Choice of immunoassay to evaluate porcine cytokine levels

Background In order to adequately monitor cytokines in experimental models, currently available methods and commercially available kits should be compared. Aim To compare the plasma and tissue concentrations of IL-1β, IL-6, IL-8, IL-10, and TNF as a measure of systemic inflammation in septic pigs. Methods Cytokines were quantified from blood and tissue samples obtained at 0, 60, 120, 180, and 240 min, and in postmortem biopsies of the liver, kidney, lung, heart, and spleen from 26 anesthetized landrace pigs. (24 with experimental sepsis, two sham controls). Porcine-specific ELISAs (R&D) and multiplex (9-plex from Thermo Fischer, 13-plex from Millipore) immunoassays were compared. Results The assays differed for the different cytokines and between blood and tissue. In blood, the highest concentration of TNF and IL-6 was in ELISA, IL-1β equal in ELISA and 13-plex, IL-8 in 13-plex and IL-10 in 9-plex. In tissue, the highest concentration of TNF and IL-1β was in ELISA, IL-6 and IL-8 in 13-plex and IL-10 in 9-plex. Conclusion The choice of analysis impacts the quantified cytokine responses in porcine models. ELISA and multiplex techniques supplement each other and our data suggest which assays to use for the quantification of the different cytokines

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

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-1β, 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

Properdin binding to complement activating surfaces depends on initial C3b deposition

Two functions have been assigned to properdin; stabilization of the alternative convertase, C3bBb, is well accepted, whereas the role of properdin as pattern recognition molecule is controversial. The presence of nonphysiological aggregates in purified properdin preparations and experimental models that do not allow discrimination between the initial binding of properdin and binding secondary to C3b deposition is a critical factor contributing to this controversy. In previous work, by inhibiting C3, we showed that properdin binding to zymosan and Escherichia coli is not a primary event, but rather is solely dependent on initial C3 deposition. In the present study, we found that properdin in human serum bound dose-dependently to solid-phase myeloperoxidase. This binding was dependent on C3 activation, as demonstrated by the lack of binding in human serum with the C3-inhibitor compstatin Cp40, in C3-depleted human serum, or when purified properdin is applied in buffer. Similarly, binding of properdin to the surface of human umbilical vein endothelial cells or Neisseria meningitidis after incubation with human serum was completely C3-dependent, as detected by flow cytometry. Properdin, which lacks the structural homology shared by other complement pattern recognition molecules and has its major function in stabilizing the C3bBb convertase, was found to bind both exogenous and endogenous molecular patterns in a completely C3-dependent manner. We therefore challenge the view of properdin as a pattern recognition molecule, and argue that the experimental conditions used to test this hypothesis should be carefully considered, with emphasis on controlling initial C3 activation under physiological conditions

Combined inhibition of complement (C5) and CD14 markedly attenuates inflammation, thrombogenicity, and hemodynamic changes in porcine sepsis

Complement and the TLR family constitute two important branches of innate immunity. We previously showed attenuating effects on inflammation and thromogenicity by inhibiting the TLR coreceptor CD14 in porcine sepsis. In the present study, we explored the effect of the C5 and leukotriene B4 inhibitor Ornithodoros moubata complement inhibitor (OmCI; also known as coversin) alone and combined with anti-CD14 on the early inflammatory, hemostatic, and hemodynamic responses in porcine Escherichia coli–induced sepsis. Pigs were randomly allocated to negative controls (n = 6), positive controls (n = 8), intervention with OmCI (n = 8), or with OmCI and anti-CD14 (n = 8). OmCI ablated C5 activation and formation of the terminal complement complex and significantly decreased leukotriene B4 levels in septic pigs. Granulocyte tissue factor expression, formation of thrombin–antithrombin complexes (p < 0.001), and formation of TNF-α and IL-6 (p < 0.05) were efficiently inhibited by OmCI alone and abolished or strongly attenuated by the combination of OmCI and anti-CD14 (p < 0.001 for all). Additionally, the combined therapy attenuated the formation of plasminogen activator inhibitor-1 (p < 0.05), IL-1β, and IL-8, increased the formation of IL-10, and abolished the expression of wCD11R3 (CD11b) and the fall in neutrophil cell count (p < 0.001 for all). Finally, OmCI combined with anti-CD14 delayed increases in heart rate by 60 min (p < 0.05) and mean pulmonary artery pressure by 30 min (p < 0.01). Ex vivo studies confirmed the additional effect of combining anti-CD14 with OmCI. In conclusion, upstream inhibition of the key innate immunity molecules, C5 and CD14, is a potential broad-acting treatment regimen in sepsis as it efficiently attenuated inflammation and thrombogenicity and delayed hemodynamic change

A complement C5 gene mutation, c.754G&gt;A:p.A252T, is common in the Western Cape, South Africa and found to be homozygous in seven percent of Black African meningococcal disease cases

Patients with genetically determined deficiency of complement component 5 are usually diagnosed because of recurrent invasive Neisseria meningitidis infections. Approximately 40 individual cases have been diagnosed worldwide. Nevertheless, reports of the responsible genetic defects have been sporadic, and we know of no previous reports of C5 deficiency being associated with a number of independent meningococcal disease cases in particular communities. Here we describe C5 deficiency in seven unrelated Western Cape, South African families. Three different C5 mutations c.55C>T:p.Q19X, c.754G>A:p.A252T and c.4426C>T:p.R1476X were diagnosed in index cases from two families who had both presented with recurrent meningococcal disease. p.Q19X and p.R1476X have already been described in North American Black families and more recently p.Q19X in a Saudi family. However, p.A252T was only reported in SNP databases and was not associated with disease until the present study was undertaken in the Western Cape, South Africa. We tested for p.A252T in 140 patients presenting with meningococcal disease in the Cape Town area, and found seven individuals in five families who were homozygous for the mutation p.A252T. Very low serum C5 protein levels (0.1–4%) and correspondingly low in vitro functional activity were found in all homozygous individuals. Allele frequencies of p.A252T in the Black African and Cape Coloured communities were 3% and 0.66% and estimated homozygosities are 1/1100 and 1/22,500 respectively. In 2012 we reported association between p.A252T and meningococcal disease. Molecular modelling of p.A252T has indicated an area of molecular stress in the C5 molecule which may provide a mechanism for the very low level in the circulation. This report includes seven affected families indicating that C5D is not rare in South Africa