Inhibition by lipid A-specific monoclonal antibodies of priming of human polymorphonuclear leucocytes by endotoxin.

Exposure to lipopolysaccharide (LPS) primes polymorphonuclear leucocytes (PMNL) for enhanced release of oxygen metabolites after subsequent stimulation. The metabolic response of human PMNL primed with LPS and stimulated with formyl-methionyl-leucyl-phenylalanine (FMLP) was measured by chemiluminescence (CL) as a parameter for endotoxic activity. Polymyxin B (PMB) and monoclonal antibodies (MAbs) with specificity for lipid A were tested for inhibition of the priming effect of Re LPS of Salmonella minnesota R595, Rc LPS of Escherichia coli J5 and smooth LPS of E. coli O111. The CL response of PMNL primed with Re LPS or Rc LPS was higher than that of PMNL primed with smooth LPS. Pre-incubation of rough or smooth LPS with PMB caused dose-dependent inhibition of priming of PMNL. Two IgM MAbs, 8-2 and 26-20, which recognise different epitopes on the hydrophobic part of lipid A, also completely prevented priming of PMNL by either rough or smooth LPS. The dose-dependent inhibitory effect of both MAbs was similar to the inhibition by PMB. These results indicate that the binding of MAbs to the hydrophobic part of lipid A is important in blocking lipid A-mediated effects.

The use of a hybridization assay for the study of host defences against herpes simplex virus.

A rapid and simple hybridization assay was developed as an alternative for virus titration for the investigation of host resistance against HSV-1 infections in vitro. The probe which was constructed for this assay was shown to be HSV-1-specific. When a monolayer of fibroblasts was infected for 24 h before hybridization, 15 PFU were detected reliably. A plateau in hybridization levels was found when the multiplicity of infection reached 1. In order to demonstrate the applicability of the probe for the study of host defences against HSV in vitro, fibroblasts were infected with HSV in the presence of different numbers of adherent cells and different concentrations of serum containing high titres of anti-HSV antibodies and complement. After 20 h of incubation, samples were lysed, spotted on Zetaprobe filter paper and hybridized with a 32P-labelled RNA probe. Spots were counted for radioactivity. The radioactivity was taken as a measure of the success of infection. Results showed that at high (10%) concentrations of serum containing high titres of anti-HSV antibodies and complement neutralization plays an important role. At low (1%) concentrations of serum containing high titres of anti-HSV antibodies and complement the phagocytic role of adherent cells becomes the dominant factor in preventing infection of the fibroblasts. However, when the number of infectious particles is increased, the protection provided by adherent cells is overwhelmed.

Effects of granulocyte colony-stimulating factor (G-CSF) treatment on granulocyte function and receptor expression in patients with ventilator-dependent pneumonia.

Considerable experimental evidence in animals suggests that treatment with G-CSF may have a beneficial effect in the management of severe infections in non-neutropenic hosts. This beneficial effect is attributed to an enhancement of granulopoiesis and neutrophil function, the latter possibly involving up-regulation of receptors on neutrophils that are involved in antibody-mediated cytotoxicity and killing of microorganisms. We compared neutrophil function and phenotype in blood and bronchoalveolar lavage fluid (BALF) of 10 patients with severe ventilator-dependent pneumonia, at baseline and following initiation of G-CSF treatment as adjunct to standard therapy. G-CSF treatment was associated with three-fold increased blood neutrophil counts at day 3 of treatment compared with baseline counts. Mean serum G-CSF concentration increased from 313 to 2007 pg/ml. After correction for lavage dilution effects, BALF G-CSF levels did not differ significantly from baseline, nor did neutrophil receptor expression (FcgammaRI, FcgammaRII, FcgammaRIII, CR3, and L-selectin) or indicators of neutrophil function such as respiratory burst activity, phagocytosis and killing of Candida albicans in BALF or blood. The mortality in this group of patients was 30% and compared favourably to the APACHE II-derived predicted mortality of 60%. We conclude that the possible therapeutic benefit of G-CSF administration in the early phase of severe bacterial pneumonia is not readily explained by its effect on baseline indicators of neutrophil function or receptor expression.