Pseudomonas aeruginosa flagellar antibodies in patients with cystic fibrosis.

An enzyme-linked immunosorbent assay specific for flagellum type (a or b) of Pseudomonas aeruginosa was used to detect serum immunoglobulin antibodies in 98 random outpatients and 14 colonized cystic fibrosis patients. Antibodies were detected to both types of flagella in addition to M-2 lipopolysaccharide. Titers to both flagellar antigens (FlAg) were 10 to 100 times higher in cystic fibrosis patients than in random outpatients of a comparable age group. Mean antibody titers against b-type FlAg were 454 for outpatients (ages newborn to 21 years), whereas the mean titer for cystic fibrosis patients (ages 6 to 21 years) was 51,520. Titers against a-type FlAg were generally lower, with mean outpatient titers of 68 and mean cystic fibrosis patient titers of 34,323. Differences were also seen in antibody titer against M-2 lipopolysaccharide, but these differences did not correspond to M-2 FlAg titers. In 98 random outpatients (ages newborn to 86 years), FlAg titers generally increased with age. To demonstrate further specificity of the enzyme-linked immunosorbent assay for flagellum antibody, Western blots were performed with selected high-titer cystic fibrosis patient sera. Sera that had a high titer (greater than 25,600) for b- or a-type FlAg showed a corresponding reactive band. These results demonstrate that flagellum antibodies are produced in humans in response to P. aeruginosa infection.     

Pseudomonas aeruginosa colonization in patients with spinal cord injuries.

The prevalence of Pseudomonas aeruginosa colonization of patients with spinal cord injury was studied annually from 1976 to 1980. The urethra, perineum, rectum, drainage bag, and urine of patients on the spinal cord injury service were cultured. A total of 224 men and 32 women were studied. Most patients were managed with an external urinary collection system or padding, with or without intermittent catheterization. P. aeruginosa was cultured from one or more body sites (urethra, perineum, or rectum) in 65% of men and 18% of women. Drainage bags on the beds were frequently colonized with P. aeruginosa (73%). Significant bacteriuria with P. aeruginosa was present in 19% of the men and 13% of the women. P. aeruginosa colonization of body sites in men was closely associated with the use of an external urinary collection system. Significantly greater urethral and perineal colonization was found in men using an external urinary collection system. P. aeruginosa serotype 11 was the predominant serotype for the first 3 years, and the number of patients colonized with serotype 11 increased with length of hospital stay. The prevalence of serotype 11 significantly decreased in the last 2 years. The antibiotic susceptibility of the strains of P. aeruginosa isolated from these patients did not change in the 5 years, except that there was increasing susceptibility to carbenicillin in later years. This increasing susceptibility to carbenicillin was a reflection of a decreased prevalence of serotype 11 in these patients, since serotype 11 was more resistant than other serotypes to carbenicillin.     

Immunoglobulin A and immunoglobulin G antibody responses to alginates from Pseudomonas aeruginosa in patients with cystic fibrosis.

Patients with cystic fibrosis have a high prevalence of mucoid, alginate-producing Pseudomonas aeruginosa that causes chronic infection of the mucosal surface of the lungs. We developed enzyme-linked immunosorbent assays (ELISAs) for determination in serum of immunoglobulin A (IgA) and IgG antibodies to alginate purified from P. aeruginosa and an ELISA for detection of IgA antibodies to a polyvalent P. aeruginosa standard antigen. Absorption experiments indicated that the assays were antigen and antibody specific and had analytical variations that ranged from 7 to 19%. Serum samples from 207 patients with cystic fibrosis, 100 healthy children, and 94 healthy adults were examined. The patients responded to P. aeruginosa infection with early IgA and IgG antibody responses that were significantly higher than in controls and noncolonized patients. Analysis of paired serum samples showed that infected patients had an increase in specific IgG and IgA antibodies that was significantly higher than in noncolonized patients. The serological data were analyzed for correlation with clinical condition; poor lung function was significantly associated with increased levels of IgA and IgG antibodies to P. aeruginosa alginate and to the standard antigen and with a relative excess of IgA antibodies to the standard antigen compared with IgA antibodies to P. aeruginosa alginate. The assays showed high predictive values if positive, but a negative test did not exclude infection, and the ELISAs should not be used for diagnostic purposes. Mucoid strains were present initially in the sputa of 28 of 54 infected patients with paired serum samples. These patients had a significant increase in anti-alginate antibodies, but it was not different from the increase seen in patients infected only with nonmucoid strains. Therefore, alginate may also be produced in vivo by nonmucoid P. aeruginosa. The study showed that early formation of IgA and IgG antibodies to P. aeruginosa alginate did not prevent development of chronic infection and that P. aeruginosa-specific IgA antibodies correlate with poor lung function.     

Opsonophagocytic killing antibody to Pseudomonas aeruginosa mucoid exopolysaccharide in older noncolonized patients with cystic fibrosis

The principal cause of morbidity and mortality in cystic fibrosis is persistent respiratory colonization with mucoid strains of Pseudomonas aeruginosa. To investigate possible mechanisms of resistance to this organism, we studied serum from 16 older (greater than or equal to 12 years) patients not colonized with mucoid P. aeruginosa, 11 older (greater than or equal to 14 years) colonized patients, 10 younger (less than or equal to 11 years) noncolonized patients, and 20 healthy adults. The samples from the older patients not colonized with mucoid P. aeruginosa contained antibody specific to the mucoid-exopolysaccharide antigen, which could mediate bacterial killing in conjunction with complement and white cells (titers of 4 to 80). These opsonophagocytic killing antibodies were not detected in samples from the 20 normal controls (P less than 0.0001 vs. noncolonized older patients) or 9 of 10 younger (less than or equal to 11 years) noncolonized patients (P = 0.0072 vs. noncolonized older patients). Although the patients with chronic colonization had higher titers of serum opsonophagocytic killing antibody than did the older noncolonized patients (P = 0.0005), these antibodies were not specific to the mucoid-exopolysaccharide antigen. We conclude that there is an association between mucoid-exopolysaccharide-specific opsonophagocytic killing antibody and a lack of detectable P. aeruginosa colonization in a subset of older, relatively healthy patients with cystic fibrosis.     

Bactericidal antibody response to Pseudomonas aeruginosa by adults with urinary tract infections.

In this investigation we found that adults with upper urinary tract infections caused by Pseudomonas aeruginosa produced serum antibodies with bactericidal activity against the bacterium. Seventeen of 20 infected adults showed bactericidal activity with a titer range of 1:10 to 1:10,000.     

Experimental immunization with Pseudomonas aeruginosa alginate induces IgA and IgG antibody responses.

We tried experimentally to induce a specific antibody response against Pseudomonas aeruginosa locally in the airways and systemically in rats by three different routes of immunization; intragastric feeding, intratracheal inoculation or subcutaneous vaccination. Three groups of rats were immunized with live mucoid P. aeruginosa PAO 579 by intragastric feeding or with killed PAO 579 intratracheally or subcutaneously. Three other groups were immunized with purified P. aeruginosa alginate either by intragastric feeding, intratracheally or subcutaneously. At weekly intervals for four weeks animals were sacrificed and serum and bronchial fluid were obtained. The specific IgA and IgG antibody response in lavage fluid and serum was measured. Only traces of antibodies could be detected in the bronchial lavage fluids. Anti-alginate IgA and IgG antibodies developed in all rats immunized with alginate but no antibodies against other P. aeruginosa antigens were detected. The highest IgA and IgG titer against alginate was induced by the subcutaneous immunization. IgA and IgG antibodies against other P. aeruginosa antigens developed in rats immunized with liver and sonicated bacteria. The highest IgA and IgG titers were obtained after intratracheal and subcutaneous immunization with sonicated bacteria. The present work has shown that IgA and IgG antibodies develop with high specificity after immunization. The different titers obtained do not necessarily reflect different degrees of protection.     

对几种含绿脓杆菌脂多糖的物质及其抗感染保护作用的初步观察

对绿脓杆菌感染防御性抗原的研究,特别是对绿脓杆菌表面抗原的研究,一直受到研究者的重视。近些年在临床应用上也有不少进展。在美国,Alexander 等研制的绿脓杆菌表面物质7价疫苗于1968年开始试用于临床,使烧伤患者因绿脓杆菌感染的死亡率下降。英国的 Jones 等研制出绿脓杆菌表面抗原多价疫苗,1976年完成系统的实验     

Polyclonal and monoclonal antibody therapy for experimental Pseudomonas aeruginosa pneumonia.

A human immunoglobulin G preparation, enriched in antibodies to lipopolysaccharide (LPS) Pseudomonas aeruginosa antigens (PA-IGIV) and murine monoclonal antibodies (MAb) to P. aeruginosa Fisher immunotype-1 (IT-1) LPS antigen and outer membrane protein F (porin), were evaluated for therapeutic efficacy in a guinea pig model of P. aeruginosa pneumonia. The concentration of antibodies to IT-1 LPS was 7.6 micrograms/ml in PA-IGIV and 478 micrograms/ml in the IT-1 MAb preparation. No antibody to IT-1 was detected in MAb to porin. For study, animals were infected by intratracheal instillation of IT-1 P. aeruginosa and then treated 2 h later with intravenous infusions of PA-IGIV, IT-1 MAb, or porin MAb. Control groups received intravenous albumin, and routinely died from pneumonia. Both PA-IGIV (500 mg/kg) and IT-1 MAb (greater than or equal to 2.5 mg/kg) treatment resulted in increased survival (P less than 0.01 to 0.001), and also improved intrapulmonary killing of bacteria. Porin MAb failed to protect from fatal pneumonia. IT-1 MAb treatment produced more survivals than did PA-IGIV treatment but only at dosages of MAb resulting in serum antibody concentrations greater than those achieved with PA-IGIV. PA-IGIV and IT-1 MAb demonstrated in vitro and in vivo (posttreatment guinea pig serum) opsonophagocytic activity for the IT-1 challenge strain. However, the polyclonal preparation required complement, whereas the MAb did not. We conclude that passive immunization with polyclonal hyperimmune P. aeruginosa globulin or with MAb to LPS antigens may be useful in the treatment of acute P. aeruginosa pneumonia. The relative efficacies of such preparations may be limited, however, by their type-specific LPS antibody concentrations.     

Experimental Pseudomonas aeruginosa Infection of the Mouse Cornea

Pseudomonas aeruginosa infection of human cornea is rare but serious. The work of previous investigators using experimental infection primarily of rabbit cornea resulted in successful therapy for 10 to 50% of clinical cases. The advantage of using the mouse is demonstrated. The methods we adapted for characterizing the untreated experimental infection included: incising the cornea to enable establishing the infection; corneal examination with a steroscopic microscope; grading corneal pathology; qualitative and quantitative monitoring of the infecting bacteria by culturing and staining sectioned and dissected tissues. The characteristics of the tissue pathology, host response, and infection were similar to those reported for other animals and man. Corneal pathology was frequently nearly maximal 1 day after infection; host response involved a progression of events of long duration; pathology persisted well beyond the period of bacterial infection. The infection was essentially noncommunicable, and invasiveness was limited to the tissues of the incised eye. The results show the possibility of tests for invasiveness of clinical isolates and for screening for therapeutic and prophylactic measures.     

Serum antibody response to Pseudomonas aeruginosa antigens during corneal infection.

Previous studies in our laboratory have indicated that naturally resistant, inbred DBA/2J mice mount a greater serum antibody response to Pseudomonas aeruginosa 19660 than susceptible C57BL/6J mice. However, the specificity of the antibody produced was not known. The present study examines the specificity and kinetics of the humoral response of these mouse strains to potential virulence factors produced by the organism during both a primary and a secondary corneal infection administered 4 weeks after the primary infection. Serum antibody levels specific for lipopolysaccharide (LPS), exotoxin A, phospholipase C (PLC), alkaline protease, elastase, and flagella were measured by enzyme-linked immunosorbent assay. Little or no antibody to either alkaline protease or elastase was detected during either primary or secondary infection. Immunoglobulin G (IgG) antibodies specific to exotoxin A, PLC, and flagella were detected 2 weeks after primary infection, and a rapid response to these antigens was measured 1 week after secondary infection. During primary infection, detectable LPS-specific antibody was only IgM, while IgG appeared only after secondary infection. The kinetics of the humoral response in susceptible C57BL/6J mice were similar to those in resistant DBA/2J mice, although the magnitude of the response varied according to the antigen tested. These results indicate that LPS, exotoxin A, PLC, and flagella are present or produced in amounts that are immunogenic during corneal infection by P. aeruginosa 19660 in the mouse strains tested.     

Characterization of antibody to the ferripyochelin-binding protein of Pseudomonas aeruginosa.

An outer membrane protein of Pseudomonas aeruginosa was previously shown to bind 59Fe-labeled pyochelin. Antibodies to the purified ferripyochelin-binding protein (FBP) were characterized by using a variety of assays. Anti-FBP cross-reacted with several P. aeruginosa isolates in an enzyme-linked immunosorbent assay. Anti-FBP significantly enhanced phagocytosis of P. aeruginosa by human polymorphonuclear leukocytes. In a serum bactericidal assay we observed no difference in viability between cells incubated with antiserum to FBP and cells incubated with preimmune serum. Anti-FBP immunoglobulin G inhibited both binding and uptake of 59Fe-labeled pyochelin by whole cells. Passive protection by anti-FBP was examined in experimental P. aeruginosa burn infections in mice. The protection provided by this antibody was strain dependent but lipopolysaccharide serotype independent.     

A monoclonal antibody specific for Pseudomonas aeruginosa amidase

Amidase from Pseudomonas aeruginosa was purified by anionic exchange chromatography and used to immunise female Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells as fusion partner. A selected IgM subclass MAb was purified from in vitro hybridoma cell line supernatant by a two-step anionic exchange chromatography. The MAb was specific for amidase from P. aeruginosa as determined by Western blotting and recognized the native and denatured forms of the enzyme.     

Fecal isolation of Pseudomonas aeruginosa from patients with cystic fibrosis.

Fecal isolation of Pseudomonas aeruginosa was observed in 8 of 10 patients with cystic fibrosis who at the time of sampling also exhibited colonization of the respiratory tract. In contrast, P. aeruginosa cells were isolated at low frequency (9.1%) from the stools of 44 patients with cystic fibrosis with no previous history of chronic colonization. The results of this study suggest that the gastrointestinal tract is not a significant chronic reservoir of P. aeruginosa prior to pulmonary colonization.     

Ecology of Pseudomonas aeruginosa in patients with cystic fibrosis

The occurrence of various Pseudomonas aeruginosa strains in the sputum of 15 patients with cystic fibrosis (CF) was monitored over periods ranging from 2 to 60 months. Isolates of P. aeruginosa were typed by four different techniques, namely serotyping, active and passive pyocin typing, and phage typing. The maximum number of different serotypes found in the patients was three (one serotype in nine patients; two serotypes in five patients; three serotypes in one patient). Pyocin and phage typing showed no marked differences between strains of the same serotype in individual patients. Exacerbations of chronic respiratory infection were not associated with changes in the sputum flora, the composition of P. aeruginosa strains in which remains constant over long periods in patients with CF.     

Virulence and the Role of Iron in Pseudomonas aeruginosa Infection

The virulence of Pseudomonas aeruginosa can be enhanced by passage in mice or rabbits. Enhanced virulence has some specificity for the host in which the passage is done. Experimental infection in the peritoneal cavity of cannulated rabbits has shown that the injection of iron compounds can lead to a rapid and fatal growth of an otherwise nonlethal dose of bacteria. In vitro the unsaturated iron-binding proteins present in the peritoneal fluid can halve the growth rate of P. aeruginosa. The restricted rate of growth is restored to normal if the iron-binding proteins are saturated with iron. Exactly the same results are achieved with purified transferrin. Both fatal and nonfatal infections with P. aeruginosa cause a sharp fall in the percentage of saturation with Fe of the plasma and peritoneal fluid. In both normal and infected animals the peritoneal fluid is invariably less saturated than the plasma. Specific antiserum not only protects against death but also against the fall in iron saturation of the plasma and peritoneal fluid. In both fatal and nonfatal infections a high proportion of viable bacteria are unphagocytized in the peritoneal cavity.     

Role of Flagella in Pathogenesis of Pseudomonas aeruginosa Pulmonary Infection

Pseudomonas aeruginosa strains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens, P. aeruginosa cells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence of fliC mutants in a neonatal mouse model of pneumonia. In the absence of fliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to a pilA mutant PAK/NP (P < 0.0001). The fliC mutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that the fliC mutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung. ¹²⁵I-labeled flagellin bound to the glycolipids GM1 and GD_1a and to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type or fliC mutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla⁺ organisms but not fliC mutants were efficiently taken up by murine macrophages. P. aeruginosa flagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.

Flagella are highly complex bacterial organelles which are unusually well conserved among diverse bacterial species. Over 50 genes are involved in the synthesis and function of flagella, suggesting that their preservation and role in chemotaxis and motility are important in the survival of many organisms (24). Flagella facilitate the acquisition of essential nutrients; thus, it seems likely that these organelles have a role in the virulence of pathogenic organisms. However, flagella are known to be highly immunogenic (16), and in certain settings Fla⁺ bacteria may be more readily cleared than Fla⁻ organisms (1).The contribution of flagella to the virulence of the opportunistic pathogen Pseudomonas aeruginosa has been examined in several animal models of infection (11, 19). Fla⁻ mutants were less invasive than motile strains in a mouse burn infection, and the administration of antiflagellum antibody had a significant protective effect (8). In a model of mucosal colonization, globally defective RpoN mutants (Pil⁻ Fla⁻) of P. aeruginosa were more deficient in their ability to colonize than to persist within the murine gastrointestinal tract (21). A rat model of Pseudomonas pneumonia was used to demonstrate the efficacy of human antiflagellum monoclonal antibody in attenuating pulmonary infection and reducing the spread of the organism within the rat lung (13). In these instrumented animals, large inocula of bacteria were instilled directly into the trachea. These studies do not directly address how flagella function in either the initial colonization of the upper respiratory tract or the subsequent infection of the lung by aspiration. Several flagellar genes, although not the flagellin structural gene fliC, have been shown to contribute to binding to respiratory mucin (2, 25). Attachment to mucin facilitates clearance from the respiratory tract via the normal mucociliary escalator. Flagella can function as ligands for macrophages and polymorphonuclear leukocytes (PMNs) which clear organisms from mucosal surfaces. In vivo selection of Fla⁻ mutants of P. aeruginosa has been demonstrated in human pulmonary infection in cystic fibrosis (CF) (14, 15). Although the first of sequential Pseudomonas isolates from a patient were motile and piliated, such as environmental strains of P. aeruginosa, genotypically identical isolates over the course of a chronic infection had an RpoN-like phenotype and failed to express functional flagella (15). There may be significant selective pressure for the emergence of Fla⁻ mutants which are less efficiently cleared by phagocytic cells (16, 29). Some respiratory pathogens such as Bordetella pertussis are nonmotile, and recent studies suggest that a bovine pathogen, Bordetella bronchoseptica, which can be motile, specifically turns off flagellar expression during the initial stages of infection (1).Since P. aeruginosa flagella are antigenically relatively homogeneous, falling into two large groups based on structural analysis and reactivity to antisera (28), they may provide a useful target for vaccine development. The ability of antiflagellum antibody to ameliorate infection in the rat lung suggests that flagella may provide a useful target for immune intervention and may be most effective as a strategy to block the initial infection of the respiratory tract by motile strains. However, the importance of normal flagellar function in the earliest stages of P. aeruginosa respiratory infection has not been established for genetically characterized strains. The purpose of this study was to examine (i) the virulence of wild-type and fliC mutant strains in the development of murine pneumonia and (ii) participation of flagella in stimulation of the subsequent immune response to the organism.     

Multifunctional Role of Human SPLUNC1 in Pseudomonas aeruginosa Infection

The human short PLUNC1 (SPLUNC1) protein has been identified as a component of the pulmonary antimicrobial response based on its structural similarity to the bactericidal/permeability-increasing (BPI) protein. Using a genetically modified mouse model, we recently verified the antimicrobial activity of SPLUNC1 against Pseudomonas aeruginosain vivo. To further define the mechanism of epithelial SPLUNC1-mediated antibacterial action, we carried out studies to determine how SPLUNC1 protects the host from acute respiratory infections. P. aeruginosa treated with recombinant human SPLUNC1 protein showed decreased growth in vitro. This antibacterial activity was due to growth inhibition, as a consequence of a SPLUNC1-induced increase in bacterial cell permeability. Removal of SPLUNC1 allowed the recovery of P. aeruginosa and suggested no permanent cell injury or direct killing of bacteria. Further investigation showed coating of bacterial cells by SPLUNC1. We suggest that this “bacterial cell coating” is necessary for the bacteriostatic function of SPLUNC1. Additionally, we demonstrated a novel role for SPLUNC1 as a chemoattractant that facilitated migration of macrophages and neutrophils. Taking the findings together, we propose synergistic roles for human SPLUNC1 as an antibacterial agent with bacteriostatic and chemotactic activities.     

Immunoglobulin allotypes and IgG subclass antibody response to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients.

Chronic Pseudomonas aeruginosa lung infection is the leading cause of death in patients with cystic fibrosis (CF). Poor prognosis correlates with a high number of anti-pseudomonas precipitins and with high levels of IgG2 and IgG3 anti-pseudomonas antibodies. Reports of several highly significant associations between certain Gm (genetic markers of IgG on human chromosome 14) and Km (k-type light chain determinants on chromosome 2) phenotypes and immune responsiveness to various antigens suggest that allotype-linked immune response genes do exist in man. Furthermore correlation between Gm types and IgG subclass levels has been reported. A group of 143 CF patients were investigated (31 non-infected and 112 chronic infected). The IgG subclass antibodies to three different P. aeruginosa antigens (P. aeruginosa standard antigen (St-Ag), alginate and LPS) were determined. Immunoglobulin allotypes were determined by haemagglutination inhibition. Samples were typed for G1m(1,2,3, and 17), G2m(23), G3m(5,21), and Km(1,3). Statistical analysis of our data demonstrate that IgG3 anti-pseudomonas antibody levels and Gm markers are related. IgG3 antibody levels to all investigated P. aeruginosa antigens are significantly higher in sera homozygous for Gm(3;5), somewhat lower in heterozygous sera, and significantly lower in sera homozygous for Gm(1,2,17;21). We suggest that genetic differences between the patients may explain the present differences in subclass patterns.