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Biomedical subjects

G Kronborg

Publications and source records attributed to G Kronborg.

28 records · Page 2Linked to original sources
PubMed1993

Complement activation by Pseudomonas aeruginosa biofilms.

In chronic infections, such as the bronchopulmonary Pseudomonas aeruginosa infection in cystic fibrosis (CF) patients, bacteria persist despite an intact host immune defense and frequent antibiotic treatment. An important reason for the persistence of the bacteria is their capacity for the biofilm mode of growth. In this study we investigated the role of biofilms in activation of complement, a major contributor to the inflammatory process. Complement activation by P. aeruginosa was examined in a complement consumption assay, production of C3 and factor B conversion products assessed by crossed immuno-electrophoresis, C5a generation tested by a PMN chemotactic assay, and terminal complement complex formation measured by ELISA. Two of the four assays showed that P. aeruginosa grown in biofilm activated complement less than planktonic bacteria, and all assays showed that activation by intact biofilms was submaximal. Factor B conversion was of low magnitude indicating the importance of the classical pathway. Complement activation by P. aeruginosa was inhibited by polymyxin B indicating that lipopolysaccharide (LPS) was the main mediator of complement activation. Immune complexes and massive influx of neutrophils are known to cause inflammatory changes in the lungs. P. aeruginosa persisting in biofilms may contribute to the constant inflammation taking place in the lungs of CF patients.

Chemotaxis, Leukocyte↗
PubMed1993

Enhancement of lipopolysaccharide-induced tumor necrosis factor secretion by hyperimmune serum from chronic infected patients.

Patients with cystic fibrosis (CF) and chronic Pseudomonas aeruginosa lung infection have a very high load of endotoxins in their lungs. However, sepsis practically never occurs in this group of patients and the presence of tumor necrosis factor (TNF)-alpha (one of the mediators of septic shock) in serum from chronically infected CF patients is contentious. The purpose of this study was to investigate the effect of hyperimmune serum from patients with CF on lipopolysaccharide (LPS, endotoxin)-induced TNF secretion from human peripheral blood mononuclear cells (PBMC). PBMC were purified from healthy donors and stimulated with a mixture of purified LPS from P. aeruginosa and serum from chronically infected CF patients or healthy controls. TNF in the cell supernatants was detected by an enzyme-linked immunosorbent assay method. CF sera showed a pronounced potentiating effect on TNF secretion from human PBMC induced by LPS from P. aeruginosa. In comparison, serum from healthy controls did not have this effect. By contrast, CF serum and serum from healthy controls showed only little potentiating effect when using LPS from Salmonella abortus equi at concentrations above 0.01 microgram/ml per 2 x 10(6) PBMC. This indicates a specific interaction between P. aeruginosa LPS and CF serum which enhances TNF secretion. The TNF responses varied depending on the sera used in the preincubation with LPS, and correlated positively to the specific IgG, IgA, and IgM anti-P. aeruginosa LPS titers of the sera. However, since TNF is hardly detectable in sera from these patients another LPS- and/or TNF-inhibitory activity may be present in these sera.

Antigen-Antibody Complex↗
PubMed1993

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.

Antibodies↗
PubMed1993

Induction of oxidative burst response in human neutrophils by immune complexes made in vitro of lipopolysaccharide and hyperimmune serum from chronically infected patients.

Purified lipopolysaccharide (LPS) from Pseudomonas aeruginosa was used as an antigen for immune complex (IC) formation in vitro together with hyperimmune sera from chronically P. aeruginosa-infected patients with cystic fibrosis (CF). P. aeruginosa LPS by itself did not induce an oxidative burst in human neutrophil granulocytes (PMN)s measured by chemiluminescence (CL). This was also the case using hyperimmune CF serum alone. In contrast, P. aeruginosa LPS together with CF serum did induce a CL response. The CL responses varied depending on the sera used for IC formation, and were reduced when protein A preabsorbed sera were used. PEG precipitation of the ICs from the mixture increased the CL response. These findings indicate that the CL responses induced by the mixture of P. aeruginosa LPS and CF serum were due to IC formation and an Fc-mediated stimulation of the PMNs. It is concluded that ICs made from sera of chronically infected CF patients and purified P. aeruginosa LPS are biologically active in terms of activating PMNs, and may contribute to the lung tissue damage seen in this group of patients.

Antibodies, Bacterial↗
PubMed1992

Determination of IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins in cystic fibrosis lung infection using immunoblotting and enzyme-linked immunosorbent assay.

IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins (OMP) were investigated in serum from cystic fibrosis (CF) patients by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Fifteen patients (eight in good and seven in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the preinfection, and early and late stages of chronic infection. Laser-scanning densitometry of photographs taken from immunoblots was used to quantify antibody level and compare with ELISA titres. The earliest anti-OMP antibodies to appear were of the IgG1 subclass. There was no significant difference in IgG subclass antibody levels to OMPs between patients in relatively good and poor clinical condition. Data presented indicate a high positive correlation among measurements of IgG subclass antibodies to P. aeruginosa OMPs using ELISA and immunoblotting.

Antibodies, Bacterial↗
PubMed1992

Antibody responses to lipid A, core, and O sugars of the Pseudomonas aeruginosa lipopolysaccharide in chronically infected cystic fibrosis patients.

Enzyme-linked immunosorbent assays were developed separately for the three main parts of the Pseudomonas aeruginosa lipopolysaccharide (LPS) molecule, namely, lipid A, core, and O polysaccharide. Anti-lipid A, anticore, and anti-O polysaccharide antibodies were measured in serum samples from 12 patients with cystic fibrosis (CF) in a longitudinal study covering the period before P. aeruginosa infection was established through at least 5 years of chronic infection. The serum antibody response to all parts of the P. aeruginosa LPS molecule increased during the course of chronic infection. The increase in anti-lipid A antibodies was specific for P. aeruginosa lipid A, since no increase in anti-Escherichia coli lipid A antibodies was seen. Immunoglobulin G, A, and M (IgG, IgA and IgM) antibodies were all involved in the specific systemic response to P. aeruginosa lipid A, core, and the O polysaccharides. IgG and IgA levels in particular increased during the course of infection and were significantly higher than the antibody increase seen with age in a healthy control group. The local immune response in the lungs was investigated by measuring IgG, IgA, and IgM antibodies to the separate parts of the P. aeruginosa LPS molecule in sputum samples from 18 CF patients with at least a 5-year history of chronic P. aeruginosa infection. Antibodies detected in sputum were mainly anti-lipid A and anti-O polysaccharide antibodies of the IgG and IgA isotypes. Very high IgA anti-lipid A titers were detected in sputum samples from some CF patients.

Adolescent↗
PubMed1992

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.

Antigen-Antibody Complex↗
PubMed1989

Determination of the components of immune complexes made in vitro with antigens derived from Pseudomonas aeruginosa.

A method for identification of the components of immune complexes would increase our understanding of the pathogenesis of chronic diseases such as Pseudomonas aeruginosa lung infection in cystic fibrosis. Capillary tube, gel diffusion and turbidimetric methods of determining immune complex formation were investigated with antigens from P. aeruginosa and the homologous rabbit antisera. Visible complexes were formed in the first two methods with flagella antigens. Purified lipopolysaccharide from P. aeruginosa would not form visible precipitates and a rapid and economical turbidimetric method was developed with 96-well microtiter plates. Larger quantities of immune complexes were formed in vitro with antigen/antibody ratios determined by the above methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting (Western blotting) were evaluated and found to be useful in determining the antigen and antibody components of these immune complexes.

Animals↗
PubMedDate not supplied

Specific IgG2 antibodies to Pseudomonas aeruginosa lipid A and lipopolysaccharide are early markers of chronic infection in patients with cystic fibrosis.

The IgG subclass antibody response to the two parts of Pseudomonas aeruginosa lipopolysaccharide; endotoxic lipid A and the O-polysaccharide, were investigated in a retrospective longitudinal study involving 16 patients with cystic fibrosis and chronic P. aeruginosa lung infection. The purpose of the study was to see if any of the IgG subclasses of either specificity could be used as prognostic markers in the development and subsequent course of the lung disease. IgG2 anti-lipid A, IgG3 anti-lipid A, and IgG2 anti-polysaccharide showed a significant positive correlation with deteriorating pulmonary function already before chronic P. aeruginosa lung infection was diagnosed as well as in subsequent years. The findings suggest antigenic exposure of the patient before chronic infection is detected by routine sputum examinations, and further support our previous findings of a critical role of the IgG subclass response in modulating the course of inflammatory lung damage in these patients.

Adolescent↗