Anti-Pseudomonas aeruginosa IgG subclass titers in patients with cystic fibrosis: Correlations with pulmonary function,neutrophil chemotaxis,and phagocytosis

To explore possible mechanisms for the association between elevated immunoglobulin levels and lower pulmonary function in cystic fibrosis patients, we measured serum IgG subclass levels and anti-P. aeruginosa IgG subclass titers and correlated levels with neutrophil phagocytosis and chemotaxis. Serum was obtained from 13 cystic fibrosis patients colonized with the same serotype ofP. aeruginosa, 12 noncolonized patients, and 12 normal volunteers. All anti-P. aeruginosa IgG subclass titers were elevated in serum from colonized patients. IgG₃ level and anti-P. aeruginosa IgG₃ titer were inversely correlated with pulmonary function. Phagocytosis ofP. aeruginosa by neutrophils correlated with serum IgG₃ level and was increased by opsonization with serum from colonized patients. Chemotactic index was increased in serum from colonized patients and inversely correlated with pulmonary function chest roentgenogram score. Chemotactic index directly correlated with anti-P. aeruginosa IgG₃ titer and serum IgG₃. These data demonstrate that cystic fibrosis patients with increased IgG₃ levels are in poorer clinical condition and that their serum enhances neutrophil function. Such patients may have increased pulmonary inflammation with subsequent lung damage.     

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.     

Detection of antibodies to Pseudomonas aeruginosa alginate extracellular polysaccharide in animals and cystic fibrosis patients by enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed to detect antibodies to sodium alginate exopolysaccharide purified from three strains of Pseudomonas aeruginosa and commercial alginate from seaweed. Good attachment of alginate occurred with polystyrene microtiter plates at pH 7.0 with 0.04 M sodium phosphate buffer. With the enzyme-linked immunosorbent assay procedure, antibodies to alginate (not previously shown to be immunogenic) could be shown in humans and after immunization of mice and rabbits. Antibody to one of the alginates cross-reacted with two other P. aeruginosa alginates and commercial seaweed alginate. In animal immunization antibody titers were maximal after a single intravenous injection with an optimal dose of P. aeruginosa 3064 alginate. Healthy controls not known to have a previous P. aeruginosa infection had low, but detectable, antibody titers to 3064 and commercial alginate. Three cystic fibrosis patients not colonized with P. aeruginosa had similar antibody levels. Twenty-eight cystic fibrosis patients colonized with P. aeruginosa formed a clearly separate group with antibody titers higher than that of the control and noncolonized cystic fibrosis patients. Antibody titers to 3064 or commercial alginate did not increase during acute P. aeruginosa bronchopneumonitis in 16 cystic fibrosis patients or after repeated episodes in 4 patients.     

Role of Pseudomonas aeruginosa exoenzymes in lung infections of patients with cystic fibrosis.

We investigated the role of Pseudomonas aeruginosa exoenzymes in cystic fibrosis lung infection in the presence and absence of specific serum antibodies. In sputa of 21 cystic fibrosis patients, concentrations of P. aeruginosa proteases and exotoxin A were determined by sensitive radioimmunoassays. In all sputa, detection of exoenzymes was negative (less than or equal to 10 ng). Positive serum antibody titers to bacterial exoenzymes were found in the majority of patients. Purified immunoglobulin G (IgG) preparations from the sera of two patients revealing specific antibody titers to the bacterial proteases neutralized these enzymes at ratios of 1,000:1 to 5,600:1 (wt/wt). Above the neutralizing capacity of IgG, proteases caused cleavage of IgG; below that level, no enzymatic activity was observed. In vitro incubation of P. aeruginosa elastase, alkaline protease, or exotoxin A with elastase derived from polymorphonuclear leukocytes showed that polymorphonuclear leukocyte elastase: (i) was cleaved by bacterial elastase, (ii) was not inactivated by alkaline protease, and (iii) inactivated exotoxin A. The results suggest that soon after the onset of P. aeruginosa lung infection in cystic fibrosis patients, bacterial proteases, but not exotoxin A, become important virulence factors. The results also suggest that exoenzymes do not directly contribute to lung damage after immune response to bacterial antigens has begun.     

Prospective study of serum antibodies to Pseudomonas aeruginosa exoproteins in cystic fibrosis.

Serum immunoglobulin G to four purified antigens from Pseudomonas aeruginosa, phospholipase C, alkaline protease, exotoxin A, and elastase, were determined in 62 patients with cystic fibrosis by enzyme-linked immunosorbent assay. The patients were followed for 12 to 24 months in a prospective study. Increased titers, i.e., titers more than 2 standard deviations above those of normal controls, were exclusively found in patients chronically colonized with P. aeruginosa and not in patients harboring only Staphylococcus aureus. The frequencies of elevated titers of antibody to the different antigens varied from 100% (phospholipase C) to 58% (alkaline protease and exotoxin A) to 15% (elastase) in the chronically colonized patients. Mean serum titer levels, expressed as multiples of the age-correlated upper normal limit (=1), were significantly higher to phospholipase C in patients with dual colonization with P. aeruginosa and S. aureus than in those colonized only with P. aeruginosa (P less than 0.001). Conversely, the other three antigens showed significantly higher serum antibody titer levels in patients harboring only P. aeruginosa (P less than 0.001). In five patients who became colonized with P. aeruginosa during the study period, serum antibodies to phospholipase C and exotoxin A increased first. Exceptions to the general pattern of antibody responses were found in three patients chronically colonized with Escherichia coli. They showed a delayed enhancement of anti-phospholipase C titers after the chronic P. aeruginosa colonization. Serum titers were not influenced by exacerbations of pulmonary infection or by antimicrobial therapy. The determination of titers of serum antibody to phospholipase C seems to be a valuable indicator of a chronic colonization with P. aeruginosa. The results further suggest that bacterial metabolism and interactions may influence the antibody response.     

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.     

Pseudomonas aeruginosa: Immune Status in Patients with Cystic Fibrosis

In order to have a better understanding of the clinical significance of Pseudomonas aeruginosa, circulating and secretory antibodies were measured. Of 100 patients diagnosed as having cystic fibrosis (CF) and an atypical mucoid P. aeruginosa cultured from their sputum, each possessed serum precipitins. These immunoprecipitates, however, were not detected in the sera of 40 CF patients, some of whom were chronically ill with pulmonary colonization by typically rough-smooth strains of P. aeruginosa. The sera of 46 CF patients and 27 CF patient parents not colonized by P. aeruginosa were negative for the precipitins. The sera from 15 of 45 chronically ill patients not having CF, however, but harboring P. aeruginosa, also possessed serum precipitins. The sera from 85 subjects not having CF and not clinically infected with P. aeruginosa were negative for precipitins. Serum hemagglutination titers as high as 1:4096 were measured in older CF patients having advanced pulmonary disease and who were infected with mucoid P. aeruginosa. Salivary titers ranged from 1:8 to 1:64. Increased levels of both circulating and secretory antibodies of the immunoglobulin A and G classes were demonstrated in patients with CF. Once a patient with CF becomes colonized with P. aeruginosa a process of conversion from the rough and smooth forms to the mucoid form is almost inevitable. Although the mucoid form predominates in the sputum, intermediates of the various colony types are often present. Serum precipitins were demonstrable only after the appearance of mucoid strains in the sputum of patients with CF. Although antibiotics tend to reduce the number of mucoid microorganisms, they are rarely, if ever, eradicated from these patients'' lungs. Recurrent episodes of servere pulmonary infection and the evidence of increasing antibody formation to mucoid strains indicates the invasiveness of these particular strains.     

Agglutinating antibody titers to members of the family Legionellaceae in cystic fibrosis patients as a result of cross-reacting antibodies to Pseudomonas aeruginosa.

The objective of this study was to evaluate the prevalence and significance of antibody titers to organisms in the family Legionellaceae in 128 serum samples collected from cystic fibrosis patients at routine examinations. Antibody titers were determined for 10 antigenic types of Legionellaceae; Legionella pneumophila serogroups 1 to 6, Fluoribacter (Legionella) bozemanae, Fluoribacter (Legionella) dumoffii, Fluoribacter (Legionella) gormanii, and Tatlockia (Legionella) micdadei. The method of antibody titer determination was the microagglutination test. Elevated titers (greater than or equal to 1:64) to one or more antigens were found in 41.3% of cystic fibrosis patients but in only 9.7% of 103 normal control subjects (P less than 0.01). Titers to 8 of the 10 antigens were directly correlated with the number of Pseudomonas aeruginosa precipitating antibodies in patient sera, as determined by crossed immunoelectrophoresis (correlation coefficients, greater than or equal to 0.74). Cross-reactions between P. aeruginosa and L. pneumophila were substantiated by crossed immunoelectrophoresis of hyperimmune rabbit serum as well as patient sera against P. aeruginosa and Legionellaceae antigens. Monospecific antibody to the "common antigen" of P. aeruginosa was used to demonstrate the presence of this antigen in L. pneumophila. The presence of cross-reacting antibodies in cystic fibrosis patients chronically infected with P. aeruginosa emphasizes the need for cautious interpretation of antibody titers to members of the family Legionellaceae.     

Increased levels of IgG subclasses specific for Pseudomonas aeruginosa exoenzyme and polysaccharide antigens in chronically infected patients with cystic fibrosis

IgG subclass levels to Pseudomonas aeruginosa alginate, alkaline proteinase, elastase and exotoxin A in sera of healthy adults, non-infected and infected cystic fibrosis patients were investigated by enzyme linked immunosorbent assay. Whereas healthy adults and non-infected cystic fibrosis patients revealed mostly negative IgG subclass levels to the four antigens, infected cystic fibrosis patients had significantly elevated IgG1, IgG2, IgG3 and IgG4 levels to both the protein antigens as well as the polysaccharide antigen. The study does not support previous findings of an impaired natural IgG2 response to polysaccharide antigen. The study does not support previous findings of an impaired natural IgG2 response to polysaccharide in chronically infected cystic fibrosis patients.     

Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis.

Although Pseudomonas aeruginosa chronically colonizes most older patients with cystic fibrosis (CF), bacterial features responsible for its persistence are understood poorly. We observed that many P. aeruginosa isolates from chronically colonized patients were nonmotile and resistant to phagocytosis by macrophages. P. aeruginosa isolates were collected from 20 CF patients for up to 10 years. Isolates from early colonization were highly motile and expressed both flagellin and pilin. However, many isolates from chronically colonized patients lacked flagellin expression and were nonmotile; a total of 1,030 P. aeruginosa CF isolates were examined, of which 39% were nonmotile. Moreover, sequential isolates recovered from several of the CF patients were consistently nonmotile for up to 10 years. Lack of motility was rare among environmental isolates (1.4%) and other clinical isolates (3.7%) of P. aeruginosa examined. Partial complementation of motility in nonmotile P. aeruginosa isolates was achieved by introduction of extra copies of the rpoN locus carried on plasmid pPT212, indicating that the alternate sigma factor, RpoN, may be involved in the coordinate regulation of virulence factors during CF infection. We hypothesize that the nonmotile phenotype may provide P. aeruginosa a survival advantage in chronic CF infection by enabling it to resist phagocytosis and conserve energy.     

Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay.

We analyzed sera from 28 patients with various types of malignancies for the occurrence of antibodies against exotoxin A of Pseudomonas aeruginosa and two Pseudomonas proteases. A total of 27 of these individuals were colonized or infected with P. aeruginosa at one time or another during the study, whereas the remaining patient was colonized with four non-P. aeruginosa species of Pseudomonas. Sera were obtained from several of these patients before P. aeruginosa colonization or infection of these individuals was detected, which provided an opportunity to evaluate their responsiveness to pseudomonal exoproducts as they acquired the organism. Exotoxin A was purified from culture supernatant fluids of strain PA-103, and the two proteases were purified from an isolate of strain JR3, a highly proteolytic strain originally recovered from the sputum of a cystic fibrosis patient. Antibodies to the exotoxin A and the two proteases were detected in these sera, and sera which contained relatively high antibody levels to exotoxin A afforded mice complete protection against lethal challenges with this substance. Statistical analyses showed that patients infected with P. aeruginosa had consistently higher antibody levels (P less than 0.005) to the exoproducts than patients who were colonized with this organism. Also, patients colonized with P. aeruginosa possessed significantly higher antibody levels (P less than 0.003) to these three exoproducts than uninfected, hospitalized patients. Parke-Davis type 1 was the strain most commonly isolated from these patients (46%), but colonization or infection due to this organism usually resulted in the production of low levels of antibody to Pseudomonas exoproducts. However, infections with Parke-David type 7 organisms were always associated with intermediate- and high-responder sera to exotoxin A. These results indicated that potentially toxic products were elaborated during the course of cancer-related colonization and infection with P. aeruginosa.     

Epidemiology of chronic Pseudomonas aeruginosa infections in cystic fibrosis.

Chronic lung infection with Pseudomonas aeruginosa is primarily responsible for pulmonary deterioration of cystic fibrosis patients. The purpose of this study was to type the P. aeruginosa isolates collected sequentially from cystic fibrosis patients, chronically colonized with P. aeruginosa, by random amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR). Sequential P. aeruginosa isolates (n: 130) that had been collected from 20 CF patients over at least 9 years were investigated. The isolates were analyzed by RAPD-PCR using two arbitrary primers. Antimicrobial susceptibility testing of all isolates was performed by the disc diffusion method. RAPD-PCR typing demonstrated that strains dissimilar in colony morphotype and of different antibiotic susceptibility patterns could be of the same genotype. Some CF patients were colonized with a rather constant P. aeruginosa flora, with strains of different phenotypes but of one genotype. However, some patients may be colonized with more than one genotype. The results also demonstrated that there might be a risk of cross-colonization between CF patients followed-up at the same center.     

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.     

Phagocytosis of Pseudomonas aeruginosa by polymorphonuclear leukocytes and monocytes: effect of cystic fibrosis serum.

It has been shown previously that serum from chronically infected patients with cystic fibrosis inhibits the phagocytosis of Pseudomonas aeruginosa by both normal and cystic fibrosis alveolar macrophages. In the present study, the ability of peripheral monocytes and polymorphonuclear leukocytes from normal volunteers and cystic fibrosis patients to phagocytize P. aeruginosa was shown not to be inhibited in the presence of serum from cystic fibrosis patients.     

Isolation and characterization of a relevant Aspergillus fumigatus antigen with IgG- and IgE-binding activity

An immunologically relevant antigen fraction was isolated from a cytoplasmic extract of Aspergillus fumigatus mycelium. The fraction was obtained by concanavalin-A affinity chromatography and hydrophobic interaction chromatography. Two protein bands were discernible in polyacrylamide gel electrophoresis while two precipitin peaks were detected in crossed immunoelectrophoresis. When tested against sera from patients with Aspergillus-induced diseases, the fraction showed both IgG and IgE antibody-binding activity. All of the sera studied from patients with allergic bronchopulmonary aspergillosis (ABPA) showed high IgG and IgE antibody titers, while sera from patients with aspergilloma and cystic fibrosis with ABPA demonstrated high IgG titers and only a moderate increase in IgE titers. None of the other groups showed any significant antibody titers against this antigen in their sera. Because of its binding to both IgG and IgE antibodies this fraction was found to be useful in the immunodiagnosis of Aspergillus-induced diseases.     

Serum bactericidal effect on Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The bactericidal activity against Pseudomonas aeruginosa strains isolated from cystic fibrosis patients was determined in a 10% concentration of normal serum or autologous cystic fibrosis serum. Of the 167 strains tested, 77 (46%) were sensitive (greater than 95% killed) in normal serum. Mucoid strains were more frequently sensitive than nonmucoid strains. Twenty-three sensitive strains tested in ethyleneglycoltetraacetic acid-chelated serum were resistant (less than 10% killed), suggesting only classical pathway activation. Absorption of cystic fibrosis serum with the autologous P. aeruginosa strain resulted in decreased killing by that serum. All sera, including the chelated and absorbed sera, had comparable total hemolytic complement levels. Patients in poor clinical condition (5 out of 12), in contrast to patients in good or moderate condition(1 out of 30), were more likely to have P. aeruginosa strains that were serum resistant in autologous serum but sensitive in normal serum. Sera from these five patients in poor clinical condition were capable of killing heterologous P. aeruginosa strains. These results suggest the presence of a protective or "blocking" activity in serum from some patients in poor clinical conditions. This association of a blocking activity with clinical condition may signal a transition point in the progression of cystic fibrosis lung disease and thus may be another contributory factor in the failure of the cystic fibrosis host to control infection.     

The levels and bactericidal capacity of antibodies directed against the UspA1 and UspA2 outer membrane proteins of Moraxella (Branhamella) catarrhalis in adults and children

The UspA1 and UspA2 proteins from Moraxella catarrhalis share antigenic epitopes and are promising vaccine candidates. In this study, the levels and bactericidal activities of antibodies in sera from healthy adults and children toward UspA1 and UspA2 from the O35E strain were measured. Human sera contained antibodies to both proteins, and the levels of immunoglobulin G (IgG) antibodies were age dependent. Adult sera had significantly higher titers of IgG than child sera (P < 0.01). The IgG3 titers to the UspA proteins were higher than the IgG1 titers in the adults' sera, while the IgG1 titers were higher than the IgG3 titers in the children's sera (P < 0.05). The IgG antibodies in the sera from 2-month-old children appeared to be maternally derived, since the mean titer was significantly higher than that in sera from 6- to 7-month-old children (P < 0.05). Serum IgA antibodies to both UspA1 and UspA2 were low during the first 7 months of age but thereafter gradually increased along with the IgG titers. Analysis of sera absorbed with UspA1 or UspA2 showed that the antibodies to UspA1 and UspA2 were cross-reactive with each other and associated with serum bactericidal activity. Examination of affinity-purified human antibodies confirmed that naturally acquired antibodies to UspA1 and UspA2 were bactericidal and cross-reactive. These results support using UspA1 and UspA2 in a vaccine to prevent M. catarrhalis infections.     

Antiphagocytic Effect of Slime from a Mucoid Strain of Pseudomonas aeruginosa

Mucoid strains of Pseudomonas aeruginosa produce a viscid slime when grown on the surface of agar media. These strains are known to colonize persistently the tracheobronchial tree of children with cystic fibrosis. Colonization may result from inhibition of phagocytosis due to slime produced by the organism. Slime separated from one mucoid strain was examined to determine whether it possessed antiphagocytic activity in vitro. Cells of P. aeruginosa, Escherichia coli, and Staphylococcus aureus were rapidly phagocytized by rabbit polymorphonuclear leukocytes when mixtures were rotated for 2 hr at 37 C in the absence of slime. The addition of relatively small amounts of slime to bacteria and leukocytes inhibited phagocytosis as measured by phagocytic killing of the organisms. Inhibition was found to be most complete with P. aeruginosa.     

Pseudomonas Aeruginosa Allergy in Cystic Fibrosis

Basophil histamine release by P. aeruginosa standard antigen was examined in cystic fibrosis patients chronically infected with mucoid P. aeruginosa (CF +P) and with pronounced antibody response against these bacteria, and in patients without P. aeruginosa infection (CF +P). All the patients showed eosinophil counts and total IgE, which did not differ significantly from that of normal persons. In the absence of patient's sera, histamine release was only found in two patients in the CF +P group, indicating that type I allergy to P. aeruginosa is not predominating in cystic fibrosis. In the presence of patients' sere significantly more of the CF+P patients responded to P. aeruginosa with histamine release compared with the CF-P patients. The response was lost by complement inactivation and regained by reconstitution of the complement activity. The involvement of a type III-mediated complement-dependent histamine release is therefore suggested in the pathogenesis of lung damage in cystic fibrosis.     

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.