Neisseria meningitidis accumulate in large organs during meningococcal sepsis.

Background: Neisseria meningitidis (Nm) is the cause of epidemic meningitis and fulminant meningococcal septicemia. The clinical presentations and outcome of meningococcal septic shock is closely related to the circulating levels of lipopolysaccharides (LPS) and of Neisseria meningitidis DNA (Nm DNA). We have previously explored the distribution of Nm DNA in tissues from large organs of patients dying of meningococcal septic shock and in a porcine meningococcal septic shock model. Objective: 1) To explore the feasibility of measuring LPS levels in tissues from the large organs in patients with meningococcal septic shock and in a porcine meningococcal septic shock model. 2) To evaluate the extent of contamination of non-specific LPS during the preparation of tissue samples. Patients and methods: Plasma, serum, and fresh frozen (FF) tissue samples from the large organs of three patients with lethal meningococcal septic shock and two patients with lethal pneumococcal disease. Samples from a porcine meningococcal septic shock model were included. Frozen tissue samples were thawed, homogenized, and prepared for quantification of LPS by Pyrochrome® Limulus Amoebocyte Lysate (LAL) assay. Results: N. meningitidis DNA and LPS was detected in FF tissue samples from large organs in all patients with meningococcal septic shock. The lungs are the organs with the highest LPS and Nm DNA concentration followed by the heart in two of the three meningococcal shock patients. Nm DNA was not detected in any plasma or tissue sample from patients with lethal pneumococcal infection. LPS was detected at a low level in all FF tissues from the two patients with lethal pneumococcal disease. The experimental porcine meningococcal septic shock model indicates that also in porcinis the highest LPS and Nm DNA concentration are detected in lungs tissue samples. The quantification analysis showed that the highest concentration of both Nm DNA and LPS are in the organs and not in the circulation of patients with lethal meningococcal septic shock. This was also shown in the experimental porcine meningococcal septic shock model. Conclusion: Our results suggest that LPS can be quantified in mammalian tissues by using the LAL assay.

Analyzing User-Generated Web-Based Posts of Adolescents' Emotional, Behavioral, and Symptom Responses to Beliefs About Depression: Qualitative Thematic Analysis.

BACKGROUND: Depression is common during adolescence. Early intervention can prevent it from developing into more progressive mental disorders. Combining information technology and clinical psychoeducation is a promising way to intervene at an earlier stage. However, data-driven research on the cognitive response to health information targeting adolescents with symptoms of depression is lacking. OBJECTIVE: This study aimed to fill this knowledge gap through a new understanding of adolescents' cognitive response to health information about depression. This knowledge can help to develop population-specific information technology, such as chatbots, in addition to clinical therapeutic tools for use in general practice. METHODS: The data set consists of 1870 depression-related questions posted by adolescents on a public web-based information service. Most of the posts contain descriptions of events that lead to depression. On a sample of 100 posts, we conducted a qualitative thematic analysis based on cognitive behavioral theory investigating behavioral, emotional, and symptom responses to beliefs associated with depression. RESULTS: Results were organized into four themes. (1) Hopelessness, appearing as a set of negative beliefs about the future, possibly results from erroneous beliefs about the causal link between risk factors and the course of depression. We found beliefs about establishing a sturdy therapy alliance as a responsibility resting on the patient. (2) Therapy hesitancy seemed to be associated with negative beliefs about therapy prognosis and doubts about confidentiality. (3) Social shame appeared as a consequence of impaired daily function when the cause is not acknowledged. (4) Failing to attain social interaction appeared to be associated with a negative symptom response. In contrast, actively obtaining social support reduces symptoms and suicidal thoughts. CONCLUSIONS: These results could be used to meet the clinical aims stated by earlier psychoeducation development, such as instilling hope through direct reattribution of beliefs about the future; challenging causal attributions, thereby lowering therapy hesitancy; reducing shame through the mechanisms of externalization by providing a tentative diagnosis despite the risk of stigmatizing; and providing initial symptom relief by giving advice on how to open up and reveal themselves to friends and family and balance the message of self-management to fit coping capabilities. An active counseling style advises the patient to approach the social environment, demonstrating an attitude toward self-action.

Transcriptomic changes in the large organs in lethal meningococcal shock are reflected in a porcine shock model.

Background: Fulminant meningococcal sepsis with shock and multiple organ failure is associated with a massive systemic inflammatory response involving solid organs. We have previously established a porcine model of the disease to study pathophysiologic and possible therapeutic strategies. Objective: This study examined whether the organ specific gene expression profile in such a large animal model reflects the profile seen in patients with fulminant meningococcal sepsis. Patients and methods: Data from gene expression profiles induced in organs from patients (n=5) and the porcine model (n=8) were imported into the Ingenuity pathway analysis (IPA) software for comparison analysis. The number of meningococci in the organs were quantified by real time-PCR. Results: The all-over transcriptional activation between different organs revealed a striking concordance between the patients and the pigs regarding the pattern of transcriptional activation and activated pathways. Comparison analysis demonstrated similar pattern of upregulation of genes being associated with a large range of inflammatory biofunctions in the patients and the porcine model. Genes associated with biofunctions such as organismal death, morbidity and mortality were similarly downregulated in the patients and the porcine model. Comparison analysis of main predicted canonical pathways also demonstrated a high degree of similarity regarding up- and downregulation in both groups. Core analysis revealed different top-upstream regulators in the different organs in the patients. In the patients pro-inflammatory regulators were most activated in the lungs. In the other organs up-stream factors that regulate signaling pathways involved in development, growth, repair and homeostasis and triglyceride synthesis were most activated. In the porcine model, the top-upstream regulators were pro-inflammatory in all organs. The difference may reflect the shorter duration of the porcine experiment than the duration of the patient's infection before death. Conclusion: The inflammatory responses measured on the transcriptomic level in organs in patients with fulminant meningococcal sepsis is reproduced in the porcine model of the disease, although some differences may exist regarding the top-upregulated factors in individual organs. Thus, this large animal model reproduces important immunological features of meningococcal sepsis and can be a valuable tool in further investigations of inflammatory aspects and possible treatment options.

Finding Relevant Psychoeducation Content for Adolescents Experiencing Symptoms of Depression: Content Analysis of User-Generated Online Texts.

BACKGROUND: Symptoms of depression are frequent in youth and may develop into more severe mood disorders, suggesting interventions should take place during adolescence. However, young people tend not to share mental problems with friends, family, caregivers, or professionals. Many receive misleading information when searching the internet. Among several attempts to create mental health services for adolescents, technological information platforms based on psychoeducation show promising results. Such development rests on established theories and therapeutic models. To fulfill the therapeutic potential of psychoeducation in health technologies, we lack data-driven research on young peoples' demand for information about depression. OBJECTIVE: Our objective is to gain knowledge about what information is relevant to adolescents with symptoms of depression. From this knowledge, we can develop a population-specific psychoeducation for use in different technology platforms. METHODS: We conducted a qualitative, constructivist-oriented content analysis of questions submitted by adolescents aged 16-20 years to an online public information service. A sample of 100 posts containing questions on depression were randomly selected from a total of 870. For analysis, we developed an a priori codebook from the main information topics of existing psychoeducational programs on youth depression. The distribution of topic prevalence in the total volume of posts containing questions on depression was calculated. RESULTS: With a 95% confidence level and a ±9.2% margin of error, the distribution analysis revealed the following categories to be the most prevalent among adolescents seeking advice about depression: self-management (33%, 61/180), etiology (20%, 36/180), and therapy (20%, 36/180). Self-management concerned subcategories on coping in general and how to open to friends, family, and caregivers. The therapy topic concerned therapy options, prognosis, where to seek help, and how to open up to a professional. We also found young people dichotomizing therapy and self-management as opposite entities. The etiology topic concerned stressors and risk factors. The diagnosis category was less frequently referred to (9%, 17/180). CONCLUSIONS: Self-management, etiology, and therapy are the most prevalent categories among adolescents seeking advice about depression. Young people also dichotomize therapy and self-management as opposite entities. Future research should focus on measures to promote self-management, measures to stimulate expectations of self-efficacy, information about etiology, and information about diagnosis to improve self-monitoring skills, enhancing relapse prevention.

Extensive Changes in Transcriptomic "Fingerprints" and Immunological Cells in the Large Organs of Patients Dying of Acute Septic Shock and Multiple Organ Failure Caused by Neisseria meningitidis.

Background: Patients developing meningococcal septic shock reveal levels of Neisseria meningitidis (106-108/mL) and endotoxin (101-103 EU/mL) in the circulation and organs, leading to acute cardiovascular, pulmonary and renal failure, coagulopathy and a high case fatality rate within 24 h. Objective: To investigate transcriptional profiles in heart, lungs, kidneys, liver, and spleen and immunostain key inflammatory cells and proteins in post mortem formalin-fixed, paraffin-embedded (FFPE) tissue samples from meningococcal septic shock patients. Patients and Methods: Total RNA was isolated from FFPE and fresh frozen (FF) tissue samples from five patients and two controls (acute non-infectious death). Differential expression of genes was detected using Affymetrix microarray analysis. Lung and heart tissue samples were immunostained for T-and B cells, macrophages, neutrophils and the inflammatory markers PAI-1 and MCP-1. Inflammatory mediators were quantified in lysates from FF tissues. Results: The transcriptional profiles showed a complex pattern of protein-coding and non-coding RNAs with significant regulation of pathways associated with organismal death, cell death and survival, leukocyte migration, cellular movement, proliferation of cells, cell-to-cell signaling, immune cell trafficking, and inflammatory responses in an organ-specific clustering manner. The canonical pathways including acute phase response-, EIF2-, TREM1-, IL-6-, HMBG1-, PPAR signaling, and LXR/RXR activation were associated with acute heart, pulmonary, and renal failure. Fewer genes were regulated in the liver and particularly in the spleen. The main upstream regulators were TNF, IL-1ß, IL-6, RICTOR, miR-6739-3p, and CD3. Increased numbers of inflammatory cells (CD68+, MPO+, CD3+, and CD20+) were found in lungs and heart. PAI-1 inhibiting fibrinolysis and MCP-1 attracting leukocyte were found significantly present in the septic tissue samples compared to the controls. Conclusions: FFPE tissue samples can be suitable for gene expression studies as well as immunostaining of specific cells or molecules. The most pronounced gene expression patterns were found in the organs with highest levels of Neisseria meningitidis DNA. Thousands of protein-coding and non-coding RNA transcripts were altered in lungs, heart and kidneys. We identified specific biomarker panels both protein-coding and non-coding RNA transcripts, which differed from organ to organ. Involvement of many genes and pathways add up and the combined effect induce organ failure.

Molecular studies of meningococcal and pneumococcal meningitis patients in Ethiopia.

Neisseria meningitidis infections in sub-Saharan Africa usually present with distinct symptoms of meningitis but very rarely as fulminant septicemia when reaching hospitals. In Europe, development of persistent meningococcal shock and multiple organ failure occurs in up to 30% of patients and is associated with a bacterial load of >106/ml plasma or serum. We have prospectively studied 27 Ethiopian patients with meningococcal infection as diagnosed and quantified with real-time PCR in the cerebrospinal fluid (CSF) and serum. All presented with symptoms of meningitis and none with fulminant septicemia. The median N. meningitidis copy number (NmDNA) in serum was < 3.5 × 103/ml, never exceeded 1.8 × 105/ml, and was always 10-1000 times higher in CSF than in serum. The levels of LPS in CSF as determined by the limulus amebocyte lysate assay were positively correlated to NmDNA copy number ( r = 0.45, P = 0.030), levels of IL-1 receptor antagonist, ( r = 0.46, P = 0.017), and matrix metallopeptidase-9 (MMP-9; r = 0.009). We also compared the inflammatory profiles of 19 mediators in CSF of the 26 meningococcal patients (2 died and 2 had immediate severe sequelae) with 16 patients with Streptococcus pneumoniae meningitis (3 died and 3 with immediate severe sequelae). Of 19 inflammatory mediators tested, 9 were significantly higher in patients with pneumococcal meningitis and possibly linked to worse outcome.

Correction: Impact of extensive antibiotic treatment on faecal carriage of antibiotic-resistant enterobacteria in children in a low resistance prevalence setting.

[This corrects the article DOI: 10.1371/journal.pone.0187618.].

Whole-blood incubation with the Neisseria meningitidis lpxL1 mutant induces less pro-inflammatory cytokines than the wild type, and IL-10 reduces the MyD88-dependent cytokines.

Levels of bacterial LPS, pro-inflammatory cytokines and IL-10 are related to the severity of meningococcal septicaemia. Patients infected with a Neisseria meninigitidis lpxL1 mutant ( Nm-mutant) with penta-acylated lipid A present with a milder meningococcal disease than those infected with hexa-acylated Nm wild type ( Nm-wt). The aim was to compare the pro-inflammatory responses after ex vivo incubation with the heat-inactivated Nm-wt or the Nm-mutant in citrated whole blood, and the modulating effects of IL-10. Concomitantly, we measured intracellular IL-6, IL-8 and TNF-α to elucidate which cell types were responsible for the pro-inflammatory responses. Incubation with Nm-wt (106/ml;107/ml;108/ml) resulted in a dose-dependent increase of the MyD88-dependent pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α), which were mainly derived from monocytes. In comparison, only 108/ml of the Nm-mutant significantly increased the concentration of these cytokines. The MyD88-independent cytokines (IP-10, RANTES) were evidently increased after incubation with the Nm-wt but were unaffected by the Nm-mutant. Co-incubation with IL-10 significantly reduced the concentrations of the MyD88-dependent cytokines induced by both the Nm-wt and the Nm-mutant, whereas the MyD88-independent cytokines were almost unaffected. In summary, the Nm-mutant is a weaker inducer of the MyD88-dependent/independent cytokines than the Nm-wt in whole blood, and IL-10 attenuates the Nm-stimulated increase in MyD88-dependent pro-inflammatory cytokines.

Large-scale reduction of tyrosine kinase activities in human monocytes stimulated in vitro with N. meningitidis.

N. meningitidis induces extensive gene expression changes in human monocytes, suggesting that complex networks of signaling pathways are activated during meningococcal sepsis. These effects are modulated by the anti-inflammatory cytokine interleukin-10 (IL-10). To further study changes in signal transduction suggested by mRNA data, we used kinase substrate arrays to identify composite kinase activities induced by lysates from a primary human monocyte model system. Cell lysates were prepared from monocytes treated with the following experimental conditions: 106 N. meningitidis/mL, 25 ng/mL IL-10, 106 N. meningitidis/mL in combination with 25 ng/mL IL-10, and vehicle. Lysates were subjected to kinase activity profiling with Tyrosine Kinase PamChip® arrays containing 144 kinase peptide substrates. In our experimental model, we were not able to detect a statistically significant large-scale change in ex vivo array peptide phosphorylation by lysates from monocytes treated for 15 minutes. Targets of the IL-10 anti-inflammatory response were not identified. A profound inhibition of array peptide phosphorylation by monocytes treated for 60 minutes was identified, suggesting low activity of a large number of kinases associated with different signaling pathways and immune cell functions, including STAT3 activity, Nf-κB and VEGF signaling, and PTEN signaling activity. The peptide representing ZBTB16, which was reduced in phosphorylation by lysates from all three experimental conditions, was in Ingenuity Pathway Analysis identified to be linked to reduced cytokine release and mRNA levels of tumor necrosis factor (TNF), IL-6, and CXCL10. Further studies should investigate changes in tyrosine kinase-mediated signal transduction in human immune cells, in order to evaluate the potential clinical application of kinome profiling in the study of systemic inflammatory responses to pathogens.

Impact of extensive antibiotic treatment on faecal carriage of antibiotic-resistant enterobacteria in children in a low resistance prevalence setting.

We prospectively studied the consequences of extensive antibiotic treatment on faecal carriage of antibiotic-resistant enterobacteria in a cohort of children with cystic fibrosis (CF) and a cohort of children with cancer compared to healthy children with no or low antibiotic exposure. The study was conducted in Norway in a low resistance prevalence setting. Sixty longitudinally collected faecal samples from children with CF (n = 32), 88 samples from children with cancer (n = 45) and 127 samples from healthy children (n = 70) were examined. A direct MIC-gradient strip method was used to detect resistant Enterobacteriaceae by applying Etest strips directly onto agar-plates swabbed with faecal samples. Whole genome sequencing (WGS) data were analysed to identify resistance mechanisms in 28 multidrug-resistant Escherichia coli isolates. The prevalence of resistance to third-generation cephalosporins, gentamicin and ciprofloxacin was low in all the study groups. At inclusion the prevalence of ampicillin-resistant E. coli and trimethoprim-sulfamethoxazole-resistant E. coli in the CF group compared to healthy controls was 58.6% vs. 28.4% (p = 0.005) and 48.3% vs. 14.9% (p = 0.001), respectively, with a similar prevalence at the end of the study. The prevalence of resistant enterobacteria was not significantly different in the children with cancer compared to the healthy children, not even at the end of the study when the children with cancer had been treated with repeated courses of broad-spectrum antibiotics. Children with cancer were mainly treated with intravenous antibiotics, while the CF group mainly received peroral treatment. Our observations indicate that the mode of administration of antibiotics and the general level of antimicrobial resistance in the community may have an impact on emergence of resistance in intestinal enterobacteria during antibiotic treatment. The WGS analyses detected acquired resistance genes and/or chromosomal mutations that explained the observed phenotypic resistance in all 28 multidrug-resistant E. coli isolates examined.

Transcriptomic data from two primary cell models stimulating human monocytes suggest inhibition of oxidative phosphorylation and mitochondrial function by N. meningitidis which is partially up-regulated by IL-10.

BACKGROUND: Biological interpretation of DNA microarray data may differ depending on underlying assumptions and statistical tests of bioinformatics tools used. We used Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) to analyze previously generated DNA microarray data from human monocytes stimulated with N. meningitidis and IL-10 ("the model system"), and with meningococcal sepsis plasma before and after immunodepletion of IL-10 ("the patient plasma system"). The objectives were to compare if the two bioinformatics methods resulted in similar biological interpretation of the datasets, and to identify whether GSEA provided additional insight compared with IPA about the monocyte host response to meningococcal activation. RESULTS: In both experimental models, GSEA and IPA identified genes associated with pro-inflammatory innate immune activation, including TNF-signaling, Toll-like receptor signaling, JAK-STAT-signaling, and type I and type II interferon signaling. GSEA identified genes regulated by the presence of IL-10 with similar gene sets in both the model system and the patient plasma system. In the model system, GSEA and IPA in sum identified 170 genes associated with oxidative phosphorylation/mitochondrial function to be down-regulated in monocytes stimulated with meningococci. In the patient plasma system, GSEA and IPA in sum identified 122 genes associated with oxidative phosphorylation/mitochondrial dysfunction to be down-regulated by meningococcal sepsis plasma depleted for IL-10. Using IPA, we identified IL-10 to up-regulate 18 genes associated with oxidative phosphorylation/mitochondrial function that were down-regulated by N. meningitidis. CONCLUSIONS: Biological processes associated with the gene expression changes in the model system of meningococcal sepsis were comparable with the results found in the patient plasma system. By combining GSEA with IPA, we discovered an inhibitory effect of N. meningitidis on genes associated with mitochondrial function and oxidative phosphorylation, and that IL-10 partially reverses this strong inhibitory effect, thereby identifying, to our knowledge, yet another group of genes where IL-10 regulates the effect of LPS. We suggest that relying on a single bioinformatics tool together with an arbitrarily chosen filtering criteria for data analysis may result in overlooking relevant biological processes and signaling pathways associated with genes differentially expressed between compared experimental conditions.

Traceability and distribution of Neisseria meningitidis DNA in archived post mortem tissue samples from patients with systemic meningococcal disease.

BACKGROUND: The pathophysiology and outcome of meningococcal septic shock is closely associated with the plasma level of N. meningitidis lipopolysaccharides (LPS, endotoxin) and the circulating level of meningococcal DNA. The aim of the present study was to quantify the number of N. meningitidis in different formalin-fixed, paraffin-embedded (FFPE) tissue samples and fresh frozen (FF) tissue samples from patients with systemic meningococcal disease (SMD), to explore the distribution of N. meningitidis in the body. METHODS: DNA in FFPE and FF tissue samples from heart, lungs, liver, kidneys, spleen and brain from patients with meningococcal shock and controls (lethal pneumococcal infection) stored at variable times, were isolated. The bacterial load of N. meningitidis DNA was analyzed using quantitative real-time PCR (qPCR) and primers for the capsule transport A (ctrA) gene (1 copy per N. meningitidis DNA). The human beta-hemoglobin (HBB) gene was quantified to evaluate effect of the storage times (2-28 years) and storage method in archived tissue. RESULTS: N. meningitidis DNA was detected in FFPE and FF tissue samples from heart, lung, liver, kidney, and spleen in all patients with severe shock. In FFPE brain, N. meningitidis DNA was only detected in the patient with the highest concentration of LPS in the blood at admission to hospital. The highest levels of N. meningitidis DNA were found in heart tissue (median value 3.6 × 107 copies N. meningitidis DNA/µg human DNA) and lung tissue (median value 3.1 × 107 copies N. meningitidis DNA/µg human DNA) in all five patients. N. meningitidis DNA was not detectable in any of the tissue samples from two patients with clinical meningitis and the controls (pneumococcal infection). The quantity of HBB declined over time in FFPE tissue stored at room temperature, suggesting degradation of DNA. CONCLUSIONS: High levels of N. meningitidis DNA were detected in the different tissue samples from meningococcal shock patients, particularly in the heart and lungs suggesting seeding and major proliferation of meningococci in these organs during the development of shock, probably contributing to the multiple organ failure. The age of archived tissue samples appear to have an impact on the amount of quantifiable N. meningitidis DNA.

Combined inhibition of C5 and CD14 efficiently attenuated the inflammatory response in a porcine model of meningococcal sepsis.

BACKGROUND: Fulminant meningococcal sepsis, characterized by overwhelming innate immune activation, mostly affects young people and causes high mortality. This study aimed to investigate the effect of targeting two key molecules of innate immunity, complement component C5, and co-receptor CD14 in the Toll-like receptor system, on the inflammatory response in meningococcal sepsis. METHODS: Meningococcal sepsis was simulated by continuous intravenous infusion of an escalating dose of heat-inactivated Neisseria meningitidis administered over 3 h. The piglets were randomized, blinded to the investigators, to a positive control group (n = 12) receiving saline and to an interventional group (n = 12) receiving a recombinant anti-CD14 monoclonal antibody together with the C5 inhibitor coversin. RESULTS: A substantial increase in plasma complement activation in the untreated group was completely abolished in the treatment group (p = 0.006). The following inflammatory mediators were substantially reduced in plasma in the treatment group: Interferon-γ by 75% (p = 0.0001), tumor necrosis factor by 50% (p = 0.01), Interleukin (IL)-8 by 50% (p = 0.03), IL-10 by 40% (p = 0.04), IL-12p40 by 50% (p = 0.03), and granulocyte CD11b (CR3) expression by 20% (p = 0.01). CONCLUSION: Inhibition of C5 and CD14 may be beneficial in attenuating the detrimental effects of complement activation and modulating the cytokine storm in patients with fulminant meningococcal sepsis.

The Neisseria meningitidis lpxL1 mutant induces less tissue factor expression and activity in primary human monocytes and monocyte-derived microvesicles than the wild type meningococcus.

Neisseria meningitidis (N. meningitidis) may cause sepsis and meningitis. N. meningitidis with a mutated lpxL1 gene has five, instead of six, acyl chains in the lipid A moiety. Compared with patients infected with the wild type (wt) meningococcus, patients infected with the lpxL1 mutant have a mild meningococcal disease with less systemic inflammation and less coagulopathy. Circulating tissue factor (TF), the main initiator of coagulation, has a central role in the development of coagulation disturbances during sepsis. To study how TF was influenced by the lpxL1 mutant, human primary monocytes and whole blood were incubated with the lpxL1 mutant or the wt meningococcus (H44/76). Monocyte and microvesicle (MV)-associated TF expression and TF-dependent thrombin generation were measured. In both purified monocytes and whole blood, our data show that the lpxL1 mutant is a weaker inducer of monocyte and MV-associated TF compared with the wt. Our data indicate that low levels of circulating TF may contribute to the reduced coagulopathy reported in patients infected with lpxL1 mutants.

Massive Organ Inflammation in Experimental and in Clinical Meningococcal Septic Shock.

Fulminant meningococcal sepsis is characterized by a massive growth of bacteria in the circulation, regarded as the primary inflammatory site, with no specific solid organ focus. Here we aimed to study the local inflammatory response in organs using a porcine model of fulminant meningococcal septic shock challenged with exponentially increasing doses of heat inactivated Neisseria meningitidis. The results were compared with those obtained in organs post mortem from three patients with lethal meningococcal septic shock. Nine patients with lethal pneumococcal disease and 14 patients with sudden infant death syndrome served as controls. Frozen tissue were thawed, homogenized and prepared for quantification of bacterial DNA by real-time polymerase chain reaction, and key inflammatory mediators were measured by ELISA in the pig material and by multiplex in the human material. In addition, gene expression assayed by Affymetrix gene expression profiling was performed in the pig study. The porcine model revealed a major influx of N. meningitidis in lungs, liver, spleen, and kidneys accompanied with major production of cardinal inflammatory mediators including tumor necrosis factor, interleukin (IL)-1ß, IL-6, and IL-8, far exceeding the amount detected in blood. Genes encoding for these mediators revealed a similar profile. By comparing the wild-type with a lipopolysaccharide (LPS) deficient meningococcal strain, we documented that LPS was the dominant group of molecules inducing organ inflammation and was required for IL-8 production. IL-10 production was predominantly stimulated by non-LPS molecules. The massive organ inflammation in the porcine model was present in the three patients dying of meningococcal shock and differed markedly from the patients with lethal pneumococcal infections and sudden infant death syndrome. In conclusion, in meningococcal sepsis, a massive local inflammatory response occurs in specific organs.

IL-10 immunodepletion from meningococcal sepsis plasma induces extensive changes in gene expression and cytokine release in stimulated human monocytes.

The severity of systemic meningococcal disease (SMD) correlates to plasma concentrations of LPS and IL-10, with the highest levels detected in non-survivors. Here, plasma from patients with SMD containing high and low concentrations of LPS were incubated with human monocytes before and after immunodepletion of IL-10 to study the effect of IL-10 on gene expression and cytokine release. Patient plasma containing IL-10 induced the expression of 1657 genes in human monocytes when compared with gene expression induced by low LPS plasma. After immunodepletion of IL-10, this number increased to 2260. By directly comparing the gene expression profiles induced before and after immunodepletion of IL-10, the presence of IL-10 differentially regulated 373 genes. Functional classes associated with these genes were cellular function and maintenance, cellular development, cellular growth and proliferation, cell-cell signaling and interaction and cellular movement. Immunodepletion of IL-10 resulted in down-regulation of genes of the leukocyte immunoglobulin-like receptor family, and up-regulation of genes of type I IFN signaling, TLR signaling, the inflammasomes, coagulation and fibrinolysis. Finally, immunodepletion of IL-10 increased the protein levels of IL-1ß, IL-8, TNF-α, MIP-1α and MIP-1ß. Data suggest that IL-10 in meningococcal sepsis plasma regulates a variety of genes and signaling pathways, likely leading to an overall inhibitory effect on the inflammatory response induced in meningococcal sepsis.

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock.

INTRODUCTION: The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis. MATERIALS AND METHODS: MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay. RESULTS: MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2-2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r(s)=-0.84), time to peak (rs=-0.83), peak (r(s)=0.85) and ETP (r(s)=0.83). CONCLUSIONS: MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.

Microparticle-associated tissue factor activity is reduced by inhibition of the complement protein 5 in Neisseria meningitidis-exposed whole blood.

Neisseria meningitidis causes fulminant meningococcal sepsis with a massive activation of the coagulation and complement cascades. Bacterial cell envelope molecules from N. meningitidis, particularly lipopolysaccharide (LPS), induce tissue factor (TF) expression. In meningococcal sepsis, TF can be detected on circulating monocytes and microparticles (MPs) within the bloodstream. During infection, Nm activates C5 and C5a, which also is able to induce TF. We evaluated the effect of eculizumab, a C5-blocking monoclonal antibodies (mAb), on cell- and MP-associated TF. Using a lepirudin-anticoagulated whole blood model, we activated the coagulation and complement cascades by N. meningitidis, and investigated the interaction between the cascade systems with special focus on cell-associated TF-expression (mRNA and protein) and MP-associated TF-dependent thrombin and fibrin generation in platelet-free plasma. We also examined the ability of TF-positive MPs to support clot formation in whole blood. In addition, the effect of corn trypsin inhibitor and time-dependent changes on MP-associated functional TF activity was examined. Inhibition of C5 reduced cell-associated TF expression at both gene and protein level, and reduced MP-associated TF-dependent thrombin and fibrin generation in platelet-poor plasma, MP-induced TF-dependent clot formation in whole blood, implying that the complement and coagulation cascades are interplayers in N. meningitidis-mediated activation of these cascades.

Global effect of interleukin-10 on the transcriptional profile induced by Neisseria meningitidis in human monocytes.

In meningococcal septic shock, the dominant inducer of inflammation is lipopolysaccharide (LPS) in the outer membrane of Neisseria meningitidis, while interleukin-10 (IL-10) is the principal anti-inflammatory cytokine. We have used microarrays and Ingenuity Pathway Analysis to study the global effects of IL-10 on gene expression induced by N. meningitidis, after exposure of human monocytes (n = 5) for 3 h to N. meningitidis (10(6) cells/ml), recombinant human IL-10 (rhIL-10) (25 ng/ml), and N. meningitidis combined with rhIL-10. N. meningitidis and IL-10 differentially expressed 3,579 and 648 genes, respectively. IL-10 downregulated 125 genes which were upregulated by N. meningitidis, including NLRP3, the key molecule of the NLRP3 inflammasome. IL-10 also upregulated 270 genes which were downregulated by N. meningitidis, including members of the leukocyte immunuglobulin-like receptor (LIR) family. Fifty-three genes revealed a synergistically increased expression when N. meningitidis and IL-10 were combined. AIM2 (the principal molecule of the AIM2 inflammasome) was among these genes (fold change [FC], 18.3 versus 7.4 and 9.4 after stimulation by N. meningitidis and IL-10, respectively). We detected reduced concentrations (92% to 40%) of six cytokines (IL-1b, IL-6, IL-8, tumor necrosis factor alpha [TNF-α], macrophage inflammatory protein alpha [MIP-α], MIP-ß) in the presence of IL-10, compared with concentrations with stimulation by N. meningitidis alone. Our data analysis of the effects of IL-10 on gene expression induced by N. meningitidis suggests that high plasma levels of IL-10 in meningococcal septic shock plasma may have a profound effect on a variety of functions and cellular processes in human monocytes, including cell-to-cell signaling, cellular movement, cellular development, antigen presentation, and cell death.

Microparticle-associated tissue factor activity measured with the Zymuphen MP-TF kit and the calibrated automated thrombogram assay.

There is increasing clinical interest for measuring microparticle (MP)-associated tissue factor (TF) activity owing to its possible role as a prothrombotic biomarker in a variety of diseases. However, the methods used are to various extents hampered by lack of (pre)analytical standardization as well as limited published documentation. The objective of this study was to evaluate the performance of the Zymuphen MP-TF kit and the calibrated automated thrombogram (CAT) assay in measuring MP-associated TF activity in plasma using a Neisseria meningitidis (Nm)-stimulated whole blood model. In addition, (pre)analytical variables like centrifugation procedures, freezing/thawing and the effect of addition of exogenous phosphatidylserine in plasma were evaluated in the CAT assay. Citrate-anticoagulated blood was stimulated with Nm bacteria for 4 h before platelet-poor plasma (PPP) or platelet-free plasma (PFP) were prepared and assayed with either of the two methods. Nm dose-dependently (10-10 bacteria/ml) induced TF-specific activity, measured as decreased lagtimes, in the CAT assay. The Zymuphen MP-TF kit also detected TF activity, although much higher Nm doses (10 bacteria/ml) were required to achieve measurable levels. Neither freezing/thawing nor the use of PPP vs. PFP influenced the TF activity, measured over a broad range of lagtimes, in the CAT assay. In conclusion, changes in lagtime in the CAT assay reflected levels of MP-associated TF activity in a more sensitive manner than the Zymuphen MP-TF kit did, in our Nm-stimulated whole blood system.