A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis.

Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally.

Immunohistochemical expression of inflammatory markers in sudden infant death; ancillary tests for identification of infection.

AIMS: Sudden unexpected death in infancy (SUDI) investigation requires extensive ancillary investigations, the results of which, such as postmortem microbiology, can be difficult to interpret. Markers of an inflammatory response, including interleukin 6 (IL-6), c-reactive protein (CRP) and cellular adhesion molecules are elevated in infections, yet little attention has been paid to their assessment after death. This study investigates the role of inflammatory markers in SUDI autopsies for determining cause of death. METHODS: Cases of SUDI over a 14 year period were identified from an autopsy database and 100 cases were selected for immunohistochemical staining of heart and liver for IL-6, CRP, P-selectin, VCAM-1 and ICAM-1 (CD54), with staining patterns compared between five groups, including infectious and unexplained SUDI. RESULTS: There were significant differences between groups. Cases of histological infection demonstrated strongly positive hepatocyte CRP and ICAM-1 expression and increased myocardial staining for CRP. Half of trauma-related deaths demonstrated diffuse hepatic CRP expression but without myocardial CRP staining. Staining of unexplained SUDI cases were predominantly negative, apart from a subgroup in whom Escherichia Coli was identified, who had increased expression of hepatic IL-6. CONCLUSIONS: There were distinct patterns of organ-specific CRP and ICAM-1 expression in SUDI by cause of death. These markers of inflammation were rarely present in unexplained SUDI suggesting either a non-inflammatory cause of death or a failure to mount an effective acute phase response. Immunohistochemical staining offers potential to identify infection-related deaths and provides insight into SUDI mechanisms.

MBL2 deficiency is associated with higher genomic bacterial loads during meningococcemia in young children.

Mannose binding lectin (MBL2) is a soluble pattern recognition receptor that is key to generating innate immune responses to invasive infection, including against the cardinal Gram-negative bacterium Neisseria meningitidis. Individuals homozygous or heterozygous for any of three variant alleles of MBL2 (O/O or A/O genotypes) have deficient concentrations of MBL2 in circulating blood, but previous studies linking MBL deficiency to susceptibility to meningococcal disease have not revealed a consistent association. We genotyped 741 patients with microbiologically-proven meningococcal disease and correlated MBL2 genotype with plasma bacterial load of N. meningitidis with blood samples taken during hospital admission. We show that individuals with genotypes compatible with MBL2 deficiency have higher measurable levels of bacterial plasma genomic load with the greatest effect seen in children <2 years of age. However, the overall impact of this is minor, because there was no evidence that such genotypes are more common in children with meningococcal disease compared with uninfected cohorts. The findings suggest that MBL2 supports innate immune defence against meningococcal disease in the early months of life, before acquired immunity is sufficiently robust for effective natural protection.

Management of Kawasaki disease.

Kawasaki disease (KD) is an acute self-limiting inflammatory disorder, associated with vasculitis, affecting predominantly medium-sized arteries, particularly the coronary arteries. In developed countries KD is the commonest cause of acquired heart disease in childhood. The aetiology of KD remains unknown, and it is currently believed that one or more as yet unidentified infectious agents induce an intense inflammatory host response in genetically susceptible individuals. Genetic studies have identified several susceptibility genes for KD and its sequelae in different ethnic populations, including FCGR2A, CD40, ITPKC, FAM167A-BLK and CASP3, as well as genes influencing response to intravenous immunoglobulin (IVIG) and aneurysm formation such as FCGR3B, and transforming growth factor (TGF) ß pathway genes. IVIG and aspirin are effective therapeutically, but recent clinical trials and meta-analyses have demonstrated that the addition of corticosteroids to IVIG is beneficial for the prevention of coronary artery aneurysms (CAA) in severe cases with highest risk of IVIG resistance. Outside of Japan, however, clinical scores to predict IVIG resistance perform suboptimally. Furthermore, the evidence base does not provide clear guidance on which corticosteroid regimen is most effective. Other therapies, including anti-TNFα, could also have a role for IVIG-resistant KD. Irrespective of these caveats, it is clear that therapy that reduces inflammation in acute KD, improves outcome. This paper summarises recent advances in the understanding of KD pathogenesis and therapeutics, and provides an approach for managing KD patients in the UK in the light of these advances.

Potential Trace Metal Co-Limitation Controls on N2 Fixation and [Formula: see text] Uptake in Lakes with Varying Trophic Status.

The response of N2 fixation and [Formula: see text] uptake to environmental conditions and nutrient enrichment experiments in three western U.S. lake systems was studied (eutrophic Clear Lake; mesotrophic Walker Lake; oligotrophic Lake Tahoe). We tested the effect of additions of bioactive trace metals molybdenum as Mo(V) and iron (Fe) as well as phosphate (P), N2 fixation, [Formula: see text], carbon (C) fixation, chlorophyll a (Chla), and bacterial cell counts under both natural conditions and in mesocosm experiments. We found distinct background N2 fixation and [Formula: see text] uptake rates: highest at Clear Lake (N2 fixation: 44.7 ± 1.8 nmol N L(-1) h(-1)), intermediate at Walker Lake (N2 fixation: 1.7 ± 1.1 nmol N L(-1) h(-1); [Formula: see text] uptake: 113 ± 37 nmol N L(-1) h(-1)), and lowest at Lake Tahoe (N2 fixation: 0.1 ± 0.07 nmol N L(-1) h(-1); [Formula: see text] uptake: 37.2 ± 10.0 nmol N L(-1) h(-1)). N2 fixation was stimulated above control values with the addition of Fe and Pin Clear Lake (up to 50 and 63%, respectively); with Mo(V), Fe, and P in Walker Lake (up to 121, 990, and 85%, respectively); and with Mo(V) and P in Lake Tahoe (up to 475 and 21%, respectively). [Formula: see text] uptake showed the highest stimulation in Lake Tahoe during September 2010, with the addition of P and Mo(V) (∼84% for both). High responses to Mo(V) additions were also observed at some sites for C fixation (Lake Tahoe: 141%), Chla (Walker Lake: 54% and Clear Lake: 102%), and bacterial cell counts (Lake Tahoe: 61%). Overall our results suggest that co-limitation of nutrients is probably a common feature in lakes, and that some trace metals may play a crucial role in limiting N2 fixation and [Formula: see text] uptake activity, though primarily in non-eutrophic lakes.

Randomized clinical trial of glutamine-supplemented versus standard parenteral nutrition in infants with surgical gastrointestinal disease.

BACKGROUND: Addition of glutamine to parenteral nutrition in surgical infants remains controversial. The aim of this trial was to determine whether glutamine supplementation of parenteral nutrition in infants requiring surgery would reduce the time to full enteral feeding and/or decrease the incidence of sepsis and septicaemia. METHODS: A prospective double-blind multicentre randomized clinical trial was performed in surgical infants less than 3 months old who required parenteral nutrition. Patients were allocated to treatment or control groups by means of minimization. Infants received either 0·6 g per kg per day alanyl-glutamine (treatment group) or isonitrogenous isocaloric parenteral nutrition (control group) until full enteral feeding was achieved. Primary outcomes were time to full enteral feeding and incidence of sepsis. Cox regression analysis was used to compare time to full enteral feeding, and to calculate risk of sepsis/septicaemia. RESULTS: A total of 174 patients were randomized, of whom 164 completed the trial and were analysed (82 in each group). There was no difference in time to full enteral feeding or time to first enteral feeding between groups, and supplementation with glutamine had no effect on the overall incidence of sepsis or septicaemia. However, during total parenteral nutrition (before the first enteral feed), glutamine administration was associated with a significantly decreased risk of developing sepsis (hazard ratio 0·33, 95 per cent confidence interval 0·15 to 0·72; P = 0·005). CONCLUSION: Glutamine supplementation during parenteral nutrition did not reduce the incidence of sepsis in surgical infants with gastrointestinal disease. REGISTRATION NUMBER: ISRCTN83168963 (http://www.controlled-trials.com).

Aberrant response to commensal Bacteroides thetaiotaomicron in Crohn's disease: an ex vivo human organ culture study.

BACKGROUND: Human ex vivo evidence indicating that an inappropriate immune response(s) to nonpathogenic bacteria contributes to disease pathogenesis in pediatric Crohn's disease (CD) is limited. The aim of the present study was to compare and contrast the early innate immune response of pediatric "healthy" versus CD mucosa to pathogenic, probiotic, and commensal bacteria. METHODS: "Healthy control" and CD pediatric mucosal biopsies (terminal ileum and transverse colon) were cocultured for 8 hours with E. coli O42, Lactobacillus GG (LGG), Bacteroidesthetaiotaomicron (B. theta), or stimulated with interleukin (IL)-1ß (positive control). Matched nonstimulated biopsies served as experimental controls. IL-8 was the immune marker of choice. IL-8 mRNA and protein levels were quantified by quantitative polymerase chain reaction and sandwich enzyme-linked immunosorbent assay, respectively. RESULTS: IL-8 secretion was observed when control, ileal biopsies were exposed to pathogenic O42 and probiotic LGG, with no response noted to commensal B. theta. In comparison, Crohn's ileal biopsies showed impaired ability to induce IL-8 in response to O42 and LGG. Control colonic tissue showed a limited response to O42 or B. theta and LGG significantly reduced IL-8 secretion. Unlike control tissue, however, Crohn's ileal and colonic tissue did respond to B. theta, with more enhanced expression in the colon. CONCLUSIONS: We provide the first ex vivo data to support the notion that aberrant mucosal recognition of commensal bacteria may contribute to pediatric CD. While IL-8 responses to O42 and LGG varied with disease status and anatomical location, B. theta consistently induced significant IL-8 both in ileal and colonic CD tissue, which was not seen in control, healthy tissue.

Staphylococcal toxins in sudden unexpected death in infancy: experience from a single specialist centre.

Around two thirds of sudden unexpected deaths in infancy (SUDI) remain unexplained following post-mortem examination. It has been postulated that a subset of unexplained SUDI may be caused by toxigenic Staphylococcus aureus. The aim of this study was to compare the prevalence of toxigenic S aureus strains in unexplained and explained SUDI (those in whom a cause of death is determined at autopsy). A retrospective review was performed of 546 SUDI autopsies as part of a larger review of >1,500 pediatric autopsies over a 10-year period, 1996-2005 inclusive. SUDI was defined as the sudden and unexpected death of an infant aged 7-365 days, and categorized into unexplained, explained with histological evidence of infection (bacterial infection group) or explained due to non-infective causes. Toxin gene profiling was carried out by PCR in cases in whom S aureus was isolated as part of clinical investigation. Of the 507 SUDI included in this analysis, bacteriological investigations were performed in 470, and S aureus was isolated on post-mortem culture from at least one site in 173 (37%). There were significantly more cases with S aureus isolated in unexplained SUDI (40%) compared to non-infective SUDI (21%; difference 19.0%, 95% CI 5.4% to 29.3%, P = 0.006). 46% of all cases with S aureus isolated underwent routine testing for a panel of staphylococcal toxin genes (including SEA to SEE, SEG to SEJ, TSST-1, and exfoliative toxins A and B). There were more cases with at least one toxigenic strain of S aureus in the unexplained SUDI (81%) and bacterial infection groups (77%) than in the non-infection group (63%), but these differences were not statistically significant (Fisher exact test, P = 0.44). Toxin gene-carrying S aureus is commonly detected at autopsy in SUDI, accounting for 78% of S aureus isolates submitted for toxin gene profiling in this series. There is a significantly higher prevalence of S aureus in unexplained SUDI compared to non-infective SUDI, but no significant difference in the proportion with toxigenic S aureus strains isolated between the groups. These data are consistent with the hypothesis that a subset of otherwise unexplained SUDI may be related to the presence of S aureus colonization/infection, but do not indicate routine testing for toxin-associated genotypes.

Endothelial injury and repair in systemic vasculitis of the young.

OBJECTIVE: Endothelial injury is central to the pathogenesis of vasculitis. The purpose of this study was to assess how indices of endothelial injury and repair change during different stages of disease activity in children with primary systemic vasculitis (PSV). METHODS: Fifty children with PSV, 17 children with nonvasculitic inflammatory diseases (pediatric inflammatory disease controls), 35 healthy age- and sex-matched pediatric controls, and 27 healthy adult controls were included in the study. Biomarkers examined were endothelial microparticles (EMPs), circulating endothelial cells (CECs), angiogenic growth factors, and endothelial progenitor cells (EPCs). EMP binding to annexin V, EMPs expressing CD144 or E-selectin, and EPCs expressing vascular endothelial growth factor receptor 2 (VEGFR-2), CD133, and CD34 were examined by flow cytometry. CECs were enumerated using immunomagnetic bead extraction techniques, and VEGF and angiopoietin 2 (Ang-2) were measured by enzyme-linked immunosorbent assay. RESULTS: Levels of CD144+ EMPs, CECs, VEGF, and EPCs were all significantly elevated in children with active vasculitis as compared with healthy children, and the levels declined with remission-inducing therapy in the individual patients. Treatment-naive patients with active disease had significantly higher levels of VEGF and Ang-2 than did those with active disease who were receiving treatment, although the levels of CECs and EMPs remained high in both of these groups. CONCLUSION: Elevation of the levels of CECs, EMPS, EPCs, VEGF, and Ang-2 occurs during active vasculitis in children. EPC responses to active vasculitis are different in children as compared with that observed in adults with vasculitis, and both CECs and EMPs can be used to monitor disease activity in children with vasculitis.

Mannose-binding lectin is present in the infected airway: a possible pulmonary defence mechanism.

BACKGROUND: Mannose-binding lectin (MBL) deficiency has been associated with infections of the respiratory tract and with increased disease severity in cystic fibrosis (CF). The mechanism is uncertain, and could relate either to systemic or local effects. The aim of this study was to determine, in a large cohort of children, whether MBL is present on the airway surface in health or disease. METHODS: Bronchoalveolar lavage (BAL) fluid from children with and without respiratory infection (some with underlying disease) was analysed for MBL and neutrophil elastase (NE). Levels were compared between groups, and correlations were examined with local and systemic inflammatory markers, infective organisms and load. RESULTS: 85 children were recruited to the study. MBL was absent in the lavage of all 7 children without lung infection but present in 62% (8/13) of those with acute pneumonia/pneumonitis, 23% (5/22) with recurrent respiratory tract infections, 17% (1/6) with primary ciliary dyskinesia and 8% (3/37) with CF (p<0.01). Children with acute pneumonia/pneumonitis had significantly higher levels than those in the other groups. There was no relationship with organisms cultured or systemic markers of inflammation, although in the group with detectable MBL in the BAL fluid, the levels correlated positively with levels of NE. CONCLUSIONS: MBL is undetectable in the non-infected airway but is present in a significant number of samples from children with lung infection. The levels found in the BAL fluid could be physiologically active and the protein may therefore be playing a role in host defence.

Infection and sudden unexpected death in infancy: a systematic retrospective case review.

BACKGROUND: The cause and mechanism of most cases of sudden unexpected death in infancy (SUDI) remain unknown, despite specialist autopsy examination. We reviewed autopsy results to determine whether infection was a cause of SUDI. METHODS: We did a systematic retrospective case review of autopsies, done at one specialist centre between 1996 and 2005, of 546 infants (aged 7-365 days) who died suddenly and unexpectedly. Cases of SUDI were categorised as unexplained, explained with histological evidence of bacterial infection, or explained by non-infective causes. Microbial isolates gathered at autopsy were classified as non-pathogens, group 1 pathogens (organisms usually associated with an identifiable focus of infection), or group 2 pathogens (organisms known to cause septicaemia without an obvious focus of infection). FINDINGS: Of 546 SUDI cases, 39 autopsies were excluded because of viral or pneumocystis infection or secondary bacterial infection after initial collapse and resuscitation. Bacteriological sampling was done in 470 (93%) of the remaining 507 autopsies. 2079 bacteriological samples were taken, of which 571 (27%) were sterile. Positive cultures yielded 2871 separate isolates, 484 (32%) of which showed pure growth and 1024 (68%) mixed growth. Significantly more isolates from infants whose deaths were explained by bacterial infection (78/322, 24%) and from those whose death was unexplained (440/2306, 19%) contained group 2 pathogens than did those from infants whose death was explained by a non-infective cause (27/243, 11%; difference 13.1%, 95% CI 6.9-19.2, p<0.0001 vs bacterial infection; and 8.0%, 3.2-11.8, p=0.001 vs unexplained). Significantly more cultures from infants whose deaths were unexplained contained Staphylococcus aureus (262/1628, 16%) or Escherichia coli (93/1628; 6%) than did those from infants whose deaths were of non-infective cause (S aureus: 19/211, 9%; difference 7.1%, 95% CI 2.2-10.8, p=0.005; E coli: 3/211, 1%, difference 4.3%, 1.5-5.9, p=0.003). INTERPRETATION: Although many post-mortem bacteriological cultures in SUDI yield organisms, most seem to be unrelated to the cause of death. The high rate of detection of group 2 pathogens, particularly S aureus and E coli, in otherwise unexplained cases of SUDI suggests that these bacteria could be associated with this condition.

Decreased monocyte class II MHC expression following major abdominal surgery in children is related to operative stress.

Monocyte class II major histocompatibility complex (MHC) expression is necessary for antigen presentation and stimulation of T-cells. The aim of this study was to correlate monocyte class II MHC response to operative stress in children and the possible influence of cytokines in the postoperative period. We studied 21 children undergoing elective abdominal surgery. Operative stress score (OSS) was calculated. Monocyte class II MHC expression was measured preoperatively, immediately after surgery, 24 and 48 h postoperatively, using flow cytometry. Class II MHC is expressed as mean fluorescence intensity (MFI) of monocytes expressing MHC (mean +/- SD). Cytokine levels (interleukins 1ra, 6, and 10, and tumor necrosis factor-alpha) were also measured. Data between time points were compared using repeated measures ANOVA. There was an immediate postoperative decrease in class II MHC expression, with lowest levels 24 h postoperatively (preoperative 50 +/- 23.6, 24 h 18.2 +/- 9.4, P < 0.0001 vs. preoperative). At 48 h there was partial recovery in class II MHC, but levels were still significantly lower than preoperative (23.9 +/- 11.1, P < 0.001). The degree of monocyte depression was related to the magnitude of operative stress. Patients who had OSS <10 displayed some recovery in expression at 48 h 25.5 +/- 11.1), whereas in patients with OSS > or = 10 (severe surgical stress), expression further decreased at 48 h (MFI 14.0 +/- 0.1). There was an elevation of interleukin-1ra in the immediate postoperative period in both groups. There was no elevation in the other cytokines. Abdominal surgery in children decreases monocyte MHC expression. Class II MHC depression was related to magnitude of surgical trauma, implying that more severe immuneparesis follows surgery of greater magnitude. This may predispose to postoperative infection.

Endotoxin immunity and the development of the systemic inflammatory response syndrome in critically ill children.

BACKGROUND: The systemic inflammatory response syndrome (SIRS) may be triggered by endotoxin. Humans have antibodies directed against the core of endotoxin (endotoxin core antibodies, EndoCAb) that appear to be protective following surgery and in sepsis. We hypothesised that children with elevated antibodies to endotoxin core would be less likely to develop SIRS in their initial period on intensive care. Because of the existing literature we defined two sub-groups according to the primary reason for ICU admission: infection and non-infection. METHODS: We recruited 139 consecutive patients admitted to a paediatric intensive care unit (PICU) with more than one organ failure for longer than 12 h as part of another study. Patients were classified on admission to PICU as having an infectious or a non-infections diagnosis. The occurrence of SIRS within 48 h of admission was recorded along with detailed clinical and demographic data, EndoCAb concentration and the potential confounding variables C-reactive protein and mannose-binding lectin. RESULTS: In the 71 patients admitted without infection (primarily post-operative and head injured) IgG EndoCAb was significantly lower in patients who developed SIRS than those who did not (72 vs. 131 MU/ml), independent of potential confounding variables. In patients with infection there was no significant difference in IgG EndoCAb between children developing SIRS and those who did not (111 vs. 80 MU/ml). CONCLUSION: Head injured and post-operative patients admitted to PICU who develop early SIRS have significantly lower serum IgG EndoCAb levels than those who do not.

Susceptibility to infection in patients with neutropenia: the role of the innate immune system.

Chemotherapy-induced neutropenia increases the risk of infection. There appears to be a wide variability in the severity and length of infective episodes. Susceptibility to infections is determined by the underlying malignant disease and its treatment, environmental factors (e.g. nutritional state of the patient and hygiene) and genetically determined variations of the immune system. The majority of primary immunodeficiencies are rare (c. frequency one in 10 000), whereas some genetic polymorphisms in the innate immune system, such as profound mannose-binding lectin deficiency, are much more common (c. frequency one in 10). Here, we review the potential role of the innate immune system in determining susceptibility to infections in patients with neutropenia.

Mannose-binding lectin: do we need it?

Mannose-binding lectin (MBL) deficiency has now been recognised for 15 years. Numerous studies have shown that MBL deficiency is associated with an increased susceptibility to a range of infections. However, the importance of MBL, in defence against infection, is still debated. This article discusses recent developments in MBL research and explores how MBL may be operating in the setting of modern medical practice.

Interleukin-4 increases the permeability of human endothelial cells in culture.

BACKGROUND: IL-4 plays a key role in the induction of allergic inflammation, but its role as an effector molecule is less well-established. Although some observations suggest that IL-4 may mediate increased vascular permeability, which is a characteristic feature of allergic inflammation, evidence for a direct effect on endothelial cell permeability is lacking. OBJECTIVE: To determine the effect of human IL-4 on the albumin permeability of cultured human endothelial cells. METHODS: Human umbilical vein endothelial cells were cultured on permeable membranes and the albumin permeability of endothelial monolayers was measured with and without exposure to recombinant human IL-4. Endothelial cells were exposed to various concentrations of IL-4 (0.001-100 U/mL), for various durations (6-24 h), either in the presence or absence of anti-IL-4 antibody. Recovery of endothelial barrier function following exposure to IL-4 was also examined. RESULTS: IL-4 induced a dose-dependent, reversible increase in endothelial permeability to albumin. Low concentrations of IL-4 (1 U/mL) induced a significant increase in endothelial permeability (P=0.004). IL-4-mediated endothelial leak occurred rapidly, within 6 h of exposure. CONCLUSIONS: IL-4 has the capacity to induce vascular leak by a direct effect on cultured endothelial cells, suggesting a potential effector role for IL-4 in the pathogenesis of vascular leak in allergic diseases.

High-level endothelial E-selectin (CD62E) cell adhesion molecule expression by a lipopolysaccharide-deficient strain of Neisseria meningitidis despite poor activation of NF-kappaB transcription factor.

Binding of host inflammatory cells to the endothelium is a critical contributor to the vascular damage characteristic of severe meningococcal disease and is regulated by endothelial cell adhesion molecules such as ICAM-1, VCAM-1 and CD62E. Intact meningococci induce far higher levels of CD62E than lipopolysaccharide (LPS) alone, whereas LPS is at least as potent as meningococci at inducing both VCAM-1 and ICAM-1 expression. This suggests that meningococci possess additional factors other than LPS present in whole bacteria that result in differential adhesion molecule expression. To investigate this possibility, we studied the capacity of an LPS-deficient isogenic strain of serogroup B Neisseria meningitidis H44/76 (lpxA-) to induce endothelial cell adhesion molecule expression and translocation of the transcription factor NF-kappaB, and compared it to both parent and unencapsulated strains of both B1940 and H44/76 and purified LPS. Although the LPS-deficient isogenic mutant of strain H44/76 was found to be a poor inducer of NF-kappaB, it induced higher levels of CD62E expression than LPS alone. These data provide evidence that intact meningococci induce a range of signals in the endothelium that are distinct from those seen with purified LPS alone and that they occur in a LPS-dependent and LPS-independent manner. These signals may explain the potent effects of N. meningitidis on CD62E expression on vascular endothelium and provide a basis for the complex endothelial dysregulation seen in meningococcal sepsis.

Severe meningococcal disease is characterized by early neutrophil but not platelet activation and increased formation and consumption of platelet-neutrophil complexes.

Approximately 25% of polymorphonuclear leukocytes (PMNL) circulate in heterotypic complexes with one or more activated platelets. These platelet-neutrophil complexes (PNC) require platelet CD62P expression for their formation and represent activated subpopulations of both cell types. In this study, we have investigated the presence, time course, and mechanisms of PNC formation in 32 cases of severe pediatric meningococcal disease (MD) requiring intensive care. There were marked early increases in PMNL CD11b/CD18 expression and activation, and reduced CD62L expression compared with intensive care unit control cases. Minimal platelet expression of the active form of alphaIIbbeta3 (GpIIb/IIIa) was seen. PNC were reduced on presentation and fell to very low levels after 24 h. Immunostaining of skin biopsies demonstrated that PNC appear outside the circulation in MD. In vitro studies of anticoagulated whole blood inoculated with Neisseria meningitidis supported these clinical findings with marked increases in PMNL CD11b/CD18 expression and activation but no detectable changes in platelet-activated alphaIIbbeta3 or CD62P expression. In vitro PMNL activation with N. meningitidis (or other agonists) potentiated the formation of PNC in response to platelet activation with adenine diphosphate. Therefore, in severe MD, PMNL activation is likely to promote PNC formation, and we suggest that the reduced levels of PNC seen in established MD reflect rapid loss of PNC from the circulation rather than reduced formation.