Geoffrey H. Shand

Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres

Alginate‐producing, mucoid P. aeruginosa is frequently found in the lungs of patients with cystic fibrosis (CF), where it causes a chronic infection. The importance of alginate in the pathogenesis was demonstrated by the ability to establish chronic P. aeruginosa lung infection in rats if P. aeruginosa entrapped in minute alginate‐beads were inoculated transtracheally. Alginate beads containing P. aeruginosa were formed by nebulizing a suspension of seaweed sodium‐alginate and P. aeruginosa into a calcium solution. The alginate bead method of establishing infection was compared to an agar‐bead method and proved to be quantitatively similar after 4 weeks. The ability of the two methods to induce formation of precipitins, IgA and IgG antibodies against P. aeruginosa antigens, including outer membrane proteins, flagella, exoenzymes and alginate, was assessed by crossed immunoelectrophoresis, enzyme‐linked immunosorbent assay and immunoblotting. The two methods of inducing infection were comparable and infected rats had significantly higher antibody response than rats inoculated with sterile beads. We suggest that the alginate bead model closely resembles the later stages of CF‐lung infection and that it offers the theoretical advantage of using a substance which is chemically similar to the alginate produced in vivo by P. aeruginosa.

Lipopolysaccharide is present in immune complexes isolated from sputum in patients with cystic fibrosis and chronic Pseudomonas aeruginosa lung infection

Sputum samples from seven patients with cystic fibrosis and chronic P. aeruginosa lung infection were investigated for immune complexes by PEG precipitation and in two different complement binding assays. All seven patients were immune complex positive. The components involved in immune complex formation were identified by SDS‐PAGE and immunoblotting. We found P. aeruginosa lipopolysaccharide as a major antigen. Both core and O‐specific saccharide antigens could be demonstrated. IgG and IgA were the immunoglobulins involved, with IgG2 as the dominating IgG subclass. Lipopolysaccharide has a number of biological activities and its presence in sputum may have consequences for the pathogenesis of lung disease in cystic fibrosis.

Antibodies from chronically infected cystic fibrosis patients react with lipopolysaccharides extracted by new micromethods from all serotypes of Pseudomonas aeruginosa

Micromethods were developed to extract lipopolysaccharide (LPS, endotoxin) from single bacterial colonies of the 20 recognized Pseudomonas aeruginosa type strains. The appearance of these LPSs in polyacrylamide gel electrophoresis (PAGE) and their reactivity with serum of cystic fibrosis (CF) patients chronically infected with P. aeruginosa was studied. Silver staining of LPS after PAGE showed that 13 of the P. aeruginosa LPSs had high numbers of O‐repeating units arranged in 1–4 clusters of banding. Low‐molecular‐weight LPS fractions were more prominent in six of the serotype strains, of which O:7 and O:14 appeared semi‐rough. Corresponding immunoblots using the CF sera showed LPS patterns very similar to the silver‐stained appearance, indicating an immune reaction to all P. aeruginosa LPS including that from the newly discovered O:18, O:19 and O:20. This was unexpected since only a few serotype strains (mostly O:3, O:6 and O:9) had been isolated from the patients. Absorption experiments using purified and chemically defined P. aeruginosa rough LPS and smooth LPS suggested these immune reactions were due to antibodies cross‐reactive to core/lipid A as well as to lower molecular weight O‐polysaccharides or “A‐bands”. However, in some cases O:3, O:6, and O:9 LPSs were also found to contain additional distinct O‐epitopes. Immune recognition of various polyagglutinable P. aeruginosa LPSs seemed also to be caused by cross‐reactive antibodies. The described microextraction methods, followed by PAGE and silver staining or immunoblotting, are easy and convenient techniques with which to study antibodies against LPS epitopes and to screen for LPS phenotypic appearance using only a few bacterial colonies from larger numbers of Gram‐negative bacterial strains.

Antigenic analysis of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins and demonstration of a lipopolysaccharide‐associated GroEL fraction in P. aeruginosa

Quantitative crossed immunoelectrophoresis was used to evaluate the antigenic similarity of Pseudomonas aeruginosa and Pseudomonas cepacia GroEL proteins. We found that the two proteins showed 75% identity. By using a panel of monoclonal antibodies against the P. aeruginosa GroEL protein, we identified 10 monoclonal antibodies which cross‐reacted with the P. cepacia GroEL protein and 21 monoclonal antibodies which recognized type‐specific epitopes on the P. aeruginosa GroEL protein. In crossed immunoelectrophoresis two different fractions of GroEL reactive material could be resolved. These fractions showed a reaction of partial identity. Examination of the two immunoprecipitates by Western blotting, showed that both fractions consisted of anti‐60 kDa GroEL reactive protein. One fraction, in addition, contained LPS with a characteristic ‘ladder’ reaction in modified Western blotting. We therefore conclude that this fraction represents a complex between LPS and GroEL.

Immunological properties of meningococcal lipopohsaccharide from serogroups A, B & C

The aim of the study was to measure and compare the oxidative burst, chemotaxis and cytokine production of human white blood cells, stimulated with meningococcal lipopolysaccharides (LPS) extracted from three different serogroups (A, B and C) of Neisseria meningitidis, and to evaluate whether convalescent sera from patients with meningococcal disease could modify cell stimulation of LPS. All three preparations of LPS from groups A, B and C were tested using the Limulus amoebocyte lysate assay (LAL), and the KDO concentrations of the LPS extracts were measured. Equivalent amounts of biologically active LPS, judged by LAL, and LPS with the same KDO concentration were assayed. IL‐1α, IL‐1β, 1L‐6 and TNF‐α production was stimulated by all three LPS preparations. All three preparations stimulated oxidative burst in monocytes (MNC). Only group A LPS stimulated neutrophil chemotaxis, while none of the three LPS stimulated superoxide production. Pooled convalescent sera from five patients with meningococcal disease suppressed the activity of neutrophils stimulated with LPS from groups B and C (p<0.05, Mann‐Whitney U‐test).