R.S. Heyderman

Investigation of platelet-neutrophil interactions in whole blood by flow cytometry

Evidence is increasing that platelets can initiate and propagate inflammatory processes by interacting with leucocytes and the vascular endothelium. Platelets have been shown to bind to neutrophils, existing as platelet/neutrophil complexes (PNC) within the circulation. We describe a simple flow cytometric method for assessing and investigating platelet interactions with neutrophils in small volumes of whole blood. Twenty-five percent (sd 6%) of circulating neutrophils from healthy adults were associated with platelets. Formation of these platelet-neutrophil complexes was CD62P (P-selectin) and divalent cation dependent. Platelet activation (with ADP or thrombin) caused a rapid and sustained rise in %PNC which differed from the pattern of free platelet activation as assessed by CD62P expression. F-met-leu-phe induced neutrophil activation but did not increase the percentage PNC. Platelet activation also caused increased neutrophil CD11b/CD18 expression which was most marked on neutrophils complexed with platelets. This straightforward technique is simple, reproducible, and allows assessment of platelet-neutrophil interactions and activation of neutrophils. It may also provide a method for estimating platelet activation in whole blood.

Whole blood model of meningococcal bacteraemia—a method for exploring host-bacterial interactions

An ex vivo whole blood model of meningococcal bacteraemia was developed to examine the total bactericidal activity of blood. Using a single defined donor and strains belonging to serogroups A, B and C and an unencapsulated strain, we demonstrated that the bactericidal mechanisms operating in whole blood varied with anticoagulant, serogroup and bacterial growth conditions. The choice of anticoagulant had a major effect on the survival of the serogroup A strain with 94% (SEM 7.6) survival in citrated blood compared to 19.7% (SEM 19.6) survival in heparinised blood after 60 min incubation. The serogroup C strain showed enhanced survival when grown in liquid medium compared to growth on solid medium (73.5%, SEM 7.5, and 8.2%, SEM 3.1, respectively, in citrated blood after 60 min). The pattern of survival of serogroup B and the unencapsulated strain were largely unaffected by these variables. Comparison with cell free conditions allowed the contribution of cellular components in meningococcal killing to be determined. Secreted levels of tumour necrosis factor and neutrophil elastase secreted during whole blood assays did not correlate with bacterial growth or viability indicating a lack of relationship between killing and activation of phagocytes.

Reduction of the anticoagulant activity of glycosaminoglycans on the surface of the vascular endothelium by endotoxin and neutrophils: Evaluation by an amidolytic assay

The processes that underlie the coagulopathy observed in severe infection are not fully understood, but seem to be due to an imbalance in the antithrombotic, and prothrombotic properties of the vascular endothelium. Sulphated glycosaminoglycans (GAGs) present on the vessel wall represent an important component of the non-thrombogenic nature of the endothelium. We have modified an amidolytic assay to study the functional ability of GAGs on human umbilical vein endothelial cells (HUVECS), and investigate the effect of E. coli endotoxin and neutrophils on HUVEC surface anticoagulant activity (SAA). Neither endotoxin alone, nor separated neutrophils at lower concentrations (less than 10(6) neutrophils per ml), had major effects on endothelial SAA. When activated neutrophils were incubated with HUVECS pre-stimulated with endotoxin, a significant decrease in SAA was seen using either plasma (mean percentage of control 67.8% +/- sem 7.8; p < 0.02) or purified ATIII (mean percentage of control 69% +/- sem 4.6; p < 0.001). We suggest that alterations in endothelial surface GAGs may occur during sepsis and inflammation, and that this may have important consequences for vascular function. This system will allow the further study of the role of GAGs in the intravascular thrombosis of severe sepsis, and other inflammatory diseases.

Detection of glycosaminoglycans on the surface of human umbilical vein endothelial cells using gold-conjugated poly-l-lysine with silver enhancement

SummaryEndothelial glycosaminoglycans are important in a diverse range of vascular functions. In the course of a biochemical and histological study exploring the role of glycosaminoglycans in inflammation, we have investigated the use of gold-conjugated poly-l-lysine with silver enhancement to establish the nature and physical location of glycosaminoglycans on the surface of cultured human umbilical vein endothelial cells. Cationic gold was effective in locating anionic sites in both cultured endothelial cells and in paraffin-embedded renal tissue. By manipulating pH, and by using enzymes specific for degrading glycosaminoglycans, it was found that, at pH 1.2, staining was directed primarily at glycosaminoglycans. The surface of human umbilical vein endothelial cells was found to be extensively covered in heparan sulphate, the histological appearance of which was dependent upon the fixation procedure employed. Heparan sulphate was also seen to co-distribute with the extracellular matrix protein, fibronectin, when endothelial cultures were simultaneously stained with cationic gold and an antibody to cellular fibronectin.

Modulation of the endothelial procoagulant response to lipopoly-saccharide and tumour necrosis factor-α in-vitro: The effects of dexamethasone, pentoxifylline, iloprost and a polyclonal anti-human IL-1α antibody

Endothelial expression of tissue factor (TF), a potent procoagulant molecule, is increased in response to inflammatory mediators such as lipopolysaccharide (LPS), tumour necrosis factor (TNF) and interleukin-1 (IL-1). We have examined the effects of three antiinflammatory agents and a polyclonal anti-human IL-1α antibody on the human endothelial TF response toE. coli 0111:B4 LPS and recombinant TNFα (rTNFα) in vitro. In contrast to the expected inhibitory effect, dexamethasone, pentoxyfilline and iloprost failed to block TF expression when administered simultaneously or 30 minutes prior to stimulation with either LPS or rTNFα. Inhibition of procoagulant activity was demonstrated with the anti-IL-1α antibody, suggesting that endothelial derived IL-1α is partially responsible for the TF response to the agonists employed. The failure of the antiinflammatory agents to inhibit endothelial TF expression highlights the possibility that therapeutic agents that modulate the circulating monocyte response to LPS and TNFα may not ameliorate the endothelial dysfunction that is also induced by these inflammatory mediators.

Assessment of the effect of candidate anti-inflammatory treatments on the interaction between meningococci and inflammatory cells in vitro in a whole blood model.

A wide range of immunomodulating agents are now available which may be of benefit in reducing inflammatory cell activation in meningococcal sepsis. In order to facilitate selection of candidate anti-inflammatory agents for clinical trials, we have used an in vitro whole blood model to evaluate the effects on meningococcal induced neutrophil and monocyte activation, of dexamethasone, prostacyclin, pentoxifylline and a human IgM anti-lipid A monoclonal antibody (HA-1A).Known concentrations of heat and penicillin killed meningococci were added to whole blood and the time course of cellular activation was determined. Using elastase-α1-antitrypsin (elastase-α1-AT) and TNFα production as markers of neutrophil and monocyte activation respectively, plasma levels of elastase-α1-AT and TNFα were found to increase in a dose-dependant manner. Elastase-α1-AT was detected early, with most release occurring between 15–30 min whereas TNFα was detected later, between 120–180 min.Dexamethasone, prostacyclin and pentoxifylline caused a dose dependant inhibition of TNFα release but had no effect on elastase release. HA-1A had no effect on either TNFα or elastase release.This model may be useful in determining the sequence of inflammatory cell activation and in selecting candidate anti-inflammatory agents for evaluation in clinical trials.

Detection of fibronectin expression by human endothelial cells using a enzyme-linked immunosorbent assay (ELISA): enzymatic degradation by activated plasminogen

An enzyme-linked immunosorbent assay (ELISA) has been developed to measure cellular fibronectin (cFN) in association with human umbilical vein endothelial cells (HUVEC) in culture. The expression of a number of functional domains on the cFN molecule was demonstrated using three specific murine monoclonal antibodies. This system was found to be sensitive, detecting as little as 0.156 microg/ml of cFN, and required only 1.3 x 10(5) cells per well confluent cells per experimental condition. This allowed multiple experiments to be performed on one batch of endothelial cells. cFN was detected on both viable and methanol fixed endothelial cells without significant non-specific antibody binding. The utility of this experimental model was studied by exploring the effect of urokinase activated plasminogen, a potent protease, on the expression of cFN and its functional domains.

Management of bacterial meningitis

The morbidity and mortality from bacterial meningitis has not changed over the last 2 decades despite a better awareness of the disease, the availability of increasingly potent antibiotics, and improvements in intensive care. One of the most destructive diseases of childhood, meningitis kills nearly 10% of those affected, leaving up to 30% with deafness or neurological defect. This chapter will discuss the acute management of the disease, focusing on how new concepts in our understanding of the pathogenesis of bacterial meningitis may lead to novel therapeutic strategies. taking antibiotics.