The present study, using robotized DNA isolation and quantitative PCR based on the Neisseria meningitidis -specific capsular transport A gene, demonstrates the ease, rapidity, specificity, and sensitivity of quantifying neisserial DNA in plasma ( n = 65) and cerebrospinal fluid (CSF) ( n = 12) from patients with systemic meningococcal disease. We found a close correlation between the levels of neisserial DNA and lipopolysaccharides in plasma ( r = 0.905) and in CSF ( r = 0.964). The median concentration of neisserial DNA in plasma in 23 patients with persistent shock was 2 × 10 ⁷ copies/ml, versus <10 ³ copies/ml in 42 nonshock patients. Furthermore, quantitative PCR made possible estimates of the total number of meningococci in plasma, as opposed to conventional blood cultures, suggesting about 1,000 dead meningococci for every viable bacterium. Finally, with logistic regression analyses, neisserial DNA may predict a patient's disease severity and outcome at hospital admission. The number of meningococci in plasma and CSF appears to be the main determinant of the lipopolysaccharide levels, clinical presentation, and outcome.

Chemokines are important in regulating leukocyte traffic during infection. We analyzed plasma chemokine levels of monocyte chemoattractant protein (MCP)–1, macrophage inflammatory protein (MIP)–1α, interleukin (IL)–8, and RANTES in patients with meningococcal infection and correlated these to plasma lipopolysaccharide (LPS) levels, which are closely associated with clinical presentation. In patients with fulminant meningococcal septicemia, versus distinct meningitis or mild systemic meningococcal disease, MCP-1 (both P<.0001), MIP-1α (both P<.0001), and IL-8 (P<.0001 and P=.011) were significantly higher and RANTES significantly lower (P=.007 and P=.021). MCP-1 (r=.88), MIP-1α (r=.82), and IL-8 (r=.89) were positively correlated to plasma LPS levels, whereas RANTES was negatively correlated (r=-.49). In an ex vivo whole-blood model, heat-inactivated wild-type Neisseria meningitidis purified meningococcal LPS, and (to a negligible extent) heat-inactivated LPS-deficient mutant N. meningitidis induced these chemokines. N. meningitidis LPS is the major cause of chemokine release in meningococcal disease

Lipopolysaccharide (LPS) in the outer membrane ofNeisseria meningitidisplays a dominant role as an inflammation-inducing molecule in meningococcal disease. We have used microarray analysis to study the global gene expression after exposure of human monocytes for 3 h to wild-typeN. meningitidis(10⁶), LPS-deficientN. meningitidis(10⁶and 10⁸), and purifiedN. meningitidisLPS (1 ng [33 endotoxin units]/ml) to identify LPS-inducible genes. Wild-typeN. meningitidis(10⁶) induced 4,689 differentially expressed genes, compared with 72 differentially expressed genes induced by 10⁶LPS-deficientN. meningitidisorganisms. However, 10⁸LPS-deficientN. meningitidisorganisms induced 3,905 genes, indicating a dose-response behavior of non-LPS cell wall molecules. A comparison of the gene expression patterns from 10⁶wild-typeN. meningitidisand 10⁸LPS-deficientN. meningitidisorganisms showed that 2,401 genes in human monocytes were not strictly LPS dependent. A list of “particularly LPS-sensitive” genes (2,288), differentially induced by 10⁶wild-typeN. meningitidisbut not by 10⁸LPS-deficientN. meningitidisorganisms, showed an early expression of beta interferon (IFN-β), most likely through the Toll-like receptor-MyD88-independent pathway. Subsequently, IFN-β may activate the type I IFN signaling pathway, and an unknown number of IFN-β-inducible genes, such as those for CXCL9, CXCL10, CXCL11, IFIT1, IFIT2, IFIT3, and IFIT5, are transcribed. Supporting this, human monocytes secreted significantly higher levels of CXCL10 and CXCL11 when stimulated by 10⁶wild-typeN. meningitidisorganisms than when stimulated by 10⁸LPS-deficientN. meningitidisorganisms. Plasma CXCL10, but not CXCL11, was positively correlated (r= 0.67;P< 0.01) to LPS in patients (n= 24) with systemic meningococcal disease. Thus, new circulating biomarkers in meningococcal disease may be suggested through LPS-induced gene expression changes in human monocytes.

To analyze plasma interferon-gamma and interleukin-10 concentrations in patients with systemic meningococcal disease and patients with severe Gram-positive septic shock caused by Streptococcus pneumoniae or Staphylococcus aureus. To study the in vitro cytokine (interferon-gamma and interleukin-10) responses in a whole blood model boosted with heat-killed Neisseria meningitidis, S. pneumoniae, and S. aureus before and after treatment with recombinant interleukin-10 or recombinant interferon-gamma. Experimental study. Laboratory. Plasma samples were collected from patients with systemic meningococcal disease (n = 66) and patients with severe Gram-positive septic shock caused by S. pneumoniae (n = 4) or S. aureus (n = 3). Whole blood was boosted with heat-killed N. meningitidis, S. pneumoniae, and S. aureus (1 x 106 colony forming units/mL), and plasmas were analyzed for interleukin-10 or interferon-gamma at 0, 5, 12, and 24 hrs. Furthermore, recombinant interleukin-10 or recombinant interferon-gamma was added before bacteria, and the effect on the secretion of interferon-gamma and interleukin-10, respectively, was analyzed after 24 hrs. The median concentration of interferon-gamma was 15 pg/mL and of interleukin-10 was 10,269 pg/mL in patients with meningococcal septic shock (n = 24) compared with median interferon-gamma concentration of 3400 pg/mL and interleukin-10 concentration of 465 pg/mL in patients with severe Gram-positive shock (p =.001). Increased interferon-gamma concentrations were associated with case fatality (p =.011). In a whole blood model we demonstrated that 1 x 106 colony forming units/mL of N. meningitidis induced more interleukin-10 but less interferon-gamma than S. pneumoniae. S. aureus induced minimal secretion of both cytokines. Recombinant interleukin-10 efficiently down-regulated the secretion of interferon-gamma, and vice versa, as shown in a whole blood model. We speculate whether high concentrations of interleukin-10 contribute to the low concentrations of interferon-gamma in fulminant meningococcal septicemia. In addition, it appears as if interferon-gamma plays a minor role in the pathophysiology of meningococcal septic shock.

Complement activation is closely associated with plasma endotoxin levels in patients with meningococcal infections. This study assessed complement ac

Currently, diagnosing and stratifying dry eye disease (DED) require multiple tests, motivating interest in a single definitive test. The purpose of this study was to investigate the potential for using tear fluid extracellular vesicle (EV)-RNA in DED diagnostics. With a role in intercellular communication, nanosized EVs facilitate the protected transport of diverse bioactive molecules in biofluids, including tears. Schirmer strips were used to collect tears from 10 patients presenting with dry eye-related symptoms at the Norwegian Dry Eye Clinic. The samples comprised two groups, five from patients with a tear film break-up time (TBUT) of 2 s and five from patients with a TBUT of 10 s. Tear fluid EV-RNA was isolated using a Qiagen exoRNeasy Midi Kit, and the RNA was characterized using Affymetrix Clariom^TM D microarrays. The mean signal values of the two groups were compared using a one-way ANOVA. A total of 26,639 different RNA transcripts were identified, comprising both mRNA and ncRNA subtypes. Approximately 6% of transcripts showed statistically significant differential abundance between the two groups. The mRNA sodium channel modifier 1 (SCNM1) was detected at a level 3.8 times lower, and the immature microRNA-130b was detected at a level 1.5 times higher in the group with TBUT 2 s compared to the group with TBUT 10 s. This study demonstrates the potential for using tear fluid EV-RNA in DED diagnostics.