Nigel Klein

FAMILIAL DISSEMINATED ATYPICAL MYCOBACTERIAL INFECTION IN CHILDHOOD: A HUMAN MYCOBACTERIAL SUSCEPTIBILITY GENE?

Inherited defects in specific components of the immune system have provided many clues to the immunological mechanisms underlying resistance to microbial infection. We report a familial immune defect predisposing to disseminated atypical mycobacterial infection in childhood. 6 children with disseminated atypical mycobacterial infection and no recognised form of immunodeficency were identified. Four, including two brothers, come from a village in Malta, and two are brothers of Greek Cypriot origin. They presented with fever, weight loss, lymphadenopathy, and hepatosplenomegaly. They had anaemia and an acute phase response. A range of different mycobacteria (Mycobacterium fortuitum, M chelonei, and four strains of M avium intracellulare complex) were isolated. Treatment with multiple antibiotics failed to eradicate the infection, although treatment with gamma interferon was associated with improvement. Three have died and the surviving children have chronic infection. Tumour necrosis factor-alpha production in response to endotoxin and gamma-interferon was found to be defective in affected patients and their parents. T-cell proliferative responses to mycobacterial and recall antigens were reduced in parents of affected children and gamma-interferon production was diminished in the affected patients and their parents. Clinical and immunological features suggest that these patients are phenotypically similar to Lsh/Ity/Bcg susceptible mice. Understanding of this defect may provide insights into the mechanisms responsible for susceptibility to mycobacteria.

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.

Endotoxin‐Induced Neutrophil Adherence to Endothelium: Relationship to CD11b/CD18 and L‐Selectin Expression and Matrix Disruption

The injury to vascular endothelium seen in severe bacterial infection may be mediated by neutrophil-derived enzymes. Neutrophil adhesion to endothelium, a prerequisite for this process, is mediated sequentially by the leukocyte adhesion molecules L-selectin and the beta 2 integrins, including CD11b/CD18. We have explored the relationship between expression of these molecules, neutrophil adherence, endothelial activation, and consequent endothelial injury, as assessed in vitro by changes to HS and FN matrices that colocalize. Endothelial prestimulation with LPS (endotoxin) caused an increase in adherence and an inversely proportional disruption in the HS matrix; disruption of the FN matrix only occurred on the further addition of fMLP. Although maximal changes in these matrices were associated with elevation of neutrophil CD11b/CD18 and reduction in L-selectin expression, these changes did not determine either the nature or extent of endothelial damage. CD11b/CD18 expression was similar in both adherent and nonadherent neutrophils, while L-selectin was shed in association with adherence in the absence of other stimuli. These changes in expression were thus independently regulated. This model may provide further insights into the interrelationship between neutrophil adhesion and activation and endothelial damage in infection with gram-negative bacteria.

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.