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The potential for IL-5 to regulate human B cells is controversial despite its well established role as a regulatory factor for murine B cells. We hypothesized that the mechanism by which human B cells were stimulated would, as with murine B cells, determine their potential to respond to IL-5. Since Staphylococcus aureus Cowan strain I (SAC) and Moraxella catarrhalis (MCat) stimulate human B cells by distinct interactions with cell-surface Ig, we compared their potential to induce an IL-5-responsive state by human B cells purified to homogeneity. Neither SAC alone nor SAC plus IL-5 stimulated Ig production, although microgram quantities of IgM were produced with SAC plus IL-2. In contrast, MCat induced microgram quantities of IgM by B cells in the absence of exogenous cytokines, and IL-5 significantly increased IgM production over twofold in the majority of donors. Synergism of IL-5 and IL-2 was detected using suboptimal concentrations of IL-2 with MCat-, but not SAC-, stimulated B cells. Donor B cells unresponsive to IL-5 when stimulated with MCat, became IL-5 responsive in the presence of IL-2. Since message for the IL-5R alpha, IL-5R beta, and soluble IL-5R alpha chains was detected in freshly isolated B cells, we further investigated whether IL-5 responsiveness to MCat, but not SAC, was due to their differential regulation of IL-5R mRNA. Surprisingly, stimulation by either MCat or SAC, without or with IL-2, increased both IL-5R alpha and IL-5R beta mRNA and decreased soluble IL-5R alpha mRNA. These studies demonstrate that, as with murine B cells, human B cells express message for IL-5R but can respond to IL-5 only if appropriately stimulated to undergo terminal differentiation.

Bacterial antibodies were studied in acute, intermediate and convalescent phase sera (mean duration from first to last sample 36 days) of 121 children hospitalized for acute lower respiratory tract infection. Antibody responses were observed in 45% of all cases and in 29% of the 21 children < 1 year old. A total of 15 responses to Streptococcus pneumoniae (pneumolysin), 20 to Haemophilus influenzae, 9 to Moraxella catarrhalis, 3 to chlamydiae and 8 to Mycoplasma pneumoniae were found. In 79 patients with 4 consecutive samples available, 52% of the 31 responses were measurable within 5 days from admission. Overall the responses were not associated with upper respiratory tract bacterial findings or acute otitis media. Significantly more responses were found in the 121 children with acute lower respiratory tract infection than in healthy controls (P < 0.007). We conclude that bacterial antibody assays provide a useful tool in the study of the etiology of acute lower respiratory tract infection in young children, even if the interval between paired serum samples is short.

The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier 'culture and spot' test. Children (age 4-13; n = 303) and patients (n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P << 0.0001). In young children (age 4-5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.

Recently, we showed that complement resistance is an important virulence factor of Moraxella (Branhamella) catarrhalis. Our study used a serum bactericidal assay to determine complement resistance in M. catarrhalis. Although the serum bactericidal assay is considered the "gold standard" for determining complement resistance, it is laborious and time-consuming and therefore not well suited for large-scale studies. Using a large number (n = 324) of M. catarrhalis isolates obtained from the sputa of patients with lower respiratory tract infections (n = 200) and young carriers (n = 124), we assessed the value of a simple "culture-and-spot" test as an alternative to the serum bactericidal assay. For both groups of isolates, the degree of concordance between the two tests used was very significant (P < 0.0001). The agreement between the two assays was estimated to be "excellent beyond chance" (as determined by Cohen's kappa test). The culture-and-spot assay is a valuable alternative to the serum bactericidal assay, not only for screening purposes as shown here but also for studying the mechanism of complement resistance in M. catarrhalis at the molecular level.

Moraxella (Branhamella) catarrhalis is a common cause of lower respiratory tract infections in adults and of otitis media in children. Little is known about the human immune response to this bacterium. In this study, immunoblot assays were performed to detect serum immunoglobulin G antibodies directed at purified outer membrane of M. catarrhalis. Twelve serum samples, two each from six patients with bronchiectasis who were persistently colonized with this organism, were tested with their homologous M. catarrhalis sputum isolates. In all the sera, the most prominent and consistent antibody response was to a minor 84-kDa outer membrane protein, OMP B1. Immunoblot adsorption assays show that these antibodies recognize surface exposed epitopes on OMP B1. Further analysis of human serum antibodies eluted from the surface of intact bacterial cells shows that these surface-exposed epitopes on OMP B1 are heterogeneous among strains of M. catarrhalis. OMP B1 is therefore an important OMP antigen on the surface of M. catarrhalis for the human immune response to infection by this bacterium.

An enzyme immunoassay (EIA) was used to determine the antibody response to different serotypes of lipopolysaccharide (LPS) antigens of Moraxella catarrhalis in adult patients with lower respiratory tract infections (LRTI). Moraxella catarrhalis was isolated from sputum or nasopharyngeal samples from 20 patients with LRTI. Sixteen of the isolates were serotype A, four were type B and none were type C. The antibody response to the different LPS serotypes was determined in paired sera from patients suffering from LRTI. In addition to the 20 patients with Moraxella catarrhalis isolated (Group 1), a group of seven patients with LRTI of unknown etiology (Group 2) and a group of ten patients with LRTI of known other bacterial etiology (Group 3) were selected for this study. An increase in antibody levels of > 1.5-fold (convalescent-/acute-phase serum) was recorded in approximately half of the patients, not only in the first group (Moraxella catarrhalis isolated) but also in the other two groups. However, in the first and second groups there was a correlation between an increase in antibody levels in the LPS EIA and in an EIA using whole bacterial cells as antigen. In the group of patients in whom Moraxella catarrhalis was isolated, the antibody response to LPS antigens was not serotype specific. The antibody response to type-A and type-B LPS was more predominant than the response to type-C LPS in most patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The oligosaccharide parts from Moraxella (Branhamella) catarrhalis serotype C lipooligosaccharides were isolated by mild acid hydrolysis followed by gel permeation chromatography. Four different oligosaccharides could be identified from strain RS26 and two from strain RS10. The structures of the O-oligosaccharides were established by methylation analyses, mass spectrometry, and NMR spectroscopy. It is concluded that the oligosaccharide O-antigens from RS26 are a mixture of octa-, deca-, and undeca-saccharides, and most likely a heptasaccharide. Strain RS10 contains the deca- and the undeca-saccharide only. The structures for the oligosaccharides are shown below. [formula: see text] OS(7) [formula: see text] OS(8) [formula: see text] OS(10) [formula: see text] OS(11) Methylation analysis of the intact lipooligosaccharides showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. It also showed that they consisted of a lipid A portion with 6-substituted glucosamine residues.

Clinical isolates of Moraxella catarrhalis (n = 86) were evaluated for their haemagglutinating activity with different types of erythrocytes. Of all the isolates tested, 12 did not agglutinate with any of the erythrocytes, whereas 65 reacted with human erythrocytes of type A, B, and 0, and 26 with erythrocytes from rabbit, guinea pig, dog, or rat. None of the isolates agglutinated with sheep and goat erythrocytes. The agglutination titres ranged from 0 to 64. Among these isolates, 13 different agglutination patterns could be distinguished. The agglutinating activity was Ca(2+)-dependent and was inhibited by proteases, by temperatures exceeding 50 degrees C and by the addition of D-glucosamine or D-galactosamine. The adherence capacity of the M. catarrhalis isolates to tracheal epithelium correlated with their agglutination titre and could be inhibited by the same treatments. These data provide strong evidence that adherence of M. catarrhalis is mediated by lectins located on the bacterial surface.

The role of Moraxella (Branhamella) catarrhalis as a respiratory tract pathogen is increasingly recognized. We looked at the human immune response against individual outer membrane proteins of M. catarrhalis and against the 81-kDa CopB protein, which has previously been shown to be a target for protective antibodies. Paired serum samples from six elderly patients with pneumonia were tested by Western blot (immunoblot) analysis by using outer membrane vesicles of M. catarrhalis 035E as antigen. All of the six convalescent-phase serum samples reacted with a protein which migrated at the position of the CopB protein and with a high-molecular-weight protein of M. catarrhalis; three serum samples also reacted with a 34-kDa outer membrane protein. Paired serum samples from 18 patients, 10 of which had M. catarrhalis infection on the basis of previous serology results, were tested by enzyme immunoassay (EIA) with the CopB protein and whole cells of M. catarrhalis 035E as antigens. Nine patients showed a significant rise in EIA titer between acute- and convalescent-phase sera when whole bacterial cells were used as antigens. Six (67%) patient samples that were positive by the EIA with the whole-cell antigen were also positive by the EIA with the CopB antigen, and six of nine patient samples negative by the EIA with the whole-cell antigen were also negative by the EIA with the CopB antigen. These results suggest that both the CopB and a high-molecular-weight protein are major targets of the immune response against M. catarrhalis, and further studies with greater amounts of patient materials are needed to elucidate the usefulness of CopB as an antigen in etiologic studies.

We searched for antibodies against Moraxella (Branhamella) catarrhalis proteins in the sera of patients with lower respiratory tract infection. Sera from 48 patients with M. catarrhalis and 39 patients without M. catarrhalis in their lower respiratory tract specimens were studied by a gel electrophoresis-immunoperoxidase technique; sera from 23 healthy adult blood donors were also included. Immunoglobulin G (IgG) antibodies against a 28-kDa protein were found significantly more frequently in patients with M. catarrhalis in lower respiratory tract specimens (71%) than in patients without M. catarrhalis in lower respiratory tract specimens (28%) or healthy adult blood donors (22%). Seroconversion, from the acute to the convalescent stages, occurred in at least eight patients with M. catarrhalis and in one patient without detectable M. catarrhalis. IgG antibodies against other M. catarrhalis proteins were found in most sera, including those obtained from blood donors. By adsorption experiments the 28-kDa protein was demonstrated to be surface exposed. IgM antibodies against an 85-kDa protein were found in serum from one patient from whom M. catarrhalis and Streptococcus pneumoniae were isolated from the lower respiratory tract, while IgA antibodies against M. catarrhalis proteins could not be detected in any serum specimen.

Previously, Gm allotypes have been shown to influence human serum Ig subclass levels as well as the Ab levels achieved after Ag stimulation. The majority of the latter studies have focused on Ab responses to polysaccharide Ags. In this study, we have investigated the relationship between Gm allotypes and naturally occurring serum Ab levels to a bacterial protein Ag, the outer membrane proteins of a common microorganism, Moraxella catarrhalis. In the sera of 160 patients having chronic/recurrent sinusitis, there was a highly significant correlation between the level of specific anti-M. catarrhalis IgG3 level and certain Gm phenotypes. After additional investigation, we found that the presence of G3m(21) homozygosity correlated significantly with lower levels of Ag-specific IgG3. Specific anti-M. catarrhalis IgG3 levels were found to be independent of total serum IgG3 concentrations, and there was no correlation between the serum level of IgG3 and any Gm phenotype. Total IgG and IgG2 that were specific for pneumococcal cell wall polysaccharide also were measured in this group of patients, and no correlation was found between the naturally occurring IgG2 subclass levels to pneumococcal cell wall polysaccharide and the interactive effect of G2m(23) (syn: G2m(n)) and Km(1). Gm allotypes may influence Ab responses other than the anti-carbohydrate responses and, therefore, should be taken into account when investigating IgG subclass responses to protein Ags.

A monoclonal antibody (MAb) to Moraxella catarrhalis O35E bound to a surface-exposed epitope of a proteinaceous antigen of this organism. The antigen, designated UspA, was present in every strain of the pathogen tested in a colony blot RIA. UspA had a molecular mass on SDS-PAGE that varied between 300 and 400 kDa, depending on the individual M. catarrhalis strain. Passive immunization of mice with the UspA-reactive Mab enhanced pulmonary clearance of M. catarrhalis. Use of this Mab to screen a M. catarrhalis genomic DNA library permitted identification of a recombinant bacteriophage expressing the M. catarrhalis UspA protein. The recombinant UspA protein was used in Western blot analysis with sera from patients with M. catarrhalis pneumonia. Convalescent-phase sera but not acute-phase sera from these patients contained antibodies to this M. catarrhalis surface protein, indicating that M. catarrhalis strains growing in vivo express this molecule.

The IgG antibody response to streptococcal M protein is distributed between the IgG1 and IgG3 subclasses, however individual sera vary with respect to the relative amounts of these two subclasses. The basis of this variation was investigated. Sera were also analysed for IgG subclass antibodies to the outer membrane proteins (OMP) of Moraxella catarrhalis, as these have also been reported to have a major IgG3 component. The mean percentage of IgG3 was higher in the antibody response to OMP and there was less variability between sera for this antigen than was seen for M protein. Non-specific binding of IgG3 in ELISA, which has been reported for some bacterial proteins (including M protein of some serotypes) was excluded as an explanation for the apparent IgG3 bias of these antibodies. The relative amount of IgG3 antibody to the two antigens showed a positive correlation, suggesting that some individuals tended to make a greater IgG3 response to unrelated antigens. Serial bleeds from two individuals maintained a relatively constant subclass profile over several months, suggesting that time since infection did not play a major role in determining the proportion of IgG1 and IgG3. Gm allotypes for the sera were determined, and found to correlate with both total serum IgG3 concentrations and with IgG subclass composition of specific antibodies. Mean serum IgG3 concentrations were highest in sera typed as Gm(fb/fb) homozygous and lowest in sera typed as Gm(ag/ag) homozygous. Similarly, in the M protein-specific antibodies, the mean percentage of IgG3 was much lower in the Gm(ag/ag) sera than in the Gm(fb/fb) homozygous sera. Sera which typed as Gm(fb/ag) heterozygous were not significantly different from the Gm(fb/fb) homozygous sera for either total serum IgG3 or for M protein-specific IgG3. Moreover, both Gm(fb/fb) homozygous and Gm(fb/ag) heterozygous sera included samples in which IgG1 was the predominant antibody subclass and the percentage of IgG3 was very low. In contrast to the M protein-specific antibodies, for the OMP-specific antibodies there was no correlation between Gm phenotype and the proportion of IgG3. The data suggest that Gm allotype may influence the IgG subclass composition of antibody responses to bacterial surface protein, but that other factors are also likely to be involved.

Otitis media was introduced in chinchillas by direct transbullar inoculation with either viable or formalin-inactivated Moraxella (Branhamella) catarrhalis. Both groups of animals developed middle ear fluids (MEF) and severe inflammatory changes in the tubotympanum. Pure cultures of M. catarrhalis were recovered for up to 5 days after inoculation from those animals inoculated with viable bacteria. Significantly elevated anti-M. catarrhalis antibody titers were detected in post-inoculation sera and in MEF or bullar lavages, and an increased number of IgG-bearing cells was also observed in the tubotympanum of these animals. Control chinchillas inoculated with pyrogen-free sterile saline failed to show any signs of otitis media. Our data indicate that viable M. catarrhalis induce acute otitis media in the chinchilla but that the bacteria are rapidly eliminated from the middle ear, precluding its usefulness as a model to study acute otitis media. Moreover, the same concentration of nonviable organisms also induces severe inflammatory changes in the middle ear. These data indicate that the chinchilla may be a useful model for studying the role of nonviable M. catarrhalis or its cell wall components on the induction and persistence of inflammation in the middle ear.

Antibodies against nonencapsulated Haemophilus influenzae and Moraxella (Branhamella) catarrhalis were measured by ELISA in paired sera from 158 adult patients with pneumonia. A mixture of 10 clinical isolates of each species was used as antigen. Eleven patients (7%) showed significant increases in antibody to H. influenzae. In 3 of them, the organism was isolated from transtracheal aspirate and in another 7 from sputum, nasopharynx, or both. Six patients with nonencapsulated H. influenzae in transtracheal aspirate cultures did not show any antibody increase. Six patients had significant increases in antibody to M. catarrhalis. The organism was isolated in transtracheal aspirates from 1 of them and in sputum and nasopharynx (or both) from another 3. Two patients with M. catarrhalis in transtracheal aspirate cultures showed no antibody response. In conclusion, the serologic methods increased the possibility to diagnose infections caused by the two agents but had low sensitivity.

An enzyme-linked immunosorbent assay (ELISA) for determination of serum immunoglobulin G (IgG) antibodies to Moraxella catarrhalis was developed, with an ultrasonic extract of M. catarrhalis immobilized on polystyrene microtiter plates serving as the antigen. The specificity was determined by adsorption tests. All of the 541 women tested showed a high level of maternal IgG antibodies to M. catarrhalis in umbilical cord blood specimens. One hundred eighty-nine children aged 0 to 15 years were examined. A low level of IgG antibodies to M. catarrhalis in serum was found in children aged up to 1 year; in older children, the levels increased with age. Levels in the same range as maternal IgG antibody levels were reached at the age of 10 years. The level of antibodies in children did not correlate with the state of colonization with M. catarrhalis or with the state of acute lower respiratory tract infection. Pairs of acute-phase and convalescent-phase serum samples did not discriminate between the children with M. catarrhalis in pure culture and those with mixed cultures of M. catarrhalis, Haemophilus influenzae, and Streptococcus pneumoniae. In adult women, high IgG antibody levels and low colonization rates with M. catarrhalis were found, whereas in small children, low IgG antibody levels and high colonization rates were found.

Opsonic antibody activity against Moraxella catarrhalis was determined in sera from children with otitis media. The antibody was determined with a new assay utilizing outer membrane antigen-coated latex beads. Antigen-coated beads opsonized in heat-inactivated pooled human serum phagocytosed 47.5 +/- 36.1 beads per 100 neutrophils compared to 15.6 +/- 10.2 beads per 100 neutrophils opsonized in hypogammaglobulinemic serum (p < .025). Antigen-coated beads opsonized in homologous sera from 11 children with M. catarrhalis otitis media demonstrated increased opsonic activity in convalescent sera (34.6 +/- 27.1) compared to acute sera (15.5 +/- 6.7; p < .05). These data suggest that infection with M catarrhalis is associated with the development of opsonic antibody.

The polysaccharide of the Moraxella (Branhamella) catarrhalis serotype A lipopolysaccharide was prepared by mild acid hydrolysis followed by gel permeation chromatography. The structure was established by methylation analysis, mass spectrometry, and NMR spectroscopy. It is concluded that the O-antigenic polysaccharide has the following structure. [formula see text] Methylation analysis of the intact lipopolysaccharide showed that the lipid A portion consisted of 6-substituted glucosamine residues. Methylation followed by methanolysis showed that two Kdo residues were present, one terminal and one 4,5-substituted residue. A terminal Kdo thus substitutes the branch-point Kdo in the 4-position.

Recurrent acute otitis media (AOM) is an extremely prevalent disease in young children. Epidemiologic associations suggest that primary prevention or reduction of AOM frequency may be achieved with breast-feeding during infancy, elimination of household tobacco smoking, and use of small rather than large day-care arrangements for infants and toddlers. Secondary antimicrobial prophylaxis with amoxicillin or sulfisoxazole reduces the frequency of recurrent AOM by about 50%, but it does not appear to reduce the duration of otitis media with effusion (OME). Tympanostomy tube insertion is not as effective as amoxicillin in reducing AOM frequency in children without OME. Adenoidectomy appears to be warranted for children who develop recurrent AOM after extrusion of tubes. Vaccines against the common bacteria and viruses causing AOM hold the greatest promise of preventing AOM and blocking the sequence of pathologic events leading to chronic OME and middle ear sequelae. The greatest progress has been made recently with pneumococcal protein conjugate vaccines, and clinical testing is in progress.

Immunohistochemical analysis of challenge sites such as skin and the peritoneal cavity has identified neutrophils as virtually the sole cellular participants in acute bacterial inflammation, peak influx occurring 24-48 h in advance of mononuclear cell populations associated with adaptive immunity. This study challenges the general applicability of this paradigm. We demonstrate here that the earliest detectable cellular response after inhalation of Moraxella catarrhalis organisms is the recruitment of putative class II major histocompatibility complex-bearing dendritic cell (DC) precursors into the airway epithelium, the initial wave arriving in advance of the neutrophil influx. Unlike the neutrophils which rapidly transit into the airway lumen, the DC precursors remain within the epithelium during the acute inflammatory response where they differentiate, and develop the dendriform morphology typical of resident DC found in the normal epithelium. During the ensuing 48-h period, these cells then migrate to the regional lymph nodes. No comparable DC response was observed after epidermal or intraperitoneal challenge, and it may be that mucosal surfaces are unique in their requirement for rapid DC responses during acute inflammation. We hypothesize that the role of the DC influx during acute inflammation may be surveillance for opportunistic viruses, and that this covert protective mechanism is operative at a restricted number of mucosal tissue sites.

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

The mechanism of resistance to human complement-mediated killing in Moraxella catarrhalis was studied by comparing different complement-sensitive and complement-resistant M. catarrhalis strains in a functional bystander hemolysis assay and an enzyme-linked immunosorbent assay (ELISA) for soluble terminal complement complexes. Complement-resistant stains appeared to activate complement to the same extent as, or even slightly better than, complement-sensitive strains. This indicates that complement-resistant strains do not inhibit classical or alternative pathway activation but interfere with complement at the level of membrane attack complex formation. A clear difference in dose-response curves for resistant and sensitive strains was observed both in the bystander hemolysis assay and in the ELISA. Complement-resistant strains showed optimum curves, whereas complement-sensitive strains gave almost linear curves. We conclude that resistant strains bind and/or inactivate one of the terminal complement components or intermediates involved in membrane attack complex formation. Trypsin, known to abolish complement resistance, changed the optimum dose-response curve of a resistant strain to a linear one, which strongly suggests that complement resistance is mediated by an M. catarrhalis-associated protein. This protein acts directly or through the binding of a terminal complement inhibitor present in serum.

In 284 Swedish children with community-acquired, roentgenologically verified pneumonia, antibodies to Moraxella (Branhamella) catarrhalis were determined in paired serum samples with an enzyme immunoassay using a whole-cell antigen preparation from 10 strains of M. catarrhalis. Only 9 children (3%) had significant increases in antibodies to M. catarrhalis. Among these 9 children, 11-39 months of age, 6 had serologic evidence of concurrent infection with other respiratory pathogens such as S. pneumoniae, non-capsulated H. influenza, RS virus and adenovirus. In 6 (67%) of the 9 children with antibody response and in 74 (27%) of the 275 children without antibody response to M. catarrhalis, nasopharyngeal cultures yielded growth of this bacterium. M. catarrhalis seems to be a common commensal in the upper respiratory tract, but a rare cause of pneumonia in children.

The significance of the isolation of Moraxella catarrhalis from sputum or nasopharynx was studied in patients treated at an infectious disease clinic. A whole-cell enzyme immunoassay was used to detect a specific antibody response to M. catarrhalis during infection. In all, 27 patients with respiratory tract infections and 4 with other infections were studied. Titre rises were recorded in 11/23 patients with lower respiratory tract infections, whereas patients with common cold or infections elsewhere all had negative serology. In patients with acute bronchitis, 7/10 patients responded with a significant titre rise. Patients with a low titre in their acute serum sample were those who responded with a titre increase during infection. The findings indicate that isolation of M. catarrhalis from sputa and nasopharyngeal samples in adults is of value for the etiological diagnosis of acute bronchitis and other lower respiratory tract infections, and is therefore important for the choice of drug for treatment, as many isolates are resistant to beta-lactam antibiotics.

Solid phase assays for the measurement of functional antibody affinity are increasingly being used in both clinical and research settings. The majority of such assays employ a chemical reagent to disturb antibody binding but relatively little is known about the properties of such reagents and the basis of their effect on antigen-antibody binding. We have evaluated the diethylamine (DEA) ELISA procedure for the measurement of functional antibody affinity in two independent assays, one for functional human IgG subclass affinity to an organism, Moraxella catarrhalis, and the other for measuring functional affinity of mouse monoclonals specific for the cat allergen Fel d I. DEA was shown to increase the pH of the buffering solution and it was this rise in pH that affected antibody binding. Alkaline buffer and DEA were equally efficient in the inhibition of binding of both the human IgG subclasses and the two mouse monoclonal antibodies to the solid phase. In contrast, pH was shown to have no role in the chaotropic effect of the ion, thiocyanate.

A major outer membrane protein (CopB) of Moraxella catarrhalis is a target for antibodies that enhance clearance of this organism from the lungs of mice. A mini-Tn10kan transposon was inserted into the cloned copB gene from M. catarrhalis O35E, and an isogenic mutant unable to express the CopB protein was constructed by transforming this mutated gene into the wild-type strain. The mutant grew at the same rate as the wild-type parent strain in broth. Unlike the serum-resistant parent strain, this mutant was sensitive to killing by normal human serum, and its ability to survive and grow in the lungs of animals was impaired. Genetic restoration of CopB protein expression resulted in the simultaneous acquisition of wild-type levels of serum resistance and the ability to resist pulmonary clearance in vivo. Thus, the CopB protein of M. catarrhalis may be important in the interaction between this organism and the defense mechanisms of the respiratory tract.

A murine model for pulmonary clearance of Moraxella catarrhalis was used to determine whether immunization could enhance clearance of this organism from the lungs. Animals actively immunized with outer membrane vesicles of M. catarrhalis cleared an endobronchial challenge with the homologous strain more quickly than did sham-immunized control animals. Western blot analysis of both this immune mouse serum and rabbit antiserum raised against outer membrane vesicles of M. catarrhalis indicated that antibodies were present to both outer membrane protein and lipooligosaccharide antigens. Passive immunization of mice with the immune rabbit serum resulted in enhanced pulmonary clearance of both homologous and heterologous strains of M. catarrhalis, indicating the involvement of serum antibody in this clearance process and the existence of conserved surface antigens in the two different M. catarrhalis strains. These results suggest that this model system may be useful for the identification of vaccine candidates among the surface antigens of M. catarrhalis.

A murine immunoglobulin G monoclonal antibody (MAb) raised against outer membrane vesicles of Moraxella catarrhalis 035E was shown to bind to a surface-exposed epitope of a major outer membrane protein of this organism. This outer membrane protein, which had an apparent molecular weight of approximately 80,000 in sodium dodecyl sulfate-polyacrylamide gels, was designated CopB. MAb 10F3, reactive with CopB, bound to a majority (70%) of M. catarrhalis strains tested. More importantly, mice passively immunized with MAb 10F3 exhibited an enhanced ability to clear a bolus challenge of M. catarrhalis from their lungs, a result which suggested that CopB might have potential as a vaccine candidate. The M. catarrhalis gene encoding CopB was cloned in Escherichia coli, and nucleotide sequence analysis of the copB gene indicated that the CopB protein was synthesized with a leader peptide, a finding confirmed by N-terminal amino acid sequence analysis of the mature CopB protein purified from M. catarrhalis 035E. Southern blot analysis showed that chromosomal DNA from seven different M. catarrhalis strains hybridized with a probe comprising the majority of the copB structural gene from strain 035E. Additional data emphasizing the extent of conservation of the CopB protein among M. catarrhalis strains were obtained from Western immunoblot analyses with polyclonal antisera raised against CopB proteins from different M. catarrhalis strains used to probe the recombinant form of the CopB protein from strain 035E. The ability of the CopB protein to function as a target for biologically active antibodies and its apparent conservation among M. catarrhalis strains warrant further investigation of this outer membrane protein as a potential vaccine candidate.

Oropharyngeal bacteria belonging to different species were tested for serological cross-reactions with Moraxella catarrhalis using sera from immunized rabbits. Sera were tested using immunofluorescence, an enzyme immunoassay (EIA) and immunoblotting. On immunofluorescence, significant cross-reactions were demonstrated with beta-hemolytic streptococci group A and group G, as well as with streptococci of the viridans group. Some cross-reactions were also noted with Neisseria meningitidis. In the EIA, strong cross-reactions were demonstrated with beta-hemolytic streptococci. No cross-reactions were obtained with Streptococcus pneumoniae, Haemophilus influenzae or common oral Neisseria. The results are of importance for the interpretation of serological tests to detect infections with Moraxella catarrhalis, and for the development of methods for detection of antigens in samples from the respiratory tract.

The proportions of secretory IgA (SIgA)-, IgG- and C3b-coated bacteria obtained from a well-defined area on the posterior wall of the nasopharynx (NPH) close to the Eustachian tube were determined. Samples taken from 25 otitis-prone (OP) and 25 non-otitis-prone (NOP) children with normal serum levels of IgA and IgG were evaluated using an immunofluorescence assay. Both groups harboured significantly more nasopharyngeal bacteria coated with IgG than with SIgA (p < 0.001). The OP children had significantly fewer SIgA-coated bacteria (p < 0.05) but more C3b-coated bacteria (p < 0.01) in the NPH than the NOP children had. No significant difference was noted between the two groups regarding IgG coating. The occurrence of Branhamella catarrhalis in the NHP was more pronounced in the OP group (p < 0.05). No significant differences in the occurrence of other middle ear pathogens (Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus) or quantitative dominance of pathogens were noted between the two groups. Deficiency in SIgA coating of the nasopharyngeal bacteria may contribute to the otitis-prone condition.

Two monoclonal antibodies (G6 and 7B), generated against a 63-kDa stress protein (GSP63) from Neisseria gonorrhoeae strain VP1, were used to investigate the antigenic heterogeneity of GSP63 among the Neisseriaceae and its antigenic relationship with the Hsp60 heat-shock protein family. Immunoblotting experiments demonstrated antibody reactivity with all pathogenic Neisseria tested and with some of the commensal strains. One of the antibodies (7B) cross-reacted with the 65-kDa M. bovis BCG heat-shock protein and with 14 out of the 21 similarly sized proteins in other bacterial species. The other antibody (G6) specifically recognized neisserial GSP63 homologues. These results demonstrate that GSP63 is a conserved neisserial antigen bearing both a unique neisserial B-cell epitope and a more widely distributed Hsp60 epitope.

The immunomodulatory properties of several lipopolysaccharides (LPS) derived from clinical isolates of Pseudomonas aeruginosa, Branhamella catarrhalis, and Bordetella pertussis were evaluated for their capacity to influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III), which is known to be regulated by suppressor and amplifier T cells (Ts and Ta, respectively). The administration of LPS, two days after immunization resulted in a significant increase in the antibody response. Such enhancement may be due mainly to the ability of the lipid A moiety of LPS to abolish the negative effects of activated Ts, thereby enabling Ta function to be more fully expressed; however, B cell mitogenicity of the LPS molecule also may be involved. By contrast, treatment with LPS at the time of immunization with SSS-III induces significant suppression of the SSS-III-specific antibody response; such suppression is not induced by LPS or lipid A derived from Escherichia coli and Salmonella minnesota, and is independent of the capacity of LPS to activate B cells polyclonally, an activity generally attributed to the lipid A fraction of LPS. Studies conducted with the LPS of P. aeruginosa indicated that the suppression induced is T cell dependent and mediated by the polysaccharide (PS) fraction of LPS; it appears to be due-at least in part-to the capacity of PS to expand or increase the size of the precursor pool of Ts, activated in response to SSS-III. The significance of these findings to the pathogenesis of certain gram-negative infections is discussed.

For serological diagnosis of infection with Moraxella (Branhamella) catarrhalis it is important to determine if there is variability of antigenic properties among different strains. Cross-reactions of nine strains were investigated by an immunofluorescence test using sera from immunized rabbits. All titres but one were 1:256 or higher, the highest being 1:4096. Thus a high degree of antigenic similarity was demonstrated among different strains of Moraxella catarrhalis. However, the homologous titres of six sera were 2 to 16 times higher than the titres for other strains indicating strain variations in antigenic properties of some surface components. There was no correlation between lipopolysaccharide type and titre in the immunofluorescence test.

Moraxella (Branhamella) catarrhalis organisms have been shown to be particularly efficient in inducing in a pure population of bone marrow-derived mononuclear phagocytes secretory and cellular activities. In the present study, the ability of peptidoglycan from this Gram-negative organism to trigger a macrophage response was compared with that elicited by peptidoglycan from Staphylococcus aureus and Bacillus subtilis. The results show that the three peptidoglycans were similarly active in triggering the secretion of tumour necrosis factor and tumouricidal activity but differed considerably in their ability to induce the generation of nitrite in macrophages; in this respect, peptidoglycan from M. catarrhalis was particularly potent. The impressive capacity of M. catarrhalis peptidoglycan to induce in low concentration the secretion of tumour necrosis factor and nitrite and tumouricidal activity may, in addition to its lipopolysaccharide, contribute to the extraordinary potential of this organism to trigger the functional activities of macrophages.

The systemic and local antibody responses to homologous strains of Moraxella catarrhalis were investigated in 14 children with otitis media. A total of 8 children (57%) demonstrated a rise in serum antibody of the immunoglobulin G (IgG) (5 of 14), IgM (5 of 14), or IgA (6 of 14) classes of immunoglobulin to outer membrane antigens. Local antibody consisted of IgG (100%), IgM (29%), and IgA (71%). The IgG and IgA specific antibody present in middle-ear effusions appeared to represent local production rather than passive diffusion from the systemic circulation. These data suggest that young children develop an antibody response to M. catarrhalis in the middle ear during otitis media but fail to develop systemic antibody in a uniform manner.

Anticarbohydrate monoclonal antibodies were tested for their ability to bind to various strains of Neisseria. A monoclonal antibody that binds to the ganglio-series glycosphingolipid, ganglio-N-triaosylceramide, also bound to strains of Neisseria gonorrhoeae but not to other species of Neisseria. An antibody specific for the globo-series glycosphingolipid, globotriaosylceramide, also bound to strains of N. gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, and Branhamella catarrhalis but not to any other strains of nonpathogenic Neisseria.

We characterized lipopolysaccharides (LPSs) from respiratory pathogenic Branhamella catarrhalis (BC) strains, and evaluated the protective property of anti-BC LPS antibody in BC respiratory infections. LPSs from four strains of BC were lipooligosaccharide having no O-side chain and a M(r) of 3 KDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All of them produced different patterns, showing two to four bands on SDS-PAGE. We found high level of anti-BC IgG antibody in convalescent sera from a patient with BC respiratory tract infection by ELISA. This IgG antibody recognized BC LPSs on Western blots. Two respiratory pathogens of BC (strains; 87-122, 88-23) were tested in a bactericidal assay employing a convalescent sera. 87-122 strain was susceptible to antibody-dependent, complement-mediated killing, while 88-23 strain was resistant. The killing of 87-122 strain was inhibited by addition of the homologous BC LPS to the convalescent sera in a dose-dependent manner. These data support that anti-BC LPS antibody may mediate complement-lysis of some strains of BC, and play a protective role in BC respiratory infections.

The virulence mechanisms of Moraxella catarrhalis that are involved in producing pulmonary infection are unknown. A well-characterized murine model was used to study the pulmonary clearance of M. catarrhalis and analyze the histopathologic changes and the role of phagocytic cells in the infected lungs. Ten strains of M. catarrhalis from various isolation sites were evaluated for their ability to resist pulmonary clearance. The rates of clearance of these strains, based on the percentage of the original inoculum remaining at 6 h after challenge, varied considerably. Histopathologic examination of lungs infected with 2 strains that exhibited very different clearance rates revealed similar pathologic responses. Analysis of the phagocytic cell response to these 2 strains revealed significant alveolar recruitment of granulocytes at 3, 6, and 24 h after bacterial challenge. However, granulocyte recruitment in response to strain B22, which was cleared readily, was significantly greater than to strain 035E, which resisted pulmonary clearance. This model system should facilitate investigation of the molecular basis of the interaction between M. catarrhalis and the lower respiratory tract.

Branhamella catarrhalis is a common cause of otitis media in children and of respiratory infections in adults with chronic bronchitis. Little is known about the antigenic structure of the outer membrane proteins (OMPs). In this study, two murine monoclonal antibodies, 7D6 and 5E8, were developed and used to characterize the major heat-modifiable OMP (OMP C/D) of B. catarrhalis. Immunoblot assays indicated that OMP C/D is heat modifiable, having a molecular mass of 55 kDa at room temperature and a mass of 60 kDa when heated under reducing conditions. Expression of the epitopes is independent of growth phase and growth media. Both epitopes are present in 51 of 51 strains of B. catarrhalis tested and are highly specific for Branhamella strains, being absent from a variety of other gram-negative species. Antibody 5E8 recognizes an epitope which is expressed on the surface of the intact bacterium. We conclude that OMP C/D is a major, heat-modifiable OMP antigen that expresses at least one stable, conserved epitope on the surface of B. catarrhalis. Future studies should focus on the role of OMP C/D in pathogenesis and on its potential role as a vaccine antigen.

Immunoglobulin (Ig) class switching appears to be preceded by induction of germ-line transcripts. In this report, we demonstrate that transforming growth factor beta (TGF-beta) induces germ-line transcripts of both the IgA subclasses (IgA1 and IgA2) in Branhamella catarrhalis (BC)-activated human spleen B cells. Two germ-line bands, one of approximately 1.85 kb and the other of approximately 1.6 kb, could be seen in cultures treated with TGF-beta. The approximately 1.85 kb band contains mRNA for a germ-line transcript of the membrane form. This band co-migrates with the productive secreted form of alpha mRNA. The other, shorter form of approximately 1.6 kb did not correlate in size with any known form of productive alpha mRNA and contained the secreted form of germ-line alpha mRNA. The induction of alpha germ-line transcripts was accompanied by a concomitant suppression of mu and gamma mRNA. We have also identified the location of a putative I alpha sequence (designated according to the generally accepted nomenclature) within approximately 0.5 kb upstream to the switch alpha (S alpha) region. The relative proportions of IgA-subclass-specific mRNA in TGF-beta-stimulated spleen B cells are concordant with the distribution pattern seen in pokeweed mitogen (PWM)-stimulated spleen mononuclear cells (MNC), which was 89 and 11% for the IgA1 and the IgA2 mRNA respectively. These results suggest a role of TGF-beta in regulating IgA class switching in human B lymphocytes.

Lipopolysaccharides (LPS) were extracted and purified from the type strain and from a clinical isolate of Branhamella catarrhalis. Chemical analysis revealed the presence of glucose, galactose, and glucosamine in different molar proportions in the LPS from these two isolates, whereas there was no difference between the two isolates in the ratios of ketodeoxyoctonate, phosphate, and the fatty acids C12, 3-OH-C12, and 3-OH-C11 present. Heptose or 3-OH-C14 was not detectable in either preparation. LPS from both strains appeared semirough according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, presenting a core polysaccharide plus one repeating unit. Immunoblotting, passive hemolysis, and hemolysis inhibition assays using anti-LPS antibodies from immunized rabbits demonstrated cross-reactivity between the LPS preparations; however, antigenic dissimilarities were also found, suggesting that more than one serotype may exist. The lipid A isolated from the two LPS was serologically identical and exhibited cross-reactivity with lipid A of members of the family Enterobacteriaceae. The B. catarrhalis LPS were biologically active, causing lethality in D-galactosamine-sensitized C57/BL6 mice and inducing Limulus amoebocyte lysate gelation.

Moraxella (Branhamella) catarrhalis is now a well-recognized pathogen in lower respiratory tract infections, particularly in the setting of chronic lung disease. The ability to produce beta-lactamase, which now characterizes most clinical strains, appears to be a recently acquired trait. The most common clinical syndrome caused by this organism is exacerbation of chronic bronchitis; this syndrome has been well described in Europe, Japan, and the United States, particularly from centers with a large elderly population with chronic lung disease. The syndrome of pneumonia is less common, and suppurative complications and bacteremia are rare.

The occurrence of IgG, IgM and IgA class antibodies against a type-specific capsular polysaccharide of Streptococcus pneumoniae (Pn) and against a whole cell antigen of Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) was studied using the ELISA method on middle ear effusion (MEE) samples of 85 patients and paired serum samples of 40 patients during the course of acute otitis media (AOM). Although specific antibodies to all of these three bacteria appeared in MEE during the course of an AOM episode, antibodies against the infecting bacteria of that particular AOM episode were more often prominent. The antibodies were also detectable in the MEE without simultaneous presence in the serum. The middle ear infection was prolonged more often if specific antibodies to the infecting bacterium could not be detected in the MEE obtained at the beginning of the AOM attack. The present study indicates that AOM caused by Pn, Hi or Br may induce both a systemic and a local production of specific antibodies against the causative organisms during the course of otitis media. The occurrence of such antibodies in MEE seems to play a major role in the resolution of AOM.

The occurrence of specific secretory antibodies against the type-specific capsular polysaccharide of Streptococcus pneumoniae (Pn) and against the whole cell antigen of Haemophilus influenzae (Hi) and Branhamella catarrhalis (Br) were measured by the ELISA method in 211 middle ear effusion (MEE) samples of 85 children with acute otitis media (AOM) during the course of the disease. Antibodies against at least one of those bacteria were detected at the initial visit in 33.6% of the ears and later in 20%. All in all, such antibodies could be found in 50% of the ears during the follow-up. Pneumococcal secretory antibodies were found in 5 out of 116 ears only, anti-Hi antibodies in 52 and anti-Br antibodies in 42 ears. The specific secretory antibodies were detected against all these bacteria regardless of the bacterial etiology of the AOM attack in question. The AOM attack was prolonged more often if such antibodies were not found in the MEE sample taken at the initial visit. The appearance of such antibodies during the disease seemed to imply termination of the AOM episode in question. The conclusions of this study are that during an AOM attack a local production of antibodies in middle ear against the three most common bacteria. Pn, Hi and/or Br, causing AOM may be induced. The appearance of such antibodies in MEE seems to be beneficial for the resolution of AOM.

Clinical isolates of Branhamella catarrhalis from patients with respiratory infections were used in this study. Electron microscopic observation after treating Branhamella catarrhalis with immune serum and ruthenium red revealed the capsule. In the phagocytosis test, most organisms were not ingested by human polymorphonuclear neutrophils in the presence of normal rabbit serum (NRS), while organisms were primarily cell associated and apparently ingested in the presence of immunized rabbit serum (IRS). The capsule may be one of the virulence factors in this bacteria. This study demonstrates the possible presence of a capsule in Branhamella catarrhalis.

By using hot acid extract and double-diffusion studies, an antigen isolated from clinical strains of Moraxella catarrhalis, designated the C antigen, was studied. The antigen is labile, relatively trypsin insensitive, and either polysaccharide or glycoconjugate in nature. At least two serologically distinct C antigens have been identified.

C5-sufficient Swiss-Webster mice (C5+) and C5-deficient DBA/2J mice (C5-) when challenged endotracheally with 2 x 10(7) cfu of Branhamella catarrhalis rapidly clear the lungs of viable bacteria in 48 h. This rapid clearance correlates with a striking influx of polymorphonuclear neutrophils that is of equal magnitude in both C5+ and C5- animals. Supernatant from Todd-Hewitt broth culture of B. catarrhalis exhibits in vivo chemotactic activity in the murine lung and in vitro chemotactic activity in Boyden chambers. The ability of B. catarrhalis to recruit granulocytes, independent of complement, may suggest a role for this organism in promoting protease-mediated lung destruction.