervous system infection after premature discontinuation of the drug. Linezolid appears to be an effective alternative for the treatment of nocardiosis.

Infections occur in childhood and adolescent athletes just as they do in all children and adolescents. Because of the sports environment, and in some instances the sport itself, athletes can be prone to infections that will alter their athletic performance or present risks to other athletes. Recognition of the infectious risks related to sports and the options for their treatment or, better yet, prevention, can help young athletes perform to their utmost potential.

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., (Copyright 2001 Wiley-Liss, Inc.)

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., (Copyright 2001 Academic Press.)

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.

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.

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.

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.

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.

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.

We used column chromatography, affinity binding, and bioassay methods to address whether the soluble tumor necrosis factor (TNF)-alpha receptors present in human colostrum and milk bind to and modify TNF-alpha bioactivity. In gel chromatography experiments, soluble TNF-alpha receptor I (sTNFRI) and sTNFRII in human colostrum sequentially increased their molecular sizes from 49 kD to 71 kD and 60 kD, respectively, after addition of increasing molar excesses of recombinant TNF-alpha. Application of colostrum to a TNF-alpha affinity matrix followed by washing and elution resulted in 2925-fold enrichment of sTNFRI, consistent with sTNFRI binding to the TNF-alpha affinity matrix. In other samples of colostrum and milk, the content of both sTNFRI and sTNFRII decreased significantly after passage over the matrix, but the material eluted from the matrix lost the ability to rebind to the TNF-alpha and was not active in a WEHI-13var bioassay for TNF-alpha. Specimens of human colostrum and milk diluted 1:16 shifted the LD50 for TNF-alpha 4-fold in this bioassay, and milk protection of WEHI-13var cells against TNF-alpha was significantly diminished after passage down the TNF-alpha affinity matrix (p < 0.001). Affinity purification of milk sTNFRI using polyclonal anti-sTNFRI produced fractions containing proteins of 30 kD, which could be visualized by Western blot using polyclonal anti-sTNFRI. Addition of this fraction to the WEHI-13var bioassay reversed the effects of 10 pg/mL TNF-alpha in the assay. These data demonstrate that sTNFRI and II from human colostrum and milk bind to TNF-alpha, that both colostrum and milk interfere with the bioactivity of TNF-alpha, and that affinity-purified sTNFRI from human milk blocks the bioactivity of TNF-alpha. These effects may contribute to the anti-inflammatory character of human colostrum and milk.

Objective: To determine changes in the incidence of candidemia in a neonatal intensive care unit (NICU) during a 15-year period (1981 to 1995) and to compare the prevalence and case fatality rates of Candida albicans and Candida parapsilosis infections., Methods: A retrospective study was conducted of candidemia occurring in infants in a NICU between January 1, 1981, and December 31, 1995. Cases were identified through computerized searching of a microbiology blood culture database. Candidemia was considered contributory to mortality if death occurred within 3 days of positive blood cultures or if there was autopsy evidence of disseminated candidiasis., Results: One hundred eleven cases of candidemia occurred in 107 infants, representing 1% of all NICU patients during the study period. The rate of candidemia in the NICU increased from 2.5 cases per 1000 admissions in 1981 to 1985, to 4.6 per 1000 admissions in 1986 to 1990 and to 28.5 per 1000 in 1991 to 1995 (P = 0.001). C. albicans was the predominant cause of candidemia between 1981 and 1990. C. parapsilosis was the most prevalent species between 1991 and 1995, causing 53 of 89 cases (60%). The mortality from C. albicans, 13 of 50 cases (26%), was significantly higher than the mortality from C. parapsilosis, 2 of 54 (4%) (P = 0.002; relative risk, 7; 95% confidence interval, 1.7 to 30)., Conclusions: The rate of candidemia in our neonatal intensive care unit increased >11-fold in the 15 years from 1981 to 1995; the prevalent Candida species shifted from C. albicans to C. parapsilosis; and candidemia associated with C. albicans has significantly higher mortality than with C. parapsilosis.

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX +/- 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had no effect on apoptosis in the absence of the analogue. In contrast, site-selective cAMP analogues that specifically activated the type I cAPK, but not type II cAPK, synergistically attenuated apoptosis. Exposure to 8-CPT-cAMP delayed, in parallel, the activity of caspase-3 (CPP-32beta), whereas mitogen-activated protein kinase kinase (MAPKK) inhibitor, PD98059, had no effect on CHX-induced apoptosis +/- 8-CPT-cAMP. Together these results indicate that type I cAPK activation is necessary and sufficient to mediate cAMP-induced delay in human neutrophil apoptosis induced by several mechanisms and suggest that one of the major sites of cAPK action is upstream of caspase-3 (CPP-32beta) activation.

We examined the effects of a human milk diet on rats with chemical colitis induced with a 4% acetic acid enema. Colonic myeloperoxidase activity was used as a surrogate marker for neutrophil infiltration. Control rats fed rat chow had little colonic myeloperoxidase activity; geometric mean, 0.27 U/g of tissue. Rats with colitis fed rat chow had significantly increased colonic myeloperoxidase activity (geometric mean, 6.76 U/g, p < 0.01 versus no colitis), as did rats with colitis fed infant formula or Pedialyte (geometric mean, 6.92 and 8.13 U/g, respectively, both p < 0.01 versus no colitis). Animals with colitis fed human milk had significantly lower colonic myeloperoxidase activity (geometric mean, 2.34 U/g) than did animals with colitis fed either chow or infant formula (p < 0.001). Similar effects were seen in rats with colitis fed infant formula supplemented with recombinant human IL-1 receptor antagonist (geometric mean, 1.95 U/g). These data show that orally administered human milk has an antiinflammatory effect on chemically induced colitis in rats, which may be mediated in part by IL-1 receptor antagonist contained in human milk.

To determine whether human milk contained soluble receptors and cytokine antagonists that might contribute to its anti-inflammatory properties, ELISA and enzyme-amplified sensitivity immunoassay methods were used to quantitate soluble intercellular and vascular cell adhesion molecules, soluble E-selectin, soluble IL-6 receptor, IL-1 receptor antagonist, and soluble tumor necrosis factor-alpha (TNF-alpha) receptors I and II in human milk and colostrum. Soluble adhesion receptors (soluble intercellular and vascular cell adhesion molecules and soluble E-selectin) were present in colostrum at levels approximately equal to serum, whereas milk levels were significantly lower. Both colostrum and milk contained soluble IL-6 receptor, but the levels present were significantly lower than that reported for serum. The colostrum contents of IL-1 receptor antagonist (672 +/- 202 pg/mL), TNF-alpha receptor I (> 3703 +/- 305 pg/mL), and TNF-alpha receptor II (> 4507 +/- 770 pg/mL) were significantly elevated over serum/plasma levels. Milk levels of IL-1 receptor antagonist and TNF-alpha receptor I were also greater than serum/ plasma levels, but lower than colostrum levels. Examination of sequential milk specimens collected from seven women over a period of 2-6 mo showed that IL-1 receptor antagonist and TNF-alpha receptors I and II persisted throughout lactation. Column chromatographic fractionation of colostrum and milk demonstrated that soluble TNF-alpha receptors I and II had molecular sizes up to 60 kD, suggesting that they might be associated with other molecules. Antigen assays for TNF-alpha in colostrum and milk, as well as chromatographic fractionation experiments, showed that, although present, most TNF-alpha was not "free" in colostrum or milk, consistent with the observed content of soluble TNF-alpha receptors I and II. These studies demonstrate that human milk and colostrum contain soluble receptors and cytokine antagonists, materials which could contribute to their anti-inflammatory properties.

Human colostrum, the first product of lactation, has antioxidant properties and inhibits selected enzyme and bactericidal activities of human neutrophils. We examined the subsequent product of lactation, mature human milk, with respect to its antioxidant activities, its effects on neutrophil enzyme activities (myeloperoxidase, beta-glucuronidase, and lysozyme), and its effects on neutrophil bactericidal and phagocytic activities. Mature human milk displayed antioxidant characteristics similar to those of human colostrum, reducing cytochrome c and consuming H2O2. Mature milk also displayed colostrum-like characteristics in depressing neutrophil myeloperoxidase and beta-glucuronidase activities, but not in altering lysozyme activity. Neutrophil bactericidal activity against Staphylococcus aureus was depressed by both mature milk and colostrum, without dramatic effects on phagocytic activity. These data show that mature milk shares characteristics with human colostrum that may result in anti-inflammatory effects, but the magnitude of these effects is generally smaller.

Although "secretory" type II 14-kDa phospholipase A2 (sPLA2) activity has been described in neutrophils, direct evidence of enzyme secretion has been elusive. We have used immunogold electron microscopy with polyclonal and monoclonal antibodies to sPLA2 to demonstrate localization of the enzyme to granules of resting human neutrophils and translocation to phagolysosomes. Soluble stimuli such as calcium ionophore A23187 stimulate loss of cell-associated enzymatic activity. Supernatant fluids from stimulated neutrophils lack measurable PLA2 but contain proteases which inactivate exogenous sPLA2. The use of alpha-1-antitrypsin as a protease inhibitor permitted this first demonstration of secretion of PLA2 activity from stimulated human neutrophils.

Oxidants generated by the NADPH oxidase of activated neutrophils can react with a number of tissue targets to form toxic metabolites such as 4-hydroxynonenal (4-HNE). 4-HNE is a lipid peroxidation product generated by free radical attack on omega-6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4-HNE-protein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X-linked, flavocytochrome b-deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4-HNE-protein adducts and gp91-phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillation-dried neutrophils. No 4-HNE-protein adducts were detected in flavocytochrome b-deficit cells from the homozygous patient or from the heterozygous CGD carrier. However, in gp91-phox-positive cells from both the normal and heterozygous CGD carrier, significant 4-HNE-protein adduct labeling was observed, primarily in the phagosomes. When data from single- and double-labeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carrier were 71% 4-HNE-protein adduct-positive and 56% gp91-phox-positive, while cells from the normal father were > 97% positive for both 4-HNE-protein adducts and gp91-phox. These results confirmed the nitroblue tetrazolium tests of 100%, 60 +/- 2%, and 0% positive for the father's, mother's, and son's cells, respectively, and demonstrated that 4-HNE-protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4-HNE and resulting 4-HNE-protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins.