Fibronectin-cleaving activity in bronchial secretions of patients with cystic fibrosis

In cystic fibrosis, colonization of the airways with Pseudomonas aeruginosa follows colonization with Staphylococcus aureus and is related to accelerated deterioration of pulmonary function. Because P. aeruginosa adheres better to cell surfaces devoid of fibronectin, we searched for fibronectin-cleaving activity in bronchial secretions and saliva from 24 patients with cystic fibrosis who were followed up for 4.5 y and from two control groups. Proteolytic activity against 125I-labeled fibronectin was continuously present in cystic fibrosis bronchial secretions; significantly higher fibronectin-cleaving activity was found in older vs. younger patients, in patients with advanced disease stages determined by a five-stage scoring system, and in those colonized with P. aeruginosa. The fibronectin-cleaving activity was due to neutrophil elastase and cathepsin G. Cystic fibrosis bronchial secretions had proteolytic activity against surface fibronectin of airway mucosal cells. Thus fibronectin-cleaving activity of bronchial secretions rather than of saliva may favor P. aeruginosa colonization of the upper respiratory tract in individuals with cystic fibrosis.     

Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis patients

Specific serum antibodies could be helpful in defining the status of Pseudomonas aeruginosa infection as well as the response to early intervention treatment in patients with cystic fibrosis (CF). We used 1,791 serum samples from 375 European CF patients with known respiratory microbiology status to define titers of P. aeruginosa antibodies directed against alkaline protease (AP), elastase (ELA), and exotoxin A (ExoA). Pseudomonas antibody titers were also measured in a separate cohort of 56 patients undergoing antibiotic treatment for eradication of P. aeruginosa. At a specificity of 97.5%, the sensitivity was highest for antibodies against AP (85.4%), followed by ELA (76.2%) and ExoA (72.0%). AP, ELA, or ExoA antibody titers were significantly higher (P < 0.001) in patients chronically infected with P. aeruginosa compared to patients with negative cultures. The sensitivity of the combined three ELISAs was higher than that for any single ELISA alone. Based on the newly defined cut-off levels, positive serum antibody titers against at least one of the three antigens were present in 43% of patients with new onset of P. aeruginosa infection. Longitudinal assessment of antibody titers assessed before and after inhaled antibiotic therapy in patients with first P. aeruginosa isolation showed a significant decrease in antibody titers against AP and ExoA in patients clearing P. aeruginosa infection, whereas titers increased in patients in whom antibiotic therapy failed to eradicate the organism. Antibody testing against AP, ELA, and ExoA offers high sensitivity and specificity for the presence of P. aeruginosa in respiratory cultures of CF patients. Although serum antibody titers are on average low at the time of first P. aeruginosa isolation from respiratory specimens, they may be useful to monitor response to therapy. However, because variability between patients is considerable, treatment decisions should not be based on P. aeruginosa antibody levels alone.     

Granulocyte neutral proteases and Pseudomonas elastase as possible causes of airway damage in patients with cystic fibrosis

We studied the possible role of granulocyte neutral proteases as mediators of airway destruction in patients with cystic fibrosis (CF) who were infected with Pseudomonas aeruginosa. We measured the enzymatic activities of bronchial secretions on purified radioactively labeled complement component three (C3), elastin, and a granulocyte elastase-specific substrate. Bronchial secretions from 18 patients with CF who were infected with P aeruginosa had a significantly higher mean value for C3 cleaving, elastolytic, and granulocyte elastase-like activity than did two control groups. High enzymatic activities were observed in patients with CF who have advanced bronchial disease (that had been determined by a clinical scoring system). Kinetics of proteolysis of radioactively labeled C3 and inhibition profiles of the activities of the three enzymatic activities studied suggest that they are mainly derived from granulocytes. In addition, 20 of 31 strains of P aeruginosa isolated from patients with CF inactivated purified alpha 1-antiprotease in vitro. We postulate that granulocyte neutral proteases and P aeruginosa may act synergistically in the airways of patients with CF and may contribute to the destruction of elastin and inactivation of C3.     

Levels of free granulocyte elastase in bronchial secretions from patients with cystic fibrosis: effect of antimicrobial treatment against Pseudomonas aeruginosa

Large amounts of free granulocyte elastase (GE), an enzyme capable of mediating airway damage, have been found in bronchial secretions of patients with cystic fibrosis who are infected with Pseudomonas aeruginosa. This finding indicates an imbalance between GE and its antiproteases, alpha 1-proteinase inhibitor (alpha 1-PI) and bronchial mucosal inhibitor (BMI), in the airways of these individuals. The effect of intravenous antimicrobial treatment against P. aeruginosa on activity and concentration of GE, BMI, and alpha 1-PI was evaluated in 30 treatment courses of 20 patients with cystic fibrosis. Although sputum volume and level of immunoreactive GE decreased and concentrations of alpha 1-PI and BMI increased significantly (P less than .05), a high level of free GE persisted. No active alpha 1-PI and BMI were detectable after treatment. High levels of GE correlated with a poor pulmonary condition (rs = .98, P less than .001). In vitro, elastolytic activity of bronchial secretions from patients with cystic fibrosis was significantly inhibited by eglin C and an oxidation-resistant variant of alpha 1-PI, both compounds currently produced by recombinant DNA technology.     

Respiratory tract colonization with Pseudomonas aeruginosa in cystic fibrosis: correlations between anti-Pseudomonas aeruginosa antibody levels and pulmonary function

Chronic Pseudomonas aeruginosa respiratory tract colonization in patients with cystic fibrosis is associated with development of antibodies to the organism. In contrast to the protection usually afforded by humoral immunity to a bacterial pathogen, the immune response to P. aeruginosa may help perpetuate infection and contribute to pulmonary damage in cystic fibrosis. To determine if specific anti-P. aeruginosa antibody levels correlated with pulmonary dysfunction, we measured antibodies to seven P. aeruginosa serotypes, and correlated the geometric mean titer with pulmonary function tests. Patients were divided into groups without P. aeruginosa colonization (n = 20), with recent colonization (n = 20), and with chronic colonization (n = 60). Noncolonized patients had normal pulmonary function or mild obstructive lung disease, and low anti-P. aeruginosa titers. Pulmonary function tests in recently colonized patients were not different from those of noncolonized patients, but antibody titers were higher. Following colonization FEV1 declined and titers increased rapidly. Patients with chronic colonization had worse pulmonary function and higher titers, but while the former were stable the latter gradually increased. An inverse correlation was found between anti-P. aeruginosa titer and FVC, FEV1, and FEF25-75 (P less than 0.001) in these patients; age was not a factor. The strong correlation between severity of lung disease and anti-P. aeruginosa titer demonstrates that an exaggerated immune response to P. aeruginosa is associated with pulmonary damage in patients with cystic fibrosis.     

IgG proteolytic activity of Pseudomonas aeruginosa in cystic fibrosis

To study how fragmented IgG antibodies might arise within the respiratory secretions of individuals with cystic fibrosis (CF), we screened protease extracts from CF polymorphonuclear leukocytes and mucoid and nonmucoid transformants of Pseudomonas aeruginosa from patients with CF for IgG proteolytic activity. All strains of P. aeruginosa tested exhibited IgG proteolytic activity. Incubation for 7 hr at 37 C was required to demonstrate generation of free Fc gamma immunoreactivity. Further analysis of these cleavage products of CF IgG demonstrated generation of Fc gamma polypeptides with 4S sedimentation coefficients and F(ab')2 fragments with 5S coefficients. Bacterial IgG proteolytic activity was inhibited by EDTA and was associated with levels of bacterial elastase exceeding 5 micrograms/mg of total protein. Pseudomonas elastase was significantly more active on IgG1 and IgG3; IgG2 and IgG4 were more resistant. This bacterial exoproduct appears to digest IgG molecules into Fab gamma, F(ab')2 fragments, and a free Fc gamma piece with a molecular weight of 40,000.     

Spread of a multiresistant strain of Pseudomonas aeruginosa in an adult cystic fibrosis clinic

We initiated a prospective surveillance study to investigate possible Pseudomonas aeruginosa cross-infection in our cystic fibrosis centre. We characterised isolates by pyocin typing and pulsed-field gel electrophoresis. 22 (14%) of 154 patients with chronic P aeruginosa had isolates with similar and new pyocin and pulsed-field gel electrophoresis types. The shared isolates showed unusual phenotypic features: they were non-pigmented, non-motile, and resistant to a number of antipseudomonal antibiotics. Cross-infection by a multiresistant P aeruginosa strain has therefore occurred in patients attending our cystic fibrosis centre. We recommend microbiological surveillance in other cystic fibrosis centres.     

Quantitation and identification of antibodies to outer-membrane proteins of Pseudomonas aeruginosa in sera of patients with cystic fibrosis

Chronic, overwhelming pulmonary infection with Pseudomonas aeruginosa is a frequent problem in patients with cystic fibrosis. Titers of antibody to the outer-membrane proteins of P aeruginosa were 10(1)-10(8) (as measured by an enzyme-linked immunosorbent assay) in the sera of 32 patients with cystic fibrosis. Fifteen patients who had been colonized with P aeruginosa for 18 months to nine years had a geometric mean antibody titer of 1.3 X 10(5)--a value approximately 500-fold higher than that for 13 patients with cystic fibrosis who had never been colonized or for 16 healthy adults without cystic fibrosis (P less than 0.001). A significant correlation was observed between the presence of antibody to outer-membrane proteins and the presence of antibody to mucoid exopolysaccharide (P less than 0.002). Nineteen serum specimens from the patients with cystic fibrosis were allowed to react with Western electophoretic blots of separated outer-membrane proteins. All of these sera contained antibodies to porin protein F. In addition, antibodies to outer-membrane proteins E, H2, and I and to a variety of minor protein components were observed in many sera.     

Immunochemical characterization of the mucoid exopolysaccharide of Pseudomonas aeruginosa

Alginic acid-like mucoid exopolysaccharide was isolated from three strains of Pseudomonas aeruginosa obtained from the sputa of patients with cystic fibrosis. Purified mucoid antigens were greater than 99% uronic acid. With a hemagglutination assay, antibody responses to the mucoid exopolysaccharide were documented after immunization of rabbits with either whole mucoid organisms or purified mucoid exopolysaccharide. The mucoid antigen from one strain (no. 2192) was composed predominantly of a single serologic epitope shared among 40 alginate exopolysaccharides from different clinical isolates. The mucoid exopolysaccharide from the other two strains (nos. 1 and 258) had a serotype-specific determinant in addition to the common epitope. Analyses of antibody in sera from normal adults, children, and patients with cystic fibrosis culture-positive and culture-negative for mucoid P. aeruginosa showed a highly significant (P less than 0.001) association between increased hemagglutination titers and positive cultures for mucoid P. aeruginosa.     

Pseudomonas aeruginosa cross-infection among patients with cystic fibrosis during a winter camp

Twenty-seven patients with cystic fibrosis from our Danish Cystic Fibrosis Center went to a winter camp for 1 week in November of 1990. This study is based on 22 of these patients. Prior to attending camp, 17 out of 22 patients harbored Pseudomonas aeruginosa in their sputum, but 5 patients did not. After returning from camp, all 22 patients harbored P. aeruginosa in the sputum, including the 5 patients whose sputum was free of P. aeruginosa before they went. Epidemiological typing used pulsed-field gel electrophoresis of the P. aeruginosa isolates was performed. The typing results showed that the 5 cystic fibrosis patients who were free of P. aeruginosa in their sputum prior to the winter camp had acquired P. aeruginosa isolates identical to the P. aeruginosa strains isolated from the other 17 cystic fibrosis patients. This constitutes a cross-colonization rate of 100%, the highest rate ever detected among patients with cystic fibrosis. We conclude that separate holiday camps based on the infection status of the patients with cystic fibrosis are necessary to avoid cross-infection of patients not infected with P. aeruginosa.     

Concentration of tobramycin given by aerosol in the fluid obtained by bronchoalveolar lavage

Whereas previous studies have used only bronchial secretions and sputum, in the present study, bronchoalveolar (BAL) fluid was analysed for tobramycin levels after aerosolization of this antibiotic. In 20 adult patients with a variety of lung disorders, the concentration of tobramycin obtained in the first aliquot of the bronchoalveolar fluid varied from less than 0.1 to 9.2 micrograms ml-1 (mean 2 +/- 2.26 micrograms ml-1) with 18 samples above 0.4 micrograms ml-1. In most of the cases, the concentration of tobramycin achieved values of tobramycin in excess of the minimal inhibitory concentration for most of the microorganisms. Thus, sampling fluids by the bronchoalveolar technique offers a suitable method to study antibiotic levels at the site of broncho-pulmonary infection. These results may help explain why aerosol antibiotic treatment appears to be useful in selected patients, especially in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa.     

Proteolytic inactivation of alpha 1-proteinase inhibitor in infected bronchial secretions from patients with cystic fibrosis

The chronic, progressively destructive bronchitis of patients with cystic fibrosis (CF) is characterized by an important imbalance between tissue destroying granulocyte proteases such as granulocyte elastase (GE) and its physiological inhibitors in bronchial secretions. Recent in vitro studies suggest, that proteases derived from bacteria or endogenous proteases may contribute to inactivation of physiological inhibitors of GE. Since only trypsin-unreactive alpha 1-proteinase inhibitor (alpha 1-PI) was detected in CF bronchial secretions, we attempted to identify the mechanism of inactivation of alpha 1-PI. We found a heat stable, serine protease-like enzymatic activity capable of degrading 125I-labelled alpha 1-PI extensively in 22 infected but not in one non-infected CF bronchial secretion. In infected secretions, only degraded alpha 1-PI, which did not migrate like oxidized alpha 1-PI in tandem-crossed immunoelectrophoresis, was detectable. We conclude, that free GE in excess as well as GE bound to bronchial mucosal inhibitor may partly account for the alpha 1-PI-cleaving activity, but that other yet unknown bacterial or host serine proteases also contribute to alpha 1-PI inactivation.     

Immunoglobulin-G subclasses in cystic fibrosis. IgG2 response to Pseudomonas aeruginosa lipopolysaccharide

Pulmonary macrophage phagocytosis of Pseudomonas aeruginosa is defective when this pathogen is opsonized with IgG antibodies isolated from serum samples from patients with cystic fibrosis (CF). To evaluate this defect further, IgG subclasses in the serum and lung fluids of patients with CF were quantitated. The pattern of IgG subclasses in serum specimens from patients with CF (n = 15) and in patients without CF but with chronic obstructive airway disease and recurrent P. aeruginosa infection (n = 4) was significantly altered from that found in normal subjects (n = 31). Immunoglobulin-G2 and IgG3 expressed as percentages of total IgG subclasses or in micrograms per milliliter of serum were significantly elevated in the serum specimens of these patients (p less than 0.05), and IgG1 was significantly decreased (p less than 0.01). It appears that the increase in IgG2 in the serum of patients with CF and those without CF but with chronic P. aeruginosa infection may be in response to chronic antigenic stimulation by P. aeruginosa lipopolysaccharide. Evidence presented to support this includes: (1) IgG2 is not increased in CF serum if a history of P. aeruginosa infection is absent, (2) IgG2 levels expressed as percentages of total IgG subclasses in CF lung fluids were positively correlated (r = 0.73) with the number of colony-forming units of P. aeruginosa present in CF sputum specimens, and (3) IgG antibodies specifically eluted from P. aeruginosa lipopolysaccharide ligands on affinity gels were largely restricted to IgG2. The opsonic index, ([IgG3] + [IgG1]) divided by ([IgG2] + [IgG4]), is inverted in CF lung fluids (0.73:1; normal, 2:1). Because pulmonary macrophages show surface receptors binding primarily with IgG3 and IgG1, it may be that such an alteration in IgG subclasses in the respiratory secretions of patients with CF further inhibits opsonin-mediated clearance of P. aeruginosa.     

Pseudomonas aeruginosa and cystic fibrosis: correlation between exoenzyme production and patient's clinical state

In this study, we investigated the correlation between the production by Pseudomonas aeruginosa isolates of four exoenzymes (protease, elastase, neuraminidase, and phospholipase C (PLC)) and the clinical state of cystic fibrosis (CF) patients. We studied 212 P. aeruginosa isolates from 22 CF patients chronically infected with this bacterium. Patients were classified into three clinical groups according to a modified Shwachman-Kulczycki-Khaw (SKK) scoring system. The production of enzymes by isolates from patients in the three populations was analyzed and compared using four statistical tests: chi-square, Mann-Whitney U, principal component analysis, and discriminant analysis. Isolates from patients with excellent or good clinical status (group I, SKK score >/=71) had higher elastase and neuraminidase activities than isolates from the other patients. In contrast, PLC activity, a common characteristic of CF isolates, was higher in isolates from patients with poor or weak clinical status (group III, SKK score 相似文献   

Antibodies to proteases and exotoxin A of Pseudomonas aeruginosa in patients with cystic fibrosis: Demonstration by radioimmunoassay.

Sera from 33 patients with cystic fibrosis and two pediatric patients being treated for chronic pulmonary infections not related to cystic fibrosis and six sera or serum pools from uninfected individuals were tested with a microtiter radioimmunoassay for reactivity against exotoxin A and two proteases from Pseudomonas aeruginosa. Exotoxin A was purified from a low-protease strain of P. aeruginosa and shown to have adenosine diphosphate-ribose transferase activity and mouse lethality. Proteases were purified from an isolate of P. aeruginosa from a patient with cystic fibrosis and had proteolytic activity against elastin and collagen in an assay employing dimethylated protein substrates. The antibody responses of the patients detected using 125I-labeled antibody to human immunoglobulin were correlated with clinical evaluations expressed as a composite score based on pulmonary findings, case histories, growth and nutrition, and chest X rays. Values in the radioimmunoassay for patients' sera were compared with those of a control serum pool and expressed as the ratio of counts per minute (cpm) in patient serum to the cpm in the control pool. Inverse correlations were found between these ratios for each of the pseudomonas exoproducts and clinical scores; highest ratios occurred in patients showing the lowest clinical scores. These results confirm that proteases and exotoxin A of P. aeruginosa are produced in cystic fibrosis pulmonary infections due to P. aeruginosa and suggest that they may serve as significant virulence factors in these chronic infectious states.     

Affinity constants of naturally acquired and vaccine-induced anti-Pseudomonas aeruginosa antibodies in healthy adults and cystic fibrosis patients.

Naturally acquired anti-Pseudomonas aeruginosa antibody fails to afford protection against repeated P. aeruginosa bronchopulmonary exacerbations in cystic fibrosis (CF) patients. In an effort to explain this phenomenon, the titer and affinity constants of serum anti-lipopolysaccharide (LPS) IgG were determined in five study groups: healthy adults before and after immunization with a polyvalent LPS-based vaccine, healthy noncolonized CF patients before and after immunization, nonimmunized CF patients with significantly elevated anti-LPS antibody titers without documented colonization, recently colonized CF patients before and after immunization, and nonimmunized CF patients chronically colonized with P. aeruginosa. Immunization elicited a significant rise in total anti-LPS immunoglobulin levels and affinity constants in both healthy adults and CF patients. Although chronically colonized patients had elevated levels of total anti-LPS antibody, these antibodies possessed affinities at least 100-fold less than those of vaccine-induced antibodies.     

Analysis of proteolytic enzymes and their natural inhibitors in serum and bronchial lavage fluid in atopic bronchial asthma, chronic bronchitis and pneumonia

The protease-antiprotease balance was evaluated in some respiratory tract diseases. Analysis of the protease activity and the natural inhibitors of proteolytic enzymes was carried out on 10 patients with atopic bronchial asthma, 21 with chronic bronchitis, 12 with pneumonia and 11 health volunteers. Paralelly, the inflammatory potency of bronchial lavage fluid was determined in guinea-pig skin test. In our studies of bronchial lavage fluids some selective changes of proteolytic enzyme activities were documented as follows: in pneumonia patients--the increase of acid and neutral proteases activity; in chronic bronchitis--the moderate increase of activities of all examined proteases and in atopic bronchial asthma--the increase of acid protease activity. Similarly, levels of the natural inhibitors of proteolytic enzymes were selectively elevated: alpha-1-antitrypsin in pneumonia and chronic bronchitis patients and alpha-2-macroglobulin in asthmatics. This finding may suggest that the proteolytic enzymes and their inhibitors play an important role in the respiratory tract pathology. This concept is supported by the high inflammatory response observed with bronchial lavage fluids obtained from pneumonia patients in guinea-pig skin test. The authors suggest that the determination of protease activity and level of the natural inhibitors of proteolytic enzymes in bronchial lavage fluids may be useful for clinical prognosis and pharmacological treatment.     

Effects of postural drainage, incorporating the forced expiration technique, on pulmonary function in cystic fibrosis

Detailed pulmonary function tests were performed on 12 patients with cystic fibrosis (CF) before and after 3 days treatment with postural drainage incorporating the forced expiration technique. The results following treatment showed a statistically significant improvement in FEV1 (P less than 0.001), FVC (P less than 0.001), PEFR(P less than 0.001), PIFR (P less than 0.001), and VEmax50 (P less than 0.025). The study demonstrates objective benefit from this form of physiotherapy in cystic fibrosis patients with copious bronchial secretions.     

Evidence that Pseudomonas aeruginosa elastase does not inactivate the bronchial inhibitor in the presence of leukocyte elastase. Studies with cystic fibrosis sputum and with pure proteins

Pseudomonas aeruginosa elastase has recently been shown to inactivate bronchial inhibitor, the major leukoproteinase inhibitor of the bronchial tree. To test if this in vitro finding is relevant to pathology, we have measured the leukocyte elastase inhibitory capacity and the immunoreactive levels of bronchial inhibitor in the acidified and neutralized sputum specimens from 15 patients with cystic fibrosis, 11 of whom were infected by P. aeruginosa and 10 of whom exhibited free bacterial elastase activity. The percentage of functionally active bronchial inhibitor in these acid-treated sputum specimens (i.e., concentration of active inhibitor/concentration of immunoreactive inhibitor X 100) was found to be 125 +/- 17%. It was positively correlated with the free leukocyte elastase concentration (r = 0.77, p less than 0.001) but not with the free bacterial elastase concentration (r = 0.46, p greater than 0.05). Therefore, in an in vivo situation, P. aeruginosa elastase does not necessarily inactivate the bronchial inhibitor. Experiments using pure proteins show that the inactivation process does not take place when leukocyte and P. aeruginosa elastase are added simultaneously to the inhibitor. This suggests that the bronchial inhibitor reacts preferentially with leukocyte elastase and that in its complexed form it is shielded from the inactivating action of P. aeruginosa elastase. In addition, the inhibitor recovered from the acid-induced dissociation of the leukocyte elastase-inhibitor complexes, exhibits a substantially higher specific activity than does the native molecule. This explains why the average functional activity of the bronchial inhibitor is significantly higher than 100% in sputum samples from patients with cystic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of gentamicin resistance in nosocomial infections

Characteristics of gentamicin resistance were studied in gram-negative bacilli from 50 consecutive patients with nosocomial infection, during a time when gentamicin resistance had recently become prevalent at Medical University Hospital. Burns, decubitus ulcers, and cystic fibrosis were common precipitating factors for acquisition of gentamicin-resistant organisms. Pseudomonas aeruginosa accounted for 76% and Enterobacteriaceae for 24% of isolates. There was high prevalence of cross-resistance to amikacin (61%) and tobramycin (58%). Of the P aeruginosa strains 36% possessed plasmids which were rapidly detected by agarose gel electrophoresis. None of the isolates transferred gentamicin resistance. Representative isolates failed to elaborate aminoglycoside-modifying enzymes or to take up labelled amikacin. Multiple immunotypes of P aeruginosa were identified. These data suggest that a nonplasmid mediated resistance mechanism such as impermeability was responsible for the emergence of gentamicin resistance.